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Molecular basis of calmodulin-dependent calcineurin
activation
Bin Sun1, Darin Vaughan1, Svetlana Tikunova3,
Trevor P. Creamer2, Jonathan P. Davis3and PM Kekenes-Huskey1,4
1Department of Chemistry, University of Kentucky, 2Center for Structural
Biology and Department of Molecular & Cellular Biochemistry, University of
Kentucky, 3Department of Physiology and Cell Biology, Ohio State University, 4
Department of Chemical and Materials Engineering, University of Kentucky
June 8, 2019
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1 INTRODUCTION
Abstract
Calcineurin (CaN) is a calcium-dependent phosphatase involved in numerous signaling
pathways. Its activation by Ca2+ is in part driven by binding of calmodulin (CaM) to a
Calmodulin (CaM)-recognition motif within the phosphatase’s regulatory domain (RD); how-
ever, secondary interactions between CaM and the Calcineurin (CaN) regulatory domain may
be necessary to fully activate CaN [1]. Specifically, it has been shown that the CaN regulatory
domain folds upon CaM binding and that there is a region C-terminal to the canonical CaM-
binding region, the ‘distal helix’, that assumes an αhelix fold and contributes to activation
[1]. We hypothesized in Dunlap et al [1] that this putative αhelical distal helix is capable
of binding CaM in a region distinct from the canonical CaM binding region (CaMBR) site,
whereby CaN is activated. To test this hypothesis, we utilized molecular simulations including
replica-exchange molecular dynamics, protein-protein docking and computational mutagene-
sis to model distal helix conformations. From these simulations we have isolated a potential
binding site on CaM (site D) that facilitates moderate affinity inter-protein interactions that
may attenuate CaN auto-inhibition. Further, molecular simulations of the distal helix A454E
mutation demonstrated weakened distal helix/CaM interactions that were previously shown
to impair CaN activity. K30E and G40D mutations of CaM at site D presented similar de-
creases in binding affinity predicted by simulations. The prediction was correlated with a
phosphatase assay in which these two mutants show reduced CaN activity. This study there-
fore provides a potential structural basis for the role of secondary CaM/CaN interactions in
mediating CaN activation.
1 Introduction
Calcineurin (CaN) is a phosphatase that contributes to gene expression in response to changes in
Ca2+ homeostasis. As such, it plays integral roles in physiological processes including neurological
development and maintenance, immune responses and tissue remodeling [2]. CaN is activated by
rising intracellular Ca2+ levels. It presents modest catalytic activity in response to Ca2+ alone,
but optimal phosphatase activity occurs upon binding Ca2+-saturated CaM. CaN is heterodimeric
protein consisting of two domains: chain A (57-61 kDa) contains the protein’s catalytic site, while
chain B (19 kDa) contributes to enzyme regulation [2,3]. At depressed Ca2+ levels, the enzyme
is inhibited by its auto-inhibitory domain (AID) that directly binds to the phosphatase catalytic
site. Maximal relief from auto-inhibition occurs upon the binding of CaM to CaN’s regulatory
domain.
Our current understanding of the protein’s activation and enzymatic activity has been shaped
by a number of atomic resolution structures of CaN determined by X-ray crystallography [4–9]
and nuclear magnetic resonance spectroscopy [10]. Of the many CaN structures that have been
deposited to the Protein Data Bank are structures that have revealed the protein’s auto-inhibited
state (PDB ID: 1aui [4]), a potentially non-physiological 2:2 CaM/CaN stoichiometric configura-
tion [7,11,12], and complexes of the enzyme with immunosuppressants [5,8] and transcription
factors [6,9]. However, much less is known about the structural basis of CaM-dependent regu-
lation of CaN, as atomic resolution CaM/CaN complexes are limited to the intact CaM bound
to small peptides comprising the CaM binding region (CaMBR) of the CaN regulatory domain
[13]. From those structures, it is clear that the CaM binding region (CaMBR) assumes αhelical
secondary structure when bound to CaM. Nevertheless, the paucity of structural information inclu-
sive of complete CaM and CaN proteins leaves critical details of CaM-dependent CaN regulation
unresolved.
Other experimental studies, however, have shed light on structural mechanism of CaN acti-
vation that have eluded crystallographic and NMR probes. It is increasingly understood that
CaM-dependent CaN activation depends on structural properties of the 95-residue (≈10 kDa)
CaN regulatory domain [14]. This segment is intrinsically disordered [4,13–15], which signifies
that it does not assume a well-defined fold in solution and for this reason has stymied efforts
to determine its structure. Nevertheless, indirect probes of its conformational properties in the
absence and presence of Ca2+-activated CaM that have revealed important clues about the mech-
anism of CaN regulation. It was first observed via circular dichroism (CD) by Rumi-Masante et
al that upon CaM’s binding, nearly fifty residues of the regulatory domain (RD)s folded into α
helices and only half of which could be accounted for by the CaMBR region. By using hydrogen
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1 INTRODUCTION
exchange mass spectrometry (HXMS), they further identified the region outside the CaMBR that
gained α-helicity upon CaM’s binding [14]. Dunlap et al [1] further confirmed the observation in
a mutagenesis study of that region. This region was coined the ’distal helix’ region (DH, residue
K441 - I458). Moreover, they revealed that single point mutations of three alanines within the
distal helix region into glutamic acids disrupted helix formation. Importantly, these mutations
reduced CaN’s apparent affinity for a substrate, p-nitrophenyl phosphate (pNPP), that competes
with the AID for the CaN active site [1].
Simulations of CaN have helped bridge experimental probes of its phosphatase activity [3,
16,17] with static, atomistic-resolution structural data. Li et al reported slight conformational
changes of the CaN B domain following Ca2+ binding via molecular dynamics (MD) simulation
and proposed that conformational similarity between the apo- and holo-CaN B-domain states
enables the former to regulate CaN activity independent of Ca2+ [18]. Harish et al utilized
virtual screening and MD simulations to design inhibitory peptides of CaN using the native AID
peptide as template [19]. Simulations have also been used to study the involvement of CaN residues
outside of its catalytic domain in the binding and anchoring of inhibitory immunosuppressant drugs
and analogs thereof [20–23]. Similarly, computational studies examining structural mechanisms
of CaM-dependent regulation of targets including CaN have emerged recently, including myelin
basic protein (MBP) [24] and myosin light chain kinase (MLCK) [25,26]. In complement to these
studies, we have additionally shown via molecular dynamics and Brownian dynamics simulations
that the CaMBR is highly dynamic in solution in the absence of CaM, that CaM binding to the
CaMBR is diffusion-limited, and that corresponding association rates are tuned by the charge
density of the CaN peptide [27]. Despite these contributions, the sequence of molecular events
that follow CaMBR binding and culminate in relief of CaN auto-inhibition remain unresolved.
These observations formed the basis of a working model of CaN activation whereby the folding
of the intrinsically-disordered distal helix into an αhelix-rich structure is coupled to relieving CaN
autoinhibition. However, it is still not known whether the distal helix directly binds to CaM, and
if so, where they might share protein-protein interaction (PPI) interfaces or how those PPIs are
potentially stabilized. In large part, the challenge in identifying potential PPI sites arises because
such interaction sites generally assume large, flat surfaces lacking specific interaction patterns
[28], such as grooves formed between αhelical bundles [29,30]. Computational protein-protein
docking engines have begun to address this challenge, including ZDOCK [31] and RosettaDOCK
[32], which have been used to successfully elucidate structural details of intrinsically disordered
peptide-involved regulation. For example, Hu et al utilized ZDOCK to successfully predict the
binding modes between disordered Yersinia effector protein and its chaperone partner [33]. Schiffer
et al explored the molecular mechanism of ubiquitin transfer starting from top-ranked ZDOCK
predicted binding pose between ankyrin repeat and SOCS box protein 9 (ASB9) and creatine
kinase (CK) [34]. Bui et al reported that phosphorylation of the IDP fragment of transcription
factor Ets1 leads to more binding-competent structures to its coactivator as evident by MD and
RosettaDOCK [35].
In this study, we utilized computational methods including protein-protein docking, enhanced
sampling and classical molecular dynamics (MD) simulations to identify potential interaction sites
between the distal helix and CaM. The protein-protein docking yielded several candidate inter-
action sites that we defined as sites A through D (Fig. 2(a)). Of these, site D on the CaM
solvent-accessible surface appears to stabilize the distal helix by moderate-affinity intermolecular
interactions. Among the intermolecular interactions stabilizing this putative PPI are two residues,
lysine (K30) and glycine (G40) found on the ‘back-side’ of CaM distal to where CaMBR is known
to bind. Their mutation to K30E and G40D were found to abolish enzyme activity [36] in an-
other globular CaM target, Myosin Light Chain Kinase (MLCK), that apparently relies on still
unresolved secondary interactions to initiate catalysis [37,38]. Analogously, our simulations of
CaM K30E and G40D variants indicate that the mutations substantially impair distal helix bind-
ing at site D. In complement to these simulations, we demonstrate that the distal helix A454E
variant also destabilizes the distal helix/site D interaction in agreement with reduced phosphatase
activity shown by Dunlap et al [1]. Our data strongly suggest that the site D and CaN distal
helix region are important to CaN activation, as site directed variants at site D residues K30
and G40 reduces CaN-dependent dephosphorylation of pNPP. Based on these results, we provide
an updated structural model of CaN activation by CaM that reflects specific CaM/distal helix
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1 INTRODUCTION
interaction sites (see Fig. 1).
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2 METHODS
Figure 1: Refined model of Calcineurin (CaN) activation by Calmodulin (CaM) through direct
binding of the ‘distal helix’ to CaM, based on the mechanism initially proposed in [1]. The two
chains of CaN (CaNA and CaNB) are colored in limegreen and lime, respectively. AID is colored in
red. CaM is colored in cyan, CaMBR is colored in magenta. The amino acid sequence of CaN RD
is shown at the bottom of the panel with CaMBR and the distal helix region colored in magenta
and black, respectively. In the absence of CaM, CaN is inhibited by its auto-inhibitory domain
(AID). After CaM binds the CaM binding region (CaMBR) on the CaN regulatory domain, a
secondary interaction between CaM and a ‘distal helix’ ultimately remove the AID from the CaN
catalytic domain. The activated CaN enzyme catalyzes the dephosphorylation of target proteins
essential to myriad physiological functions.
2 Methods
Our simulation protocol consisted of four primary steps. 1) replica exchange molecular dynamics
(REMD) simulations of the isolated CaN distal helix region were used to generate trial confor-
mations for protein/protein docking with CaM. 2) The ZDOCK protein/protein docking engine
yielded initial poses for putative CaM/CaN interaction sites. 3) The docking poses were refined
using extensive, microsecond-length molecular dynamics simulations. 4) Binding affinities based
on MM/GBSA were used to rank-order distal helix/CaM poses for sites A-D. We further chal-
lenged the predicted structural models by introducing mutations in the distal helix and putative
interaction site D that have been experimental probed in prior works. We describe these steps in
detail below and additionally summarize pNPP phosphatase assay.
2.1 Replica exchange molecular dynamics (REMD) sampling of the iso-
lated distal helix
In accordance with our approach in [27], we performed replica exchange molecular dynamics
(REMD) simulations of the distal helix region (K441-I458) in the absence of CaM to exhaustively
sample likely conformational that are in equilibrium. The distal helix peptide was constructed
by the auxiliary tleap program in Amber16 [39] in an extended configuration and parameterized
using the Amber ff99SBildn [40] force field. The peptide was then minimized via sander [41]in
vacuo until convergence of the energy gradient (drms ≤0.05) or the number of steps 1×105(with
first 50 steps of steepest decent and rest steps of conjugate gradients algorithm) was satisfied. The
minimized structure was then used as the starting structure for REMD simulations coupled with
the Hawkins, Cramer, Truhlar pairwise generalized born implicit solvent model [42] via the igb = 1
option in Amber. The monovalent 1:1 salt concentration was set to 0.15 M and a non-bound cutoff
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2.2 Docking of distal helix to CaM/CaMBR complex via ZDOCK 2 METHODS
of 99 Å was chosen. Ten replicas were created with temperature ranges spanning 270-453 K. The
temperature of each replica was calculated via the Patriksson et al webserver [43] to ensure the
exchange probability between neighbouring replicas was approximately 0.4, as recommended in
[44,45]. Each replica was first subjected to 1×105steps of energy minimization via pmemd with
the first 50 steps being steepest decent and rest steps being conjugate gradients. The minimized
systems were subsequently heated from 0 to their respective target temperatures over an 800 ps
interval using a timestep of 2 fs with Langevin thermostat. The equilibrated replicas were then
subjected to 100 ns of production REMD simulations under target temperature with Langevin
thermostat. The SHAKE [46] algorithms were used for REMD simulations. Clustering analysis
with hierarchical agglomerative (bottom-up) approach using cpptraj were conducted on the 300
K REMD trajectory to divide the trajectory into ten clusters; the average root mean squared
deviations (RMSD) between each cluster was around 6 Å.
2.2 Docking of distal helix to CaM/CaMBR complex via ZDOCK
The protein-protein docking webserver ZDOCK3.0.2 [31] was used to determine probable binding
poses for the REMD-generated distal helix conformations on the CaMBR-bound CaM complex.
The CaM/CaMBR complex configuration was obtained from the Protein Databank (PDB ID: 4q5u
[47]). It has been reported that 62% percent of experimentally-resolved PPIs are characterized
by the binding of an α-helical peptide within grooves formed between adjacent α-helical on the
target protein surface [30]; therefore we narrowed the ZDOCK search to four α-helical-containing
regions on the CaM solvent-exposed surface. These sites are shown in Fig. 2(a), from which we
determined a list of probable amino acid contacts as input to ZDOCK (see Table S1). During
the ZDOCK calculations, the receptor (CaM/CaMBR complex) was kept fixed while grids were
constructed around receptor with the size being 80X80X80 and spacing being 1.2 Å. The ligand
(distal helix) was then docked via fast fourier transform (FFT) algorithm on the 3D grids. The
scoring function consists of interface atomic contact energies (IFACE) [48], shape complementarity
and electrostatics with charge adopted from CHARMM19 force field [49]. The initially generated
2×103poses were subjected to a culling process to eliminate those having no contacts with residues
we specified in Table S1. After culling, there were zero, two, eighty-eight and three poses left at
sites A-D, respectively. For case in site A, ZDOCK then instead output all initial 2×103poses
and we found that the top ten poses were still near site A (Fig. 2(b)). The pose with highest score
at each site was chosen for further refinement using molecular dynamics.
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2.3 Conventional molecular dynamics (MD) simulations of ZDOCK-generated distal helix/CaM
poses 2 METHODS
Figure 2: (a) Four tentative binding sites (orange) on the surface of CaM-CaMBR complex.
CaM is colored in cyan, CaMBR is colored in magenta and Ca2+ ions are colored in yellow. (b)
ZDOCK predicted conformations of distal helix interacting with CaM/CaMBR complex at each
site. Predicted distal helix conformations from site A to D are colored as red, salmon, warmpink
and firebrick, respectively. /net/share/bsu233/CaMDH/zdock_MD/esitmate_SASA/log.pml
2.3 Conventional molecular dynamics (MD) simulations of ZDOCK-
generated distal helix/CaM poses
Explicit-solvent MD simulations were performed on the ZDOCK-predicted distal helix/CaM com-
plexes to further refine the distal helix binding poses. The amino acid sequence from CaMBR to
distal helix is shown at the bottom of Fig. 1and the sequence definition of CaMBR and distal
helix are the same as [1]. We first inserted peptide linkers for each pose between the CaMBR
C-terminus (R414) and the N-terminus (K441) of the distal helix via tleap. The initial linker
was generated via tleap and energy-minimized as done in Sect. 2.1. The minimized structures
were subsequently simulated in vacuo to heat the systems from 0 to 300 K. The last frame of
the short equilibration run was subject to additional energy minimization in vacuo to facilitate
its compliance with the distal helix and CaMBR termini. The top poses from ZDOCK presented
distal helix orientations that were all compatible with the CaMBR and linker configurations. The
optimized linker was placed adjacent to the CaMBR and distal helix; tleap was then used to
link the peptide components. The resulting structures were then subjected to energy minimiza-
tion, followed by a 100 ps heating process to raise the system temperature to 300 K for which all
atoms except the linker were fixed via the ibelly function in sander MD engine of Amber. This
minimization and heating was perform in vacuo and the purpose is to further relax the linker in
the presence of distal helix and CaM/CaMBR complex. The last frame of heating stage was used
as input configurations for explicit-solvent molecular dynamics simulations.
Each in vacuo starting configuration was solvated in a TIP3P [50] waterbox with 12 Å bound-
ary margin. K+and Cl−ions were added to neutralize the protein and establish a 0.15 M salt
concentrations. After parameterizing the system using the ff14SB force field [51] via tleap, the
system was subjected to an energy minimization, for which all atoms except hydrogens, water and
KCl ions were constrained by the ibelly functionality. The cutoff value for non-bond interactions
was set to 10 Å. A 2 fs timestep was chosen, as SHAKE [46] constraints were applied on bonds
involving hydrogen atoms. Two heating procedures were performed to heat the system from 0 to
300 K using the Amber16 sander.mpi engine [41]. In the first heating stage, the ibelly function
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2.4 MD simulations of CaM (K30E and G40D) and CaN distal helix variants (A454E)2 METHODS
was used to keep all system except the water box and KCl ions fixed. The water box was heated
to 300 K over a 100 ps interval under the NVT ensemble. For the second heating stage, the entire
system was heated from 0 to 300 K over 500 ps under the NPT ensemble, for which the back-
bone atoms of CaM, CaMBR and distal helix were constrained by an harmonic potential (force
constants of 3 kcal mol−1Å−2). Thereafter, an additional 1 ns equilibrium stage was conducted at
300 K under the same constraints, but with a reduced force constant of 1 kcal mol−1Å−2. These
equilibrium simulations were followed by 100 ns production-level MD simulations. The weak-
coupling thermostat [52] was used during the simulation. Clustering analysis was performed on
the production trajectory using the same strategy in Sect. 2.1. The average root mean squared
deviations (RMSD) between each cluster was around 6 Å. Based on the rationale that extend-
ing simulations using less-frequently sampled structures provides greater overall sampling of the
conformational space [53], we identified 5-6 low-probability states as inputs for subsequent MD
simulations. Approximately 1 µs of trajectory data were simulated in total for each site.
2.4 MD simulations of CaM (K30E and G40D) and CaN distal helix
variants (A454E)
Clustering analyses is a common used data-mining method in MD simulation which groups tra-
jectories into clusters based on structural similarity [54]. In this study, clustering analyses was
performed on the production-level MD trajectories of the distal helix/CaM configurations that
yielded the most favorable binding scores by MM/GBSA. A representative structure of the most
populated cluster was selected as an input for in silico mutagenesis in order to validate the model
against experiment. Namely, the CaM K30E and G40D variants, as well as the CaN A454E
variant, were built by replacing and regenerating the amino acid side chains using tleap. The
resulting structures were energy minimized with a stop criterion of (drms <= 0.05) for the energy,
during which all atoms except the mutated residues were fixed via the ibelly function in Amber.
The energy-minimized structure was then solvated and simulated according to the same procedure
in Sect. 2.3. All simulation cases in this study were listed in Table S3.
The binding free energy between distal helix and CaM was estimated via Molecular Mechanics-
Generalized Born and Surface Area continuum solvation (MM-GBSA) [55].
∆G=hGDH−C aM i−hGCaM i−hGDHi(1)
Where hGDH−C aM i,hGCaM iand hGDH iare ensemble-averaged free energies of distal helix-CaM
complex, CaM and distal helix, respectively. In this study, the trajectories of these three com-
ponents were extracted from MD trajectories via cpptraj at a 2 ns frequency. The generated
sub-trajectories were used as input of MMPBSA.py script shipped with Amber16 to calculate
the free energies of each part. The salt concentration was set as 0.15 M with generalized Born
model option setting as igb = 5. No quasi-harmonic entropy approximation was made during the
calculation.
2.5 Structural Analyses
Clustering analysis, root mean squared deviations (RMSD)/root mean squared fluctuations (RMSF)
calculations, hydrogen bonds and secondary structure analysis were performed via cpptraj [56]
shipped with Amber16. The reference structure of RMSD calculation was CaM/CaMBR crystal
structure (PDB ID: 4q5u [47]). Prior to RMSF calculation, the whole MD trajectory was first rms
fitted to the first frame using CaM/CaMBR backbone atoms. Secondary structure probability of
each residue was calculated using cpptraj with define secondary structure of proteins (DSSP) al-
gorithm [57]. The colvar module [58] within VMD was used to assess the total α-helix content of
REMD-generated distal helix and DHA454E conformation. The hbond command within cpptraj
was used to analyze hydrogen bonds between distal helix and CaM/CaMBR. During the hbond
analysis, the angle cutoff for hydrogen bonds was disabled while the default 3 Å cutoff between
acceptor and donor heavy atoms was used. Scripts and cpptraj input files used for above analy-
ses will be publicly available at https://bitbucket.org/pkhlab/pkh-lab-analyses/src/default/2018-
CaMDH.
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2.6 Calcineurin phosphatase assay using para-nitrophenyl phosphate (pNPP) substrate3 RESULTS
2.6 Calcineurin phosphatase assay using para-nitrophenyl phosphate
(pNPP) substrate
Materials. pNPP was obtained as the bis(tris) salt (Sigma), dithiothreitol reducing agent (Sigma),
assay buffer (80mM Tris pH 8, 200mM KCl, 2mM CaCl2), MnCl2.Preparation of Enzymes and
Proteins. The wild-type, K30E, and G40D variants were obtained from JP Davis and prepared
at a concentration range of 300 µM to 3 mM in assay buffer. Enzyme Assay. Phosphatase
assays were performed using 30 nM calcineurin, and 90 nM calmodulin in 96-well Corning Costar
microtiter plates with a reaction volume of 120 µL. Assays proceeded in the manner of [1] with
each CaM assayed in triplicate and over three plates to account for technical variation. Control
reactions absent calcineurin were added to the end of each lane with 200 mM pNPP to determine
the rate of enzyme-independent substrate hydrolysis. Kinetic Analysis. The pNPP substrate
reactions were varied over 11 concentrations, increasing from 0 mM to 200 mM for each column.
60 minute UV-Vis recordings were obtained on a Molecular Devices FlexStation 3 plate reader
using Softmax Pro 7 software at 405 nm with 10 minute read intervals. The resulting data
was inspected for appropriate Michaelis-Menten kinetics, such as proper substrate saturation, by
plotting absorbance against substrate concentration. Following this, the readings were linearized
to produce the double reciprocal Lineweaver-Burk plot for extraction of Vmax and KMbased on
the following equation:
1
V=KM
Vmax
[pN P P ] + 1
Vmax
(2)
3 Results
Prior studies [14,59] have indicated that CaM binding to the CaN’s canonical CaM-binding region
requires secondary interactions beyond this region to fully activate the phosphatase. Rather, CaN
activity is likely dependent on a secondary interaction between the CaN regulatory domain and
CaM. A study by Dunlap et al [1] suggested that a distal helix region spanning residues K441
to I458 was likely involved in CaM binding. However, it was unclear which region(s) of the
CaM solvent-exposed surface would contribute to a potential PPI. We therefore used molecular
dynamics and protein-protein docking simulations to identify plausible wild-type CaN interaction
sites on CaM, and challenge these predictions with mutagenesis. Our predicted site was validated
using a CaN pNPP phosphatase assay.
3.1 Regulatory domain (RD)-construct propensity for secondary struc-
ture formation in absence of CaM
Circular dichroism (CD) and HXMS analysis in [14] suggest that there exists α-helical structure
beyond the canonical CaMBR region after CaM’s binding. We therefore sought to assess αhe-
licity in the REMD-simulated distal helix peptides. Previously [27], we found that extensive MD
simulations of the isolated CaMBR yielded a small population of α-helical structures suitable for
binding CaM in its canonical binding pose [60]. We therefore applied a similar REMD procedure
(see Sect. 2.1) to the proposed distal helix segment of the CaN regulatory domain to assess the
propensity for the spontaneous formation of secondary structure in the absence of CaM. Here, we
performed 100 ns of REMD simulations on the wild-type (WT) distal helix as well as a A454E
variant. The latter was considered as it has been reported to exhibit reduced α-helical content
in the presence of CaM [1], which is suggestive of abolishing the distal helix/CaM interaction.
Following the REMD simulations, we performed clustering analysis to identify the predominant
conformations of the two peptide configurations. Interestingly, we observed that both the WT
distal helix and its A454E mutant partially fold into an αhelix in the absence of CaM. As shown
in Fig. 3(a), the representative structure of most populated clusters of the distal helix and A454E
mutant (83.8% and 85.3% of the total trajectory, respectively) both contain helical fragments.
While the overall α-helix contents (≈45%) of these two fragments were statistically indistinguish-
able, a contiguous helix was formed in the WT distal helix, whereas it was fragmented in the
mutant. These helicity features are further quantified as residue’s α-helix structural probability
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3.1 Regulatory domain (RD)-construct propensity for secondary structure formation in absence
of CaM 3 RESULTS
shown in Fig. 3(b): the distal helix region has the maximum probability present at middle region
while the A454E has maximums present near the two terminis. Both the simulated distal helix
and its variant therefore could adopt α-helix content in the absence of CaM, but it remains to be
determined whether the dominant structures are capable of binding the CaM surface. We note
that experimental assays of the complete RD do not detect significant secondary structure; this
discrepancy may be a result of using substantially different RD lengths (S374 to Q522 residues in
Rumi-Masante et al [14] and K441-I458 in this study). We discuss this difference in further detail
in Limitations (Sect. 4.5).
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3.2 Protein-protein interactions between RD-construct and peptide-bound CaM 3 RESULTS
Figure 3: (a) Sequence of distal helix/DHA454E and representative structures of four
most populated clusters from 100 ns REMD simulations. The structures are colored in rain-
bow with N-termini as blue and C-termini as red. (b) Secondary structure probability of each
residue calculated from REMD trajectory via cpptraj with DSSP algorithm. The lower panel
shows the total α-helix contents of two fragments calculated via the colvar module of VMD.
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/rmsf.ipynb
3.2 Protein-protein interactions between RD-construct and peptide-
bound CaM
The overwhelming majority of CaM-containing complex structures resolved to date include only
limited fragments of the bound target protein [60]. CaM-bound CaN is no exception, as the
mostly likely physiological conformation [47] consists of monomeric CaM in a canonical ’wrapped’
conformation about a target region in CaN(A391-R414) [13]; however, it is evident that secondary
interactions beyond this domain play a role in CaN activity, yet atomistic-level structural details
of these interactions have not yet been resolved. Therefore, in order to resolve potential binding
regions for the distal helix region, we seeded a protein-protein docking engine, ZDOCK [31], with
candidate α-helical structures identified through REMD simulations. The docking simulations
were performed in regions that included grooves formed between αhelices we identified at the
CaM solvent-accessible surface. We selected these regions, since such secondary structures are
believed to nucleate protein-protein interactions [61]. Furthermore, a thorough examination of
protein-protein complex structures in the Protein Data Bank in 2011 suggested that αhelices
contribute to 62% of all PPI interaction surfaces [30] between binding partners. Narrowing the
search region on CaM to those containing α-helical regions yielded four candidate sites (A-D) that
spanned nearly the entire CaM solvent-exposed surface (see Fig. 2(a)).
The most energetically-favorable distal helix/CaM poses predicted via ZDOCK at sites A-D
are summarized in Fig. S1. The docked poses reflect significant interactions of at least the distal
helix C-terminal loop with loops bridging adjacent α-helices on the CaM surface. At site A, polar
residues near N97, Y99 and D133 from two of the C-terminal CaM domain’s loops interact with
the distal helix, compared with just one EF-hand motif loop at site B (D129, D133 and D135).
The site C poses were primarily stabilized by hydrophobic interactions formed from CaN residues
L444/I458 and F16/L4 on CaM, in addition to a loop-loop interaction via CaM D64. The site
D poses reflected distal helix C-terminal loop interactions with CaM EF-hand loop residues near
N42 and K94. Most poses were unsurprisingly parallel to α-helical/α-helical ‘grooves’ on the CaM
solvent-exposed surface and were evidently anchored through interactions between the proteins’
loop regions.
In contrast, we found that the A454E variant docked poorly at sites A-D (see Fig. S2), as
assessed by the proximity of docked poses to the designated CaM sites in fact, most predicted
poses tended to localize toward site A, albeit with weak interactions. Moreover, we speculate that
the impaired binding of DHA454E may arise from its fragmented αhelical structure, in contrast to
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3.2 Protein-protein interactions between RD-construct and peptide-bound CaM 3 RESULTS
the contiguous regions for the WT variant (see Table S2 for docking scores and Fig. S1/Fig. S2 for
docking poses). Although docking scores were provided by the ZDOCK algorithm to rank order
potential poses, we did not analyze these scores in detail as we later refined these structures using
more detailed simulations and energy expressions. This refinement corrects for artifacts from the
ZDOCK algorithm, which assumes rigid conformations for both proteins that would ordinarily
be expected to relax in the bound complex. Hence, in the following section we pursue extensive
microsecond-scale all-atom MD simulations to refine and assess the predicted poses.
The docked CaN/CaM configurations from the previous section were intended as inputs for
MD-based refinement of nearly intact CaN regulatory domain complexes with CaM. Subsequent
refinement using microsecond-length MD simulations relax the rigid protein conformations as-
sumed in ZDOCK. To refine these poses, we linked the docked distal helix fragments with the
CaMBR fragment resolved in the CaM/CaN complex (PDB ID: 4q5u) from [47]. Each of the four
candidate binding sites yielded distal helix orientations that were compatible with the 26 residue-
length linker. Following initial optimizations of the linker described in Sect. 2.3, we performed
µs-length, explicit solvent simulations with the regulatory domain bound to CaM. Since the pre-
dicted A454E distal helix poses appeared to be inferior to those of the WT variant, we refined
only the WT poses and thereafter introduced A454E mutations to the refined conformations.
We first assess the integrity of the predicted binding modes based on Molecular Mechanics-
Generalized Born and Surface Area continuum solvation (MM-GBSA). MM/GBSA scoring of the
MD-generated configurations provides a coarse estimate of binding affinity without significantly
more expensive free energy methods. We reported the binding free energy of distal helix between
CaM/CaMBR as well as between CaMBR and CaM in Fig. 4. Significantly, we found that binding
of WT distal helix at the CaM site D yielded a more pronounced favorable average binding free
energy (∆G≈ −27.7 kcal mol−1) than sites A, B and C (−2.5 kcal mol−1,−17 kcal mol−1,−22.5
kcal mol−1) with P-values (1×10−4,2.8×10−3and 1.144 ×10−1, respectively) confirming that
the means are significant compared to the null hypothesis. Similarly, the binding free energies
of distal helix interactions were generally substantially weaker (−2.5to −27.5 kcal mol−1) than
those between the CaMBR and CaM (∆G <−1.20 ×102kcal mol−1) Although MM-GBSA is a
very approximate scoring method for molecular complexes, the consistent trends in numbers of
hydrogen bond contacts, RMSF amplitudes and binding scores suggests that the site D is the most
likely region for forming stable CaM/distal helix interactions.
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3.2 Protein-protein interactions between RD-construct and peptide-bound CaM 3 RESULTS
Figure 4: Approximate binding free energies between CaM and the distal helix (left) or CaMBR
regions (right) via Molecular Mechanics-Generalized Born and Surface Area continuum solvation
(MM-GBSA). Black bars correspond to wild-type CaN, whereas colored bars utilize the A454E
CaN and CaM variants. The calculation was conducted on frames extracted every 2 ns from MD
trajectories. The error bar represents standard error of mean. The values above bars in the left
panel are P values of each case with null hypothesis that their mean values are equal to site D.
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/mmgbsa.ipynb
We supplement the energy scores with structural indicators of stability, namely contacts and
RMSF. We report in Fig. 5the corresponding root mean squared deviations (RMSD) and root
mean squared fluctuations (RMSF) of the peptide backbone atoms from CaM and CaMBR. We
additionally include two CaM variants with mutations at site D, which we rationalize later in
Sect. 3.3. We found that the average RMSD values of the MD-predicted conformations relative to
the experimentally-determined CaM/CaMBR structure were at or below 2 Å; we attribute these
small fluctuations to stable CaM/CaMBR interactions that were insensitive to the distal helix
docking pose. Similar to the RMSD data, the CaM and CaMBR RMSF values are comparable
in amplitude and nearly indistinguishable between distal helix/CaN docking poses, with most
residues presenting values below 1.5 Å. The prominent peaks in excess of 5.0 Å correspond to
the CaM termini and the N-terminus of the CaMBR. We additionally observe a variable region
midway along the CaM sequence, which corresponds to the labile linker between its globular N-
and C- domains that is implicated in allosteric signaling [62].
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3.2 Protein-protein interactions between RD-construct and peptide-bound CaM 3 RESULTS
Figure 5: (a) Root mean squared deviations (RMSD) of peptide backbone atoms
of CaM and CaMBR from µs-length MD simulations. The reference structure for the
RMSD calculation was based on the CaM/CaMBR crystal structure (PDB ID: 4q5u). (b)
Root mean squared fluctuations (RMSF) of non-hydrogen atoms in CaM and CaMBR.
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/rmsf.ipynb
The small and statistically indistinguishable RMSF values for the CaM/CaMBR in Fig. 5
suggest that distal helix binding had negligible impact on binding the CaM recognition motif.
This is an important observation, as viable binding poses for the distal helix are expected to
preserve the binding between the CaMBR and CaM. We base this assumption on CD data collected
in [63] that indicated substantial alpha helical character in the CaM/CaN complex following
dissociation of the distal helix domain. Therefore, we then assessed the integrity of the distal
helix poses using RMSF analyses and measurements of inter-protein contacts. In Fig. 7we report
representative configurations of the distal helix region (red) in complex with CaM (cyan), as well as
their corresponding per-residue RMSF values in Fig. 6. To guide interpretation, we hypothesized
that RMSF values above 5 Å were indicative of poorly stabilized residues. We later rationalize
this value by comparing approximate binding energies as computed by MM-GBSA. At site A,
both the distal helix/CaMBR linker and the distal helix reflect RMSF values in excess of ∼10
and ∼15 Å, respectively. These large fluctuations arise from the breadth of binding orientations
evident in Fig. 7(a), which we interpreted as a poorly-stabilized configuration. Similarly, the site
B configurations also appear to be loosely bound, based on linker and distal helix RMSF values
beyond 10 Å. In contrast, the distal helix RMSF values at sites C and D were below 5 Å, with
the latter site reporting the smallest values among the sites we considered, which is evidence of a
stable binding configuration.
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3.2 Protein-protein interactions between RD-construct and peptide-bound CaM 3 RESULTS
Figure 6: (a-d) Representative structures of from the microsecond length MD simulations
initialized from ZDOCK-predicted distal helix poses. CaM is colored in cyan, CaMBR is colored
in magenta and Ca2+ ions are depicted as yellow spheres. The linker and distal helix regions in
site A-D are colored as red, salmon, warmpink and firebrick, respectively. (e-h) Non-hydrogen
atom RMSFs of linker and distal helix residue calculated from MD simulations of each site, as
an indicator of binding stability. The red dash line depicts RMSF value as 5 Å. * During the
MD simulations, distal helix structures initiated at site B migrated toward site D (see Fig. S3).
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/rmsf.ipynb
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3.2 Protein-protein interactions between RD-construct and peptide-bound CaM 3 RESULTS
Figure 7: Interaction between linker/distal helix of CaN and CaM at site A-D. Key residues at
the interaction surface are shown in sticks with black labels for CaM residues and red labels for
distal helix residues. See Table S1.3 for specific values.
As has been shown in other proteins regulated by disordered protein domains [64–66], there are
often multiple poses the contribute to regulation. We therefore assessed the most significant inter-
protein contacts contributing to the ensemble of distal helix binding poses at sites A-D. Among
these poses, the distal helix configurations at site D presented the lowest distal helix RMSF values
among the considered sites. Significantly, the site D distal helix configuration presented several
hydrogen bond-facilitated interactions with CaM, including two long-duration (37% and 55% of
sampled configurations) interactions between Q445 and CaM residues R37/K94, pairing of CaM
K21 with glutamic acids E453 and E450, as well as E456 with CaM residues K30 and R37.
Contacts between CaM and CaN, as well as their longevities (as assessed by the percentage of
MD frames satisfying a hydrogen bond contact cutoff of 3 Å between oxygen and nitrogen atoms)
are additionally quantified in Fig. 8(specific values are listed in Table S1.3). The latter data
indicate a modestly greater degree of hydrogen bonding of the distal helix at site D (10 h-bonds
were above 10%) versus site B (9), and a significantly greater degree relative to sites A (1) and
C (3). Furthermore, the site D pose appears to be stabilized by both the N- and C-domains of
CaM (residues D20-S38 and R90-N111, respectively). We speculate that this bi-dentate interaction
could improve CaMBR binding by locking CaM into its collapsed configuration and thereby prevent
disassembly. Although during the simulations, the distal helix at site D maintained significant
α-helix (see Fig. S3 and Fig. S4), we note that a significant percentage of the predicted structures
exhibited beta sheet character in the linker region (see Fig. S5) that was not observed in the CD
cpectra collected by Rumi-Mansante et al [14]. This persistent secondary structure was limited to
a few residues (see Fig. S5 and Fig. S6) thus may be beyond the limits of detection in earlier CD
experiments. We comment on this further in the Limitations (see Sect. 4.5). Meanwhile, site B
reflected interactions with both CaM terminal domains that were attenuated, while sites A and C
were mostly bound by interactions of their linker regions with the CaM N-domain. Interestingly,
we observed that the distal helix poses originating at site B migrated toward site D (see Fig. S3),
which likely explains the higher hydrogen bonding in site B versus sites A and C.
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3.3 Effects of putative CaN/CaM site D mutagenesis 3 RESULTS
Figure 8: Percentage of simulated frames which have hydrogen bonds formed between CaN
peptide (linker and distal helix) and CaM. The linker and distal helix are indicated by grey and
black bar, respectively.
As a result of HXMS conducted by Rumi-Masante et al [14] of the RD construct CaN in solution
with CaM, it is apparent that residues R414 through E456 are within a stretch of residues that are
somewhat protected from solvent, which suggest that relief of CaN autoinhibition entails binding
at least the distal helix region. We note that the HXMS data could not precisely distinguish which
residues were protected, as proteolysis and mass spec was conducted on short peptides. Further,
HXMS data detects only bonds involving backbone amide protons, thus we speculate that the CaN
site chain interactions with CaM may stabilize the distal helix alpha helical structure. Hence, we
suggest that CaM/CaN configurations that stabilize the distal helix region likely contribute to
CaN activation. Based on this rationale, the small RMSF values and extensive hydrogen bonding
of the CaN distal helix with the CaM site D relative to other ZDOCK identified regions suggest
that CaN is most stabilized at site D.
3.3 Effects of putative CaN/CaM site D mutagenesis
MD simulations of the WT CaN CaMBR-distal helix sequence suggest that CaM site D is a prob-
able binding region for the CaN regulatory domain. To challenge this hypothesis, we performed
MD simulations of CaN distal helix and CaM site D variants that could reduce CaN activity to
test whether the distal helix/CaM interaction was impaired. Namely, we introduced the CaN
A454E and CaM K30E and G40D mutations into the MD-optimized WT structures. We elected
to mutate the WT CaMBR/distal helix complexes with CaM, as the WT complex appeared to
have favorable stability, whereas repeating the REMD/zdock steps with the mutants may not have
yielded viable configurations. The proposed A454E CaN variant was based on CD data collected
by Dunlap et al [1] that demonstrated reduced α-helical content upon binding CaM relative to
the WT with impaired CaN activation. The CaM variants we examined in this study were based
on experimental mutagenesis studies [36] of CaM-dependent Myosin Light Chain Kinase (MLCK)
activation, for which secondary interactions beyond the canonical CaM binding motif were re-
quired for enzyme activation [37,38] (Fig. 9(a)). Although these secondary CaM interactions are
involved in directly binding the MLCK catalytic domain in contrast to CaN [37], two residues
(K30 and G40) implicated in binding [36] reside within the site D identified in our simulations.
We also reported the MM-GBSA-calculated binding free energies between distal helix and CaM
of the mutants in Fig. 4. While the WT distal helix at the CaM site D has most stable binding with
∆G≈ −27.7 kcal mol−1, the three mutations K30E, G40D and A454E have less favorable ∆Gs
as −21.8 kcal mol−1,−17.9 kcal mol−1and −24.4 kcal mol−1with P-values being 8.12 ×10−2,
5.1×10−3and 2×10−4, respectively. The MM-GBSA-energies clearly shown that mutations
would impair the binding affinity between distal helix and CaM. Accordingly, we presented linker
and distal helix RMSF data for the WT and mutants in Fig. 9(b). The distal helix RMSF values
among the two CaM variants were moderately increased compared to the WT case. Specifically,
for the WT system, the distal helix residues were entirely within 10 Å and as low as ∼2.5 Å. In
contrast, the K30E variant yielded RMSF values no smaller than approximately 5 Å, while the C-
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3.3 Effects of putative CaN/CaM site D mutagenesis 3 RESULTS
terminal half approaches values nearing 15 Å. This trend manifested in fewer long-lived hydrogen
bond contacts between the distal helix and both CaM domains (see Fig. 9). Similarly, the G40D
mutation appeared to significantly disrupt interactions with CaN, as the entire distal helix region
was characterized with RMSF values over ∼10 Å in amplitude, with corresponding decreases in
hydrogen bond contacts. We reported the MMGBSA calculated binding free energy between the
distal helix and the CaM/CaMBR in Fig. 4. Among the mutations we considered, the A454E
mutant had the most severe impact on RMSF values, as all residues comprising the linker and
distal helix regions resulted in fluctuations above 8 Å. We also reported the α-helix probability of
distal helix residue for variants in Fig. S7. It was found that all variants preserved a significant
degree of overall helicity despite evidence of impaired interactions with CaM. However, the specific
residues which formed α-helix were different among the variants: the mutation of A454 to E454
shifted the helicity to the first half of distal helix while the two CaM variants had the second half
region being αhelical. Altogether, these simulation data suggest that 1) the WT distal helix is
stabilized at the site D CaM region, 2) site D residues R37 and K30 are implicated in distal helix
binding and 3) disruption of site D binding by CaN A454E is consistent with reduced helicity and
enzyme activity measured experimentally.
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3.4 Phosphatase assays of site-directed CaM mutants 3 RESULTS
Figure 9: (a) Comparison of CaM-petide complex structure from CaN and MLCK (PDB
ID: 2lv6 [67]). K30 and G40 are labeled (shown as sticks) based on their implication in
the activation of the CaM target Myosin Light Chain Kinase (MLCK) [36] and proximity
to site D determined by our simulations. (b) Non-hydrogen RMSF of linker and distal
helix in WT and mutants. The dash line depicts RMSF value as 5 Å. (c) Percentage
of simulated frames which have hydrogen bonds formed between linker/distal helix and CaM
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/rmsf.ipynb and hbond.ipynb
3.4 Phosphatase assays of site-directed CaM mutants
To support the simulation results, namely that the distal helix region binding predominantly to
site D would impact CaN activity, we analyzed the kinetics of CaN mediated hydrolysis of pNPP.
Our hypothesis was that disruption of site D/distal helix binding would reduce the accessibility
of the catalytic site for pNPP binding which would reduce the apparent substrate affinity. This
reduction would arise from the AID competing for the catalytic site, as a result of compromised
site D/distal helix interactions. We therefore conducted pNPP assays using two site D variants,
K30E and G40D. We analyzed substrate turnover in a Michaelis-Menten model, as described in
the Methods. Phosphatase assays performed on CaM variants strongly suggest a statistically
significant reduction (p-values in Table 1) in catalytic activity by a substantial increase in KM
for K30E and G40D over the WT (27.6±1.1 mM, 46.0±2.3 mM, and 35.5±3.4 mM, respectively)
indirectly indicating weaker binding of the distal helix peptide to the mutated CaM construct.
Table 1: Kinetic parameters of pNPP dephosphorylation with WT CaM and two site D variants.
P-values given by Welch’s t-test for difference of means with unequal variance.
CaM KM(mM) SD p-value Vmax (µmol/min/mg) SD p-value
WT 27.6 1.3 - 0.01 0.001 -
K30E 46.0 2.8 0.002 0.02 0.002 0.10
G40D 35.5 2.2 0.008 0.02 0.010 0.09
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4 DISCUSSION
4 Discussion
4.1 Summary of Key Findings
We have used computational modeling to elucidate a potential mechanism for CaM-dependent
regulation of CaN activity, through binding of a ’distal helix’ region of the regulatory domain.
Our microsecond-duration MD simulations indicate that the distal helix region maintains sig-
nificant α-helical when bound to WT CaM. In contrast, we predict that an engineered variant
(A454E) disrupts the domain’s secondary structure and ability to competently bind CaM. Both
predictions are in agreement with experimental probes of CaN regulatory domain structure and
phosphatase activity [1]. Namely, among the four potential regions on CaM’s surface that were
solvent-accessible after binding the CaMBR, our data suggest that an RD region spanning the
CaMBR through the distal helix was best stabilized at a site nestled between the CaM N- and
C-terminal domains. In silico mutagenesis of two N-terminal CaM residues (K30E and G40D),
prevented distal helix binding in our model, which we suggest hinders CaN activation, similar
to identical mutations in CaM that were found to inactivate another CaM target, Myosin Light
Chain Kinase (MLCK). We additionally report that our REMD simulations suggest that the iso-
lated distal helix region spontaneously assumes significant α-helical in absence of CaM; which
differ from trends observed in the complete RD domain observed experimentally [14]. We discuss
this limitation and its implications in Sect. 4.5. Finally, we confirmed the potential CaM site D
binding site for the distal helix through site-directed K30E and G40D variants, which we found
to weaken CaN binding as reflected by an increase in KM(from 27.6 mM to 46.0 and 35.5 mM,
respectively) in a pNPP phosphatase assay.
4.2 Plausible binding modes for putative CaN distal helix with CaM
Previous studies suggest that binding of regulatory domain residues beyond the CaMBR region are
involved in CaM-dependent relief of CaN autoinhibition [1,14]. Increases in regulatory α-helical
content were reported upon binding CaM, that could not be accounted for by the CaMBR alone.
Alanine to glutamic acid mutations at RD positions (A451E, A454E and A457E) C-terminal to
the CaMBR decreased α-helical content and CaN activity. Further, HXMS studies indicate that
in a complex of CaM with a regulatory domain/AID/C-terminal domain CaN construct that the
CaMBR through distal helix regions had reduced solvent accessibility, suggestive of secondary
interactions beyond the CaMBR. We calculated the backbone hydrogen bonds formed within the
linker and distal helix region as indicator of solvent-protection and compared against experimental
HXMS data. As shown in Fig. 10, In WT, site D has 16 hydrogen bonds, the 2 dominant hydrogen
bonds (red arrow) are formed within the beta-sheet region (Fig. S5). Also in the distal helix
region, two long-lived hydrogen bonds (>40% simulation time) exist. Compared with other cases,
backbone hydrogens at site D would be most protected from HXMS due to the larger number of
hydrogen bonds and relatively longer duration. Although A454E has largest number of hydrogen
bonds, most are short-lived and the residue pairs which form hydrogen bonds are well seperated
in sequence, indicating these hydrogen bonds do not contribute to α-helix secondary structure.
Our computational modeling suggest that the putative distal helix region contains significant α
helical character when bound to CaM site D, which qualitatively resemble those of experiment,
suggesting reduced susceptibility to hydrogen/deuterium exchange. Nevertheless, compared to
experimental HXMS data showing solvent-protected hydrogens are present across the whole linker
and distal helix region, our computational backbone hydrogen bonds data indicates less degree
of solvent-protection as the majority of hydrogen bonds are present in the N terminus of linker
region and C-terminus of distal helix region in site D. This discrepancy could possibly be explained
by the different lengths of CaN constructs used in HXMS experiment and our simulations. The
construct in HXMS experiment contains the whole RD domain plus AID and C-tail while our
simulations contain residues of A391 to I458 of RD domain. As shown in Fig. S9, we started from
site D and run extra simulations with AID fragment added to distal helix region, the data clearly
shows that the presence of extra residues would enhance the number and duration of backbone
hydrogen bonds present in the linker and distal helix region.
Additionally, several long-lived hydrogen bonds between the distal helix and CaM site D were
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4.2 Plausible binding modes for putative CaN distal helix with CaM 4 DISCUSSION
found to stabilize the bound configuration, which dampened the variations of peptide position
found at other identified sites (A-C) as reported by RMSF and energetic analyses (Fig. 8).
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4.2 Plausible binding modes for putative CaN distal helix with CaM 4 DISCUSSION
Figure 10: Backbone hydrogen bond analysis in the linker and distal helix region (E415
to I458). Each arrow represents one hydrogen bond with color indicating percentage of
simulated frames with this hbond existed. Only hydrogen bonds exist >10% of simulation
time are shown (we also show hdrogen bonds with >5% in Fig. S8). The whole region
was divided into three subregions as indicated by the dashed magenta arrows below each
subpanel. The subregion definition is consistent as the experimental HXMS data in Fig-
ure 8 in [14]. The number under the magenta arrow depicts the number of hydrogen
bond in this subregion (one trans-subregion hydrogen bond contributes 0.5 to each subregion).
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/analyze the backbone Hbond.ipynb
While we believe site D is the most probable site for distal helix binding, interactions with
other potentially less-favorable sites could occur and contribute to the bound RD conformational
ensemble. Such a diverse ensemble of strongly and weakly bound conformations is increasingly
evident in complexes involving intrinsically disordered peptide (IDP)s and globular targets [27,68]
and may be adopted by CaN as well. It is also interesting that CD experiments in [1] suggested that
the distal helix contact is abolished at temperatures above 38 degrees Celsius. It is tempting to
speculate that the comparatively larger RMSFs of the bound distal helix configurations relative to
the CaMBR, in addition to the weaker interaction energies, may render the distal helix interaction
susceptible to melting.
Strengthening the case for the involvement of the CaM site D in binding the CaN distal
helix are our comparisons against two CaM variants with substantially impaired ability to relieve
enzyme auto-inhibition in another CaM target, Myosin Light Chain Kinase (MLCK) [36]. CaM
appears to relieve MLCK auto-inhibition [69] through binding the kinase’s regulatory domain [70]
and adopts a similar conformation as the CaN/CaM complex with CaM ‘wrapping’ around an α
helical CaMBR motif (see also Fig. 9(a)) [1,67]. Importantly, both appear to utilize secondary
interactions beyond the CaMBR motif and it was shown by Van Lierop et al for MLCK that
K30E and G40D mutations far from its CaMBR-binding domain prevented CaM-dependent kinase
activity. These sites are localized to the site D region we identified for the distal helix in our study.
Although the secondary interactions in MLCK likely involve CaM binding directly adjacent to the
enzyme’s catalytic domain [71], we speculated that mutagenesis of these CaM residues could also
impact CaN activation. Namely, we hypothesized that mutated these residues would destabilize
distal helix binding. We confirmed this in our computational model by demonstrating less favorable
distal helix binding scores.
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4.3 Assessment of phosphatase activity 4 DISCUSSION
4.3 Assessment of phosphatase activity
To challenge our hypothesis that impaired distal helix binding to CaM reduce CaN activity, we
used kinetic phosphatase assays with the substrate pNPP on WT and the aforementioned CaM
mutants. The Michaelis constant, Km, obtained from these experiments informs on the ability of
the catalytic site to bind and dephosphorylate pNPP. This substrate is specific to the catalytic site
due to its low molecular weight, which allows for an indirect analysis of the extent to which CaM
binding removes the AID. We reported significantly higher KMfor both K30E and G40D, thus
these mutants evidence weaker distal helix binding that impedes removal of the AID from the CaN
catalytic site. As a result, the CaM variants reduce the CaN catalysis of the dephosphorylation
reaction, which can be interpreted as the AID competing within pNPP at the catalytic site and
yielding a reduced apparent substrate affinity. This loss in affinity coincides with 40% increases
in KMreported for CaN A454E relative to WT CaN [1], which were attributed to impaired distal
helix formation. It should be noted that the small pNPP molecular is a preferable candidate for
assessing distal helix binding, as opposed to common peptide-based dephosphorylation targets
like RII [47]. Namely, the phospho-peptide binds to a site outside the active site (the LxVP site),
therefore its binding, and hence Km, would be unaffected by mutations in the distal helix region.
Rather, the effects would only be evident in the estimated Vmax values. pNPP, on the other hand,
binds directly to the active site. Mutations in the distal helix region that disrupt its folding and
allow the AID to bind to the active site would result in reduced pNPP binding (higher Km), but
no change in Vmax. This explanation has been used by earlier authors studying the inhibitory
properties of the AID as a peptide [47].
4.4 Tether-model of CaM-dependent CaN activation
We recognize that a shortcoming of our modeling approach is that it is limited to simulations
of CaM complexes with fragments of the CaN regulatory domain, whereas distal helix binding’s
effects on CaN activity are coupled to the entire regulatory domain and specifically, the AID. We
therefore discuss a qualitative description of ‘linker’ dynamics of the regulatory domain appropriate
for the AID-dependent inactivation of CaN. Specifically, we speculate that we can describe extents
of CaN inactivation based on the AID’s effective concentration at the CaN catalytic site. This
effective concentration is controlled by the tethering of the AID to CaN, which effectively confines
the AID to a smaller volume (than free diffusion) that results in a higher interaction probability
with the active site [72]. We use this effective concentration perspective to qualitatively assess
how distal helix interactions with CaM impact CaN activity, as explicit all-atom simulations of
the complete RD are prohibitively expensive. Here we leveraged previous theoretical models of
protein activation [73,74] by describing AID binding to the CaN catalytic domain as an intra-
PPI. This PPI leverages a molecular tether (the regulatory domain) to enhances the local effective
AID (p) concentration near the catalytic domain.
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4.4 Tether-model of CaM-dependent CaN activation 4 DISCUSSION
To illustrate this principle in CaN, we provide a basic extension of a linker-dependent modula-
tion model we recently applied to the calcium-dependent troponin I (TnI) switch domain binding
to troponin C (TnC) [72]. For this reaction, Ca2+ binding to TnC generates a conformation that
can facilitate TnI binding:
TnC Ca2+ Ca·TnC TnI Ca·TnC·TnI;(3)
hence, increasing the TnI concentration would promote the generation of TnC·TnI with fewer
equivalents of Ca2+. In the tethered state, we estimated that the effective switch peptide concen-
tration was an order of magnitude greater near its TnC target than would be expected for a 1:1
stoichiometric ratio of untethered (free) switch peptide to TnC. Accordingly, we experimentally
confirmed that formation of the TnC/TnI switch peptide occurred at lower Ca2+ concentrations for
the TnC-tethered TnI compared to a cleaved system in which both TnC and TnI were untethered.
In a similar vein, we created a hypothetical linker-based model of CaN activation, based on a
polymer-theory based model for the probability distribution of the linker spanning the CaMBR
and AID domains (see Fig. 11). We introduce this model with several assumptions. Firstly, we
postulate the CaN inhibition is dependent on the free AID concentration, of which the latter is
determined by the RD ’tether’ length. This tether length can assume three distributions associ-
ated with the CaM-free, CaMBR-bound CaM and CaMBR+distal helix-bound CaM, respectively.
Lastly, for simplicity we assume that distal helix binds CaM independent of the AID’s bound
state, though in reality we recognize there will be a competition between these two events.
Under these assumptions, we describe the effective [AID] at the CaN catalytic domain, based
on the RD linker length in its CaM-free, CaMBR-bound CaM and CaMBR+distal helix-bound
CaM states. We based this on an effective concentration model for tethered ligands suggested by
Van Valen et al [73],
[AID]eff =3
4πξ L 3/2
exp −3D2
4ξL (4)
, where Dis the distance between CaMBR and catalytic site, Lis linker length, and ξis the
persistence length. As stated in [73], although [AID]ef f in Eq. 4has unit of concentration, a
real meaningful unit of [AID]eff that allows accurate interpretation of experiments could only be
achieved via fitting to existing experimental data. Fortunately, experimental assays have been
conducted to investigate the competitive inhibitory effect of isolated AID peptide on CaN phos-
phate activity on substrate peptide [75,76]. In the assays, the reduction of phosphate activity was
recorded as isolated AID peptide was added to intact CaN pre-incubated with CaM and substrate
RII peptide. According to the experimental setup, there existed three competitive components
that could bind the catalytic site of CaN: substrate RII peptide, isolated AID peptide and teth-
ered AID from the intact CaN itself. Similar to Pon definition which represents the probability
of switch peptide being on under the competitive binding of free ligand and tethered ligand to
receptor in [73], we also defined a Pon which represents the percentage of CaN phosphate activity
on substrate RII peptide under competitive binding from isolated AID peptide and tethered AID:
Pon =1 + [RII]
Kd1
1 + [RII]
Kd1+[AID]
Kd2+[tAID]
Kd2
(5)
, where [RII],[AID]and [tAID]are concentrations of substrate peptide, isolated and tethered AID
peptide, respectively. [RII]is set as 5 µM according to exprimental setup and the dissociation
constant of substrate peptide Kd1is assumed to be 10 µM. Tethered AID peptide is assumed to
have same dissociation constant Kd2as isolated peptide with value being experimentally mea-
sured as 40 µM [75,76]. The fitting of Eq. 5to experimental data in [75] with [tAID]as free
pameter is shown in Fig. 11(b). [tAID]was fitted as 2.07 µM and this value is corresponding
to [AID]eff of ’CaMBR+distal helix-bound CaM’ case in our tether model. In following tether
model analysis, the [AID]ef f from Eq. 4were scaled by [tAID]to give meaningful unit of effective
AID concentration.
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4.4 Tether-model of CaM-dependent CaN activation 4 DISCUSSION
To conduct tether model analysis, we first provide a rough estimate for the linker length
through simulations of residues E415-M490 C-terminal to the CaMBR (see Fig. 11(a)). Starting
from WT/A454E site D simulations, an optimized fragment (residues K459 to M490) containing
AID built by tleap was fused to the C-termini of distal helix in the representative structure
of first two most populated clusters. The complete structures were resolvated and simulated for
≈0.7µs as that described in Sect. S1.1. These simulations indicate that the WT AID to CaM
distance is approximately 23 Å, versus approximately 40 Å for the A454E variant that precludes
distal helix binding.
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4.5 Limitations 4 DISCUSSION
Figure 11: (a) Distribution of AID center of mass (COM) relative to the CaM/CaMBR
complex. The black and red spheres represent the COMs of AID in WT and A454E cases,
respectively. The lower panel depict distance between COMs of AID and CaM. The number
above black bar are P values of WT case with null hypothesis that its values are equal to
A454E case. (b) Fitting of the competitive-inhibitor model (Eq. 5) to experimental data
from [75]. (c) Effective AID concentrations calculated via Eq. 4. The shaded green area
represents effective [AID] that leads to CaN’s activation. Right panel illustrates the as-
sumed distance between CaMBR and catalytic site. The value is set as 66 Å in this study.
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/CaN_tether_model.ipynb
Based on these data, in Fig. 11(c) we demonstrate the effective AID concentration over a range
of ligand lengths (L), predicted from Eq. 4assuming D= 66 Å for the distance between CaM
and the CaN AID binding site and ξ= 3 Å [77]. The black dot represents the CaMBR/distal
helix (DH)-bound case, which has a tethered ligand length estimated from our simulation of
approximately 23 Å or roughly 8 free amino acids. The blue dot represents free RD, which has
ligand length of 95 residues (M387 to E481). The red dot represents the CaMBR-bound (no distal
helix interaction as for the A454E case, in this case, the tethered ligand length estimated from our
simulation as 40 Å). Based on these linker lengths, the corresponding effective [AID] concentrations
for CaMBR-bound (A454E) states were 6.76 µM versus 2.07 µM for the CaMBR/distal helix-
bound case. For the free RD case, the effective [AID] is 3.20 µM. This approximate model
qualitatively captures the experimental trends in activity data reported in the literature [1,78],
namely that maximal CaN activation requires CaM binding.
There are several considerations that could improve the accuracy of this model. These include
assumptions that the linker follows a random-walk chain distribution, that the catalytic domain
does not attract and thereby bias the AID distribution and that the CaN molecule does not
sterically clash with the linker chain. Further, precise knowledge of the CaM distribution relative to
the CaN B-chain would be needed to refine the effective linker lengths. Despite these assumptions,
the model provides a qualitative basis for how RD mutations or variations in RD length could
influence the efficiency of CaN (in)activation, similar to the model systems with synthetic linkers,
as in [79].
4.5 Limitations
We observed appreciable degrees of alpha helical and beta sheet character in the regulatory domain
that were not evident in the CD data from [14]. A primary distinction between the modeling and
experimental studies is that we used a much smaller regulatory domain fragment (residue A391
to I458) than the full length domain in Rumi-Masante et al [14], owing to the computational
expense. It is possible that there are different tendencies to form secondary structure, based on
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5 CONCLUSIONS
the regulatory domain length. Since we simulated only a small fragment of the RD domain, this
might have increased the peptide’s preference for alpha helical structure than would otherwise
be observed in measurements of the entire RD. For instance, it has been shown that IDPs have
length-dependent preference of residue compositions as longer IDP has more enriched K, E and
P than short IDP [80], implying the conformational properties of IDPs which are determined by
sequence charge distribution [81] are also length-dependent. As a concrete example, Lin et al [82]
reported that the 40-residue disordered amyloid beta monomer has reduced β-hairpin propensity
when compared to the longer 42-residue monomer.
We additionally recognize that differences in ionic strength or solvent composition might influ-
ence the percentage of alpha helical character, although this seemed to be a modest effect in our
simulations of the CaMBR alone [27]. Importantly, in that study, we reported negligible alpha
helical character for that isolated CaMBR peptide, which suggests that our force field was not
artificially stabilizing alpha helices, as had been an issue in earlier modeling studies of IDPs [83,
84]. Nevertheless, the potential overestimate of alpha helical content for the isolated peptide is
probably of little consequence, since the predicted bound distal helix was shown to confirm exhibit
significant alpha helical content consistent with experiment.
We utilized a REMD approach to sample the distal helix sequence in the absence of CaM;
although REMD has been shown to perform well in terms of qualitatively describing conforma-
tional landscape, chemical shifts, α-helix stability for peptides of lengths comparable to the distal
helix [85–87], we did not have the means to experimentally validate the predicted apo ensembles.
Nevertheless, the simulations provide testable hypotheses in terms of the alpha helical content.
We additionally limited ourselves to subsets of the CaM surface for the docking search, which
represented approximately 38% of the solvent-exposed surface area. However, given that the
microsecond-length simulations were sufficient to reorient the site B configurations into the site D
site, we anticipate the docked distal helix candidates reasonably sampled the thermodynamically-
accessible regions of the CaM surfaces. Although it has been demonstrated that RD binding to
CaM is diffusion-limited, it is also possible that the intermediate complexes could be further op-
timized to form a final bound state, which would perhaps lead to more accurate assessments of
critical intermolecular contacts and energy estimates. For the latter, alchemical methods such as
thermodynamic integration may provide more accurate affinity estimates, albeit at a substantially
greater computational expense compared to ‘end point’ methods like MM/GBSA. Lastly, more
detailed simulations of the RD ensemble in the presence of the complete CaM and CaN struc-
tures are needed to more accurately characterize the effective AID distribution controlling CaN
(in)activation.
5 Conclusions
We have developed a computational strategy to elucidate potential binding poses for a secondary
interaction (the ‘distal helix’) between the CaN regulatory domain and CaM that is apparently
essential for competent CaN activation. We combined REMD simulations of isolated distal helix
peptides, protein-protein docking of the distal helix peptides to the CaMBR-bound CaM surface,
and microsecond-scale MD simulations of candidate poses to implicate a so-called CaM site D
in binding the CaN distal helix. The predicted site D region is in part stabilized through direct
interactions with K30 and indirectly through G40, which is consistent with experimental probes
of a CaM-activated enzyme, MLCK. With these data, we provide a qualitative model of AID-
dependent CaN activation, which can be used to further refine potential molecular mechanisms
governing the activation process and susceptibility to missense mutations. Given the broad range
of physiological processes mediated by CaM binding to intrinsically disordered target proteins [60],
the mechanistic details of CaN activation in this study may extend to diverse systems, including
channel and cytoskeletal regulations [60,88].
There are several compelling directions to pursue that would provide essential clues governing
CaM-dependent CaN activation. For one, we have predicted several contacts that appear to be
involved in stabilizing the distal helix region; mutagenesis of these potential ‘hotspots’ on the
CaM and measurements of subsequent CaN phosphatase could help validate this site. In addition,
more detailed characterization of the RD intrinsically-disordered conformation ensemble would
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5 CONCLUSIONS
benefit future modeling. Given the difficulty in probing ensemble properties of IDPs, it is likely
that modeling and experiment, such as fluorescence resonance energy transfer (FRET) labeling,
should work in tandem toward this goal. Furthermore, relating these RD ensemble properties to
the propensity for AID and CaN catalytic domain interactions would comprise an essential step
toward a complete model of CaM-dependent CaN activation.
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6 ACKNOWLEDGEMENT
6 Acknowledgement
We dedicate this study to the late Jeffry Madura, Ph.D., whose contributions to computational
chemistry and the scientific community as a whole will be forever cherished. Research reported in
this publication, release was supported by the Maximizing Investigators’ Research Award (MIRA)
(R35) from the National Institute of General Medical Sciences (NIGMS) of the National Insti-
tutes of Health (NIH) under grant number R35GM124977. This work used the Extreme Science
and Engineering Discovery Environment (XSEDE) [89], which is supported by National Science
Foundation under grant number ACI-1548562.
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REFERENCES 1
S1 Supplementary Information (SI): Molecular basis of
calmodulin-dependent calcineurin activation
Bin Sun1, Darin Vaughan1, Svetlana Tikunova3,
Trevor P. Creamer2, Jonathan P. Davis3and PM Kekenes-Huskey*1,4
1Department of Chemistry, University of Kentucky, 2Department of Molecular & Cellular
Biochemistry, University of Kentucky, 3Department of Physiology and Cell Biology, Ohio State
University, 4Department of Chemical and Materials Engineering, University of Kentucky,
Lexington, KY, USA 40506
* Corresponding authors. E-mail address: pkekeneshuskey@uky.edu (P. Kekenes-Huskey)
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REFERENCES 2
S1.1 Methods
The initial structure corresponding to CaN distal helix to AID region (residues 459 to 490) was
built from sequence via tleap. The initial structure was subjected to minimization and MD
simulation in vacuum according to the procedure described Sect. 2.3. The optimized structure
was then appended to the C-terminus of distal helix region from the representative structure of
site D simulations via tleap. The representative structures of the first two most populated clusters
from site D simulations were selected, making the simulation duplicate. The sytem was then
solvated in TIP3P waterbox with 0.15 M KCl ions added. The simulation details are same as
previous section in which the tleap built structure was first relaxed while rest part being fixed
during the heating and equilibrium stage. After reaching equilibrium, about 0.7 µs production
simulation was performed from each replica of the duplicate. The simulations were repeated for
the CaN A454E mutant. The total number of MD cases considered in this study is listed in
Table S3.
S1.2 Tables
Table S1: Residues at each tentative binding site on collapsed CaM used in ZDOCK to predict
distal helix interaction at each site
Tentative Site Residues
Site A R86, F89, V142, Y138
Site B R106, I125, D118, D122
Site C I9, F12, L69, F65
Site D K30 ,T34, R37, Q49
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REFERENCES 3
Table S2: ZDOCK docking scores of distal helix and DHA454E at each site. During each docking,
a culling process was applied on the initially generated 2×103poses to eliminate those having no
contacts with residues we specified in Table S1. After culling, the docking scores of highest-scored
poses (up to 10 poses) are given. In DH-site A and DHA454E-site B cases, there was no remaining
poses left after culling, we instead output the first 10 poses’ scores from the initially-generated
2×103poses.
site A site B site C site D
DH DHA454E DH DHA454E DH DHA454E DH DHA454E
pose 1 840.19 618.11 412.93 642.15 687.64 506.11 445.27 642.15
pose 2 786.35 617.84 378.87 629.03 668.06 505.23 414.33 629.00
pose 3 780.74 609.35 618.11 582.64 496.39 386.93 618.11
pose 4 726.30 608.15 617.84 572.20 482.48 617.84
pose 5 711.46 578.58 609.35 567.99 471.27 609.35
pose 6 705.07 564.20 608.15 564.11 470.38 608.15
pose 7 699.21 559.22 578.58 558.02 467.40 578.58
pose 8 681.33 550.57 567.75 550.77 465.25 567.75
pose 9 678.08 543.51 567.45 512.28 459.97 567.45
pose 10 670.06 537.14 564.20 505.47 457.62 564.20
Table S3: MD simulation cases.
Cases Starting structure Simulation length (ns)
CaN DH fragment tleap built from sequence 100 ns REMD
CaN DHA454Efragment tleap built from sequence 100 ns REMD
site A PDB 4q5u + ZDOCK-predicted 1 µs
site B PDB 4q5u + ZDOCK-predicted 1.26 µs
site C PDB 4q5u + ZDOCK-predicted 1.42 µs
site D PDB 4q5u + ZDOCK-predicted 1.38 µs
CaM K30E representative simulated structure from site D 1.22 µs
CaM G40D representative simulated structure from site D 1.22 µs
CaN A454E representative simulated structure from site D 1.1 µs
site D with AID added representative simulated structure from site D duplicates with 0.68 µs each
CaN A454E with AID added representative simulated structure from CaN A454E duplicates with 0.7 µs each
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REFERENCES 4
S1.3 Figures
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REFERENCES 5
Figure S1: Highest-ranking CaM/distal helix interaction poses predicted by ZDOCK3.0.2 [31]
webserver at each site. The color scheme is same as Fig. 2. Key residues at the interaction surface
are shown in sticks with black labels for CaM residues and red labels for distal helix residues.
Comparisons of the WT distal helix poses versus predictions for DHA454E are shown in Fig. S2.
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REFERENCES 6
Figure S2: Comparison of Zdock predicted poses of DH and DHA454E mutant at each site.
The DHA454E mutant is colored in gray. At site A and C, the poses of DH and mutant are close,
while at site B and D, the DHA454E mutant are predicted to be located near site A.
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REFERENCES 7
Figure S3: Overlap of MD simulated distal helix conformation start-
ing at site B (colored in salmon) and site D (colored in red). Dur-
ing the simulations, distal helix starting at site B migrated to site D.
go to /net/share/bsu233/CaMDH/zdock_MD/zdocksite4/traj/pdb/s4−s2−comparasion, type "pymol log.pml"
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REFERENCES 8
Figure S4: Secondary structure propensity of the distal helix during the MD simulations, as
predicted via CPPTRAJ using the DSSP algorithm. The abundance of green indicates significant
alpha helicity.
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Figure S5: Illustration of β-sheet formed in T427-G439 region from site D simulations. The
shown structures are representative structures of first four most populated clusters.
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Figure S6: Secondary structure propensity of the linker at site D during the MD simulations,
as predicted via CPPTRAJ using the DSSP algorithm. The blue region depicts the formation of
β-sheet during most of the simulation time.
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REFERENCES 11
Figure S7: α-helix structural probability of each residue in distal helix region of WT, CaM
K30E and G40D mutants and CaN A454E mutant calculated from MD simulations initiated at
site D.
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Table S4: Hydrogen bonds between distal helix and CaM in each case. Only hydrogen bonds
sustained for >=10% of the simulation duration are listed. The first residue is from distal helix
and second residue is from CaM
Site A Site B Site C Site D CaM K30E CaM G40D CaN A454E
Q442-R90 0.17 K441-G40 0.10 Q442 R90 0.17 Q442 S101 0.10 Q445 S38 0.10 S446 T34 0.14 V459 K94 0.18
K441-R90 0.27 A457 R37 0.10 E453 L4 0.13 E450 K21 0.10 Q445 K21 0.10 S446 N111 0.16 A451 K94 0.28
E445 N42 0.11 E453 Q3 0.28 E456 K30 0.11 A454 K115 0.10 E453 K30 0.19
L444 N42 0.12 Q445 H107 0.12 D455 K115 0.15 K441 N42 0.21
E456 K21 0.13 E453 K21 0.19 Q442 R37 0.19 D455 R37 0.22
E456 S38 0.17 S446 S38 0.23 L444 S38 0.29 Q442 N42 0.24
K441 R90 0.23 D455 R37 0.23 T448 T110 9.35
K441 D93 0.28 E456 R37 0.32
D455 R37 0.30 Q445 R37 0.37
Q445 K94 0.55
Figure S8: Backbone hydrogen bond analysis in the linker and distal helix re-
gion. Each arrow represents one Hbond with color indicating percentage of simulated
frames with this hbond existed. hbonds exist >5% of simulation time are shown.
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/analyze the backbone Hbond.ipynb
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REFERENCES 13
Figure S9: Comparison of backbone hydrogen bond in the linker and DH region be-
tween WT site D and WT site D with extra AID fragment added. The backbone hydrogen
bond of CaMBR from WT site D is also show as reference. 1) In one of the two runs
with AID added, the number of i,i+4 hbond (Ni,i+4) was increased by 1 due the pres-
ence of AID fragment, while the other run has value unchanged. 2) In both runs with
AID added, the backbone hbonds become more stable as the duration of hbonds are gen-
erally enhanced. 3) The helicity of linker and DH region in site D (with/without AID)
are much less stable than that of CaMBR, as indicated by smaller value of Ni,i+4, 6/7 vs. 19
https://bitbucket.org/pkhlab/pkh−lab−analyses/src/default/2018−CaMDH/analyze the backbone Hbond.ipynb
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