Crystal structures of free and ligand‐bound forms of the TetR/AcrR‐like regulator SCO3201 from Streptomyces coelicolor suggest a novel allosteric mechanism

Abstract

TetR/AcrR‐like transcription regulators enable bacteria to sense a wide variety of chemical compounds and to dynamically adapt the expression levels of specific genes in response to changing growth conditions. Here, we describe the structural characterisation of SCO3201, an atypical TetR/AcrR family member from Streptomyces coelicolor that strongly represses antibiotic production and morphological development under conditions of overexpression. We present crystal structures of SCO3201 in its ligand‐free state as well as in complex with an unknown inducer, potentially a polyamine. In the ligand‐free state, the DNA‐binding domains of the SCO3201 dimer are held together in an unusually compact conformation and, as a result, the regulator cannot span the distance between the two half‐sites of its operator. Interaction with the ligand coincides with a major structural rearrangement and partial conversion of the so‐called hinge helix (α4) to a 310‐conformation, markedly increasing the distance between the DNA‐binding domains. In sharp contrast to what was observed for other TetR/AcrR‐like regulators, the increased interdomain distance might facilitate rather than abrogate interaction of the dimer with the operator. Such a ‘reverse’ induction mechanism could expand the regulatory repertoire of the TetR/AcrR family and may explain the dramatic impact of SCO3201 overexpression on the ability of S. coelicolor to generate antibiotics and sporulate.

Full text

Crystal structures of free and ligand-bound forms of the
TetR/AcrR-like regulator SCO3201 from Streptomyces
coelicolor suggest a novel allosteric mechanism
Sebastiaan Werten
1
, Paul Waack
2,
*, Gottfried J. Palm
2
, Marie-Jo€
elle Virolle
3
and
Winfried Hinrichs
2
1 Institute of Genetic Epidemiology, Medical University of Innsbruck, Austria
2 Institute of Biochemistry, University of Greifswald, Germany
3 Institute for Integrative Biology of the Cell (I2BC), Universit
e Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France
Keywords
gene regulation; polyamine; SAXS;
transcription; X-ray crystallography
Correspondence
S. Werten, Institute of Genetic
Epidemiology, Medical University of
Innsbruck, Sch€
opfstr. 41, 6020 Innsbruck,
Austria
Tel: +43 512900370578
E-mail: sebastiaan.werten@i-med.ac.atW.
Hinrichs, Institute of Biochemistry,
University of Greifswald, Felix-Hausdorff-Str.
4, 17489 Greifswald, Germany
Tel: +49 17654172520
E-mail: winfried.hinrichs@uni-greifswald.de
Present address
*Bayer AG, Berlin, Germany
(Received 14 May 2022, revised 14 July
2022, accepted 25 August 2022)
doi:10.1111/febs.16606
TetR/AcrR-like transcription regulators enable bacteria to sense a wide
variety of chemical compounds and to dynamically adapt the expression
levels of specific genes in response to changing growth conditions. Here, we
describe the structural characterisation of SCO3201, an atypical TetR/
AcrR family member from Streptomyces coelicolor that strongly represses
antibiotic production and morphological development under conditions of
overexpression. We present crystal structures of SCO3201 in its ligand-free
state as well as in complex with an unknown inducer, potentially a polya-
mine. In the ligand-free state, the DNA-binding domains of the SCO3201
dimer are held together in an unusually compact conformation and, as a
result, the regulator cannot span the distance between the two half-sites of
its operator. Interaction with the ligand coincides with a major structural
rearrangement and partial conversion of the so-called hinge helix (a4) to a
3
10
-conformation, markedly increasing the distance between the DNA-
binding domains. In sharp contrast to what was observed for other TetR/
AcrR-like regulators, the increased interdomain distance might facilitate
rather than abrogate interaction of the dimer with the operator. Such a ‘re-
verse’ induction mechanism could expand the regulatory repertoire of the
TetR/AcrR family and may explain the dramatic impact of SCO3201 over-
expression on the ability of S. coelicolor to generate antibiotics and sporu-
late.
Introduction
Streptomyces are a genus of gram-positive, soil-
dwelling bacteria, capable of producing a wide variety
of secondary metabolites of immediate biomedical
interest. These include an estimated 80% of currently
available antibiotics, as well as numerous antitumour
agents, antifungals and antivirals [1–3]. Certain species
of Streptomyces have in addition gained popularity as
hosts for heterologous protein expression [4]. A char-
acteristic feature of streptomycetes is their complex
differentiation cycle, which involves profound changes
in metabolism as well as in morphology [5–7]. The dif-
ferentiation process is triggered by variations in the
intra- and extracellular concentrations of specific com-
pounds, particularly nutrients that constitute vital
sources of phosphorus and nitrogen [8]. Concentra-
tions of these substances are often gauged by one-
component systems [9], such as the TetR/AcrR [10]
family of transcription regulators. Members of this
Abbreviations
HTH, helix-turn-helix motif; SAXS, small-angle X-ray scattering; RMSD, root-mean-square deviation.
521The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
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family act as homodimeric repressors or activators,
which bind to palindromic sequences in the promoters
of target genes and are released by a cognate small
molecule, the inducer. TetR/AcrR-like regulators are
bipartite and consist of an N-terminal DNA-binding
domain that comprises a helix-turn-helix (HTH) motif,
followed by a C-terminal domain required for dimeri-
sation and inducer recognition [11]. In all cases where
crystal structures of DNA-bound as well as inducer-
bound forms are available, interaction with the small
molecule is found to allosterically increase the distance
between the DNA-binding domains, which then
exceeds the spacing between the half-sites in the opera-
tor [10,12]. As a consequence, DNA-binding is abro-
gated and transcription enabled [13].
SCO3201, a TetR/AcrR-like regulator from Strepto-
myces coelicolor, was originally described by Xu et al.
[14], who screened a genomic library for factors that
influence antibiotic production and development.
Overexpression of SCO3201 was found to strongly
reduce levels of actinorhodin (ACT), a polyketide
antibiotic, as well as to inhibit morphological differen-
tiation and sporulation of S. coelicolor [14]. The pro-
tein was shown to physically interact with the
promoters of at least 16 genes, including some that
encode downstream regulators, thereby directly and
indirectly affecting numerous genes that play a role in
the differentiation process [15]. An initial crystallo-
graphic analysis of SCO3201 at 2.1

A resolution
revealed a highly asymmetric homodimer, with an
unidentified small molecule bound to one of its sub-
units [15]. In this study, we describe a new crystal
form that lacks the unknown ligand, leading to an
essentially symmetric and unusually compact confor-
mation of the dimer. This compact conformation is
also observed in solution by means of small-angle X-
ray scattering (SAXS). Furthermore, we have repeated
the crystal structure determination of the asymmetric,
ligand-bound dimer at a higher resolution (1.89

A),
providing more detailed insight into the nature of the
small molecule, a possible polyamine. Taken together,
our data suggest that a novel allosteric mechanism
underlies the ligand response of SCO3201.
Results
The crystal structure of the ligand-free form of
SCO3201 reveals an unusually compact ‘closed’
state
The asymmetric unit of the ligand-free P2
1
crystal
form that we obtained contains four polypeptide
chains (A–D), which form two homodimers (AB and
CD). The overall fold of SCO3201 (Fig. 1) is typical
of the TetR/AcrR family of transcription factors [10]
and comprises an all-helical regulatory domain that
also mediates dimerisation, preceded by a DNA-
binding three-helix-bundle (a1–a3). The second and
third helix of this DNA-binding domain correspond to
a classical helix-turn-helix (HTH) motif. A distinguish-
ing feature of SCO3201 is the additional a-helix (a8)
within a solvent-exposed loop of the canonical TetR/
AcrR fold, which juts out from the core of the ligand-
binding domain (Fig. 1A). However, the most striking
aspect of the structure is arguably the exceptionally
short distance separating the HTH motifs of the
homodimer. The recognition helices (a3), which are
crucial for sequence-specific DNA binding [10], are
22.6

A apart in dimer AB and 24.5

A in dimer CD
(this is measured as the distance between the centres of
mass of the helical residues, amino acid residues 66–
72, considering main-chain atoms only). In most struc-
tures of ligand-free and DNA-bound TetR/AcrR-like
regulators the corresponding distance is within the
range 35–41

A, while even longer distances have been
observed for inducer-bound repressors [16]. The posi-
tioning of the DNA-binding domains of SCO3201,
which gives the dimer a remarkably compact (‘closed’)
appearance compared to other members of the TetR/
AcrR family, mainly arises because of a pronounced
bend in the so-called hinge helix (a4). This helix plays
a key role in the allosteric induction mechanism of
TetR/AcrR-like repressors, as it physically links the
HTH motif to the ligand-binding domain. Interest-
ingly, the equivalent of a4 in SCO3201 is a composite
helix, as its N-terminal part in direct contact with the
DNA-binding domain adopts a 3
10
-conformation. This
results in a bend of approximately 30°at the junction
with the a-helical section, in good agreement with the
average bend angle for 3
10
-atransitions (37°19°)
observed by Pal et al. [17]. Each of the four indepen-
dent protein chains in the crystal features a similarly
bent composite hinge helix and the AB and CD dimers
can be superimposed with an RMSD of 1.6

A for a
total of 358 aligned C
a
-atoms (Fig. 1B).
Ligand binding coincides with major
conformational changes
A second crystal form, belonging to space group
P2
1
2
1
2
1
, turned out to be indistinguishable from the
one that we had already analysed in earlier work [15].
However, crystals diffracted to higher resolution this
time (1.86

A versus 2.1

A). As in the earlier struc-
ture, the asymmetric unit comprises a single homod-
imer, whose subunits adopt markedly different
522 The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
The induction mechanism of SCO3201 S. Werten et al.

conformations. While chain B is virtually identical to
the four molecules in the ligand-free structure (with C
a
-
RMSD values ranging between 0.79 and 1.0

A), the
DNA-binding domain of chain A has moved in the out-
ward direction by approximately 10

A, resulting in an
increased a3–a3 distance of 34.8

A (Fig. 2). This
change coincides with the binding of an unknown
ligand to a cavity within the A-subunit (Figs 2and 3).
The movement of the DNA-binding domain is caused
by a repositioning of the composite hinge helix, which
undergoes a 10°outward rotation (away from the
ligand binding pocket) as well as a 3

A shift in the
upward direction in Fig. 2A. This movement is accom-
panied by a rearrangement of the adjacent helices a5,
a6 and a7. All of these changes result from direct con-
tacts between the helices and the ligand, which force the
binding pocket to expand. Interestingly, the upward
shift of the hinge helix would cause its outer 3
10
-
segment to collide with the adjacent a7 helix, while the
DNA-binding domain would be drawn into the ligand-
binding domain. Such clashes are avoided by an exten-
sion of the 3
10
-helix (Fig. 2B), which now includes two
additional residues belonging to the a-helical region in
the ligand-free structure (Ala81 and Pro82). The result
is not only an appreciable elongation of the hinge helix,
but also a reduction of the bend angle to approximately
15°. This straightening is a direct consequence of the
fact that the remodelled 3
10
-ajunction does not obey
the sequence requirements for composite helices that
were formulated by Pal et al. [17]. In particular, Pro82
no longer occupies the favourable second position in
the a-helix, but instead acts as the final residue of the
3
10
-helix. This precludes proper capping of the a-helix
and formation of a larger bend. Instead, Pro82 is rigidly
integrated into the structural framework of the 3
10
-helix
by hydrogen bonds between its side chain (C
d
-H) and
the carbonyl moieties of Glu78 and Ala79 (with C
d
-O
distances of 3.1 and 3.4

A, respectively).
In contrast to the extensive changes to helices
a4–a7, the main dimerisation interface consisting of
helices a9 and a10 remains essentially unaffected by
the presence of the ligand. These helices can be super-
imposed onto the corresponding part of the ligand-free
AB dimer with a C
a
-RMSD of 0.38

A (68 aligned resi-
dues). In the ligand-free dimer, however, two highly
similar salt bridges related by non-crystallographic
symmetry (Fig. 3B) connect the carboxylate of Glu155
in helix a7 to the guanidinium moiety of Arg195 in
helix a9 of the dimerisation partner. In the liganded
structure, the head group of the unknown small mole-
cule is at 2.5

A, that is well within hydrogen bonding
distance, from one of the carboxylate oxygen atoms of
Glu155. This interaction abrogates one of the hydro-
gen bonds between Glu155 and the side chain of
Arg1950, thereby breaking the electrostatic symmetry
of the two salt bridges.
Fig. 1. The structure of the ligand-free
SCO3201 homodimer. (A) Ribbon
representation of the dimer in two
orientations. Chains A and B are shown in
beige and green, respectively. The exposed
helix a8, which has no equivalent in other
TetR/AcrR regulators, is highlighted in
brown (chain A) and dark green (chain B).
Designations LBD and DBD indicate the
ligand-binding and DNA-binding domains,
respectively. The distance separating the
recognition helices (a3) of the two HTH-
motifs is indicated by an arrow. (B) Stereo
view of the superposed AB (colours as in
panel A) and CD (grey) homodimers, shown
as C
a
-traces.
523The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
S. Werten et al. The induction mechanism of SCO3201

The elusive ligand may be an aliphatic polyamine
Unfortunately, mass spectrometry analysis of dissolved
crystals failed to identify the unknown ligand. However,
owing to the markedly improved resolution of the data,
the contours of the small molecule are now clearly
defined in the electron density map (Fig. 3). Although
the identity of the ligand cannot be unambiguously
deduced from the density alone, the properties of the
binding site and the way it interacts with the ligand
necessitate a linear aliphatic chain of at least 8 heavy
(non-hydrogen) atoms, including a positively charged
(or at least polar) head group. In view of these require-
ments, aliphatic polyamines [18] seem to be likely candi-
dates. Well-balanced levels of these ubiquitous
compounds, the most abundant of which are sper-
midine, spermine and putrescine [18,19], are considered
essential for cell growth, proliferation and survival in
all organisms [20].InStreptomyces, polyamines have
been shown to play key roles in gene regulation and
metabolism [21]. On the basis of these considerations,
we have tentatively modelled spermidine into the elec-
tron density map. The observed Van der Waals contacts
to hydrophobic residues lining the binding pocket and
the short-distance interaction of the head group with
the carboxylate moiety of the Glu155 side chain are
consistent with this proposition. However, other
polyamines (including the closely related compounds
putrescine, cadaverine and spermine) are equally plausi-
ble candidates and further biochemical studies will be
required to unambiguously identify the natural ligand.
As the binding site widens towards the protein surface,
the outer region of the bound molecule may well be dis-
ordered and, as a result, remain invisible in the density
map. Consequently, the unknown molecule could be
considerably longer than the part that is actually
observed.
SCO3201 adopts the closed conformation in
solution
The markedly different conformations observed in
our crystal structures prompted us to characterise
SCO3201 in solution by means of SAXS (Fig. 4).
Comparison of the extrapolated forward scattering
(I
0
) to that of a bovine serum albumin (BSA) refer-
ence sample suggests a particle mass of 46.4 kDa,
while the concentration-independent Bayesian method
of Hajizadeh et al. [22], yields a value of 46.7 kDa.
These results are in excellent agreement with the pre-
dicted mass of the SCO3201 homodimer (46.7 kDa).
A more detailed analysis of the data using CRYSOL
[23] (Fig. 4A) indicates that the scattering from
SCO3201 is explained almost to within experimental
error (v
2
=1.2) by the CD dimer from the ligand-free
crystal structure. In contrast, using the asymmetric
liganded structure as a model (either with or without
the small molecule) leads to a considerable lack of fit
(v
2
=2.6). The same is true for a symmetrical ‘doubly
liganded’ model, constructed by superimposing a copy
of the A chain of the liganded structure onto the
dimerisation interface of the B chain and combining
that with the original A chain (v
2
=2.5). An ab initio
model calculated from the experimental data using
Fig. 2. Comparison of the ligand-bound and ligand-free SCO3201 structures. (A) Superposition of liganded (protein chains shown as beige
and green ribbons with the unknown small molecule, tentatively modelled as spermidine, represented by red spheres) and free SCO3201
(dimer AB, grey ribbons). The outward movement of one of the HTH motifs by 10

A, which coincides with ligand binding to the same pro-
tein chain, is indicated by an arrow. The portion of the hinge helix that undergoes substantial conformational changes (bright green and blue
in the respective structures) is shown in more detail in panel (B). (B) Conformational changes within the composite hinge helix upon ligand
binding. The N-terminal 3
10
-segment is extended by two residues previously part of the a-helical region (Ala81 and Pro82), leading to stretch-
ing and straightening of the hinge helix. All side-chain atoms beyond C
b
have been removed to improve clarity, except those of Pro82.
Hydrogen bonds are shown as dashed lines.
524 The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
The induction mechanism of SCO3201 S. Werten et al.

the program DAMMIN [24] closely matches the dimer
from 5EFY (Fig. 4B). We therefore conclude that, in
our SAXS experiments, SCO3201 predominantly
adopts a closed conformation similar to the one seen
in the ligand-free crystal structure. Several things may
explain why the ligand-induced conformation is not
observed here. In the first place, the basic conditions
(pH 8.5) might favour dissociation of the complex, as
the pH of the solution could have a considerable
impact on the protonation state of both the ligand
and the glutamate residue it is in contact with.
Indeed, crystals of the complex were obtained at pH
5.0, whereas the ligand-free protein crystallised at pH
8.5. Compared to the crystallisation assays, the SAXS
experiments were also carried out at much lower pro-
tein concentrations, which would be expected to shift
the binding equilibrium towards the dissociated state.
Furthermore, only a small fraction of the purified
protein may have been liganded in the first place.
While this might not prevent crystallisation of the
liganded species, the unliganded form would in this
case dominate the SAXS signal. Finally, we cannot
exclude the possibility that the unknown ligand was
not co-purified from the expression host, but con-
tained in the chemicals used during crystal structure
determination, in particular the paraffin oil (a com-
plex and poorly defined mixture of organic com-
pounds) that we used for cryoprotection. However,
mass spectrometry analysis of a protein solution
extensively equilibrated with paraffin oil also failed to
identify the unknown ligand.
Discussion
The crystallographic structure of the ligand-free form
of SCO3201 reported in the present study reveals a
domain arrangement that, to our knowledge, has not
been encountered in other members of the TetR/AcrR
family. The DNA-binding domains of the protein
dimer are unusually close to one another, mainly as a
result of a pronounced inward bend in the hinge
helices. We have several reasons to believe that this
arrangement is not merely the result of crystal packing
effects, but corresponds to the native state of the
ligand-free regulator. First, a transition from a 3
10
-
helix to an a-helix is fully expected to produce the
Fig. 3. The interaction of SCO3201 with the
inducer. (A) Stereo image of the liganded
cavity. The view is from the opening of the
cavity at the protein surface towards the
interior of the dimer, with the dyad axis
oriented vertically in the plane of the image.
Difference density (mFo-DFc) corresponding
to the inducer, contoured at 3.0 r, is shown
in blue. Nearby side chains are represented
as stick models, as is the spermidine
molecule that was modelled into the
density. The short-distance interaction
between the head group of the inducer and
the side chain of Glu155 is represented by
a dashed line. (B) The dimerisation interface
of ligand-bound SCO3201, viewed along the
dyad axis. Hydrogen bonds involving the
inducer, Glu155 and Arg195 are shown as
dashed lines, together with the
corresponding interatomic distances. As can
be seen here, the local non-crystallographic
symmetry of the dimerisation interface (the
Glu155-Arg1950and Glu1550-Arg195 salt
bridges in particular) is broken by the
interaction of Glu155 with the ligand.
525The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
S. Werten et al. The induction mechanism of SCO3201

kind of kinked junction that is observed here [17]. Sec-
ond, all four of the independent polypeptide chains in
the crystal feature a composite hinge helix with a com-
parable bend, despite the fact that each chain experi-
ences a different lattice environment. And finally, our
SAXS data corroborate the existence of the closed
conformation in solution.
Intriguingly, the compact arrangement of the two
HTH-motifs, with their recognition helices at less than
25

A from each other, would preclude binding to con-
secutive instances of the major groove on one face of
the promoter. This DNA-binding mode, which is a
hallmark of the TetR/AcrR family [10], requires an
a3–a3 distance very close to 35

A[16]. This implies
that the SCO3201 dimer has to undergo a major con-
formational change if it is to interact with the opera-
tor. Although DNA binding by members of the TetR/
AcrR family is typically accompanied by appreciable
structural changes in the regulatory domain [10], all
currently known cases involve DNA-binding domains
that are far apart in the free repressor and need to be
brought into closer proximity to enable promoter
binding. Inducers can then act simply by preventing
such rearrangements [25]. Strikingly, this is the exact
opposite of what we seem to observe for SCO3201.
In our second crystal form, interaction with a small-
molecule ligand causes a large (~10

A) increase in the
distance between the DNA-binding domains, which
would now be compatible with DNA binding. This
observation suggests that SCO3201 could possibly func-
tion by means of a ‘reverse’ induction mechanism, in
the sense that ligand binding might increase, rather than
decrease, the affinity of the regulator for DNA. To our
knowledge, such a mechanism has not yet been sug-
gested for any naturally occurring TetR/AcrR family
members. Mutagenesis studies of TetR(D), however,
have given rise to artificial variants of this repressor (re-
ferred to as ‘revTetR’) that, instead of being released
from DNA by tetracycline like the wild-type protein,
show enhanced promoter binding in the presence of the
antibiotic [26–28]. For the related LacI/GalR family of
transcription factors, cases of increased as well as
decreased DNA-binding in the presence of cognate
small-molecule ligands have been reported [29].
It is currently unclear whether or not the SCO3201
dimer is able to interact with a second inducer mole-
cule, which might further enlarge the physical distance
between the HTH motifs and lead to a complex tran-
scriptional response to increasing ligand concentra-
tions. It should be noted, however, that many TetR/
AcrR-like repressors only bind a single ligand molecule
per homodimer [10], either in a central cavity around
the symmetry axis of the dimer, as in FrrA [25],orin
one of several available pockets within the individual
subunits, for example in QacR [30] and TtgR [31].
The conformational changes that we observe in
SCO3201 are ultimately caused by a slight expansion
of the binding pocket, a direct consequence of the
bulkiness of the inducer. This effect should be virtually
independent of the precise binding specificity of the
cavity, a phenomenon that we have also observed for
the induction mechanisms of TetR(D) [32] and FrrA
[25]. As noted before, modularity of ligand recognition
and allostery is expected to result in a considerable
degree of evolutionary flexibility, allowing existing
transcriptional regulators to mutate and adapt to new
inducers and regulatory circuits while their induction
mechanism remains intact [32].
Loop-to-helix transitions within helix a4, sometimes
linked to the presence of helix-destabilising residues
(Pro, Gly) and generally triggered by direct interac-
tions of the helix with the inducer, have been reported
for several TetR/AcrR-like repressors [33,34]. The
allosteric mechanism of SCO3201, in contrast, relies
on a composite hinge helix and the elongation of its
Fig. 4. SAXS analysis of SCO3201 in solution. (A) Experimental
SAXS data (black dots) and theoretical curves calculated for the
ligand-free (red) and ligand-bound (blue) crystallographic structures.
Theoretical curves were fitted to the experimental data using CRY-
SOL, with 7PT0 and the CD dimer of 5EFY as the models, respec-
tively. (B) Ab initio model (grey surface) with the superposed 5EFY
CD dimer shown in ribbon representation (beige and green).
526 The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
The induction mechanism of SCO3201 S. Werten et al.

3
10
-segment in response to the expansion of the ligand-
binding cavity. Interconversion of 3
10
- and a-helices is
believed to be an important regulatory mechanism in
biomolecular signalling, in particular in voltage-gated
channels and other membrane-associated switches [35].
However, we are not aware of such mechanisms hav-
ing been described for transcription factors. A search
of the PDB using DALI [36] indicates that SCO3201
most strongly resembles entry 2RAE (Z-score: 16.4),
which corresponds to the 2.2

A crystal structure of a
repressor from Rhodococcus sp. RHA1. Although this
protein lacks the additional a-helix within loop a7-a8,
the relative positioning of most of its helices is roughly
comparable to that of counterparts in SCO3201
(Fig. 5A). Like SCO3201, 2RAE possesses a composite
hinge helix, with a helix-destabilising residue (Gly67)
at the transition between the a- and 3
10
-segments
(Fig. 5B). As in SCO3201, the 3
10
-segment also con-
tains a proline residue (Pro65), whose side chain con-
tributes to the 3
10
hydrogen-bonding network.
Although no ligands are present in the deposited
model, inspection of the 2mFo-DFc map reveals an
area of poorly explained electron density within the
putative binding cavity, presumably arising from the
presence of a planar organic molecule (Fig. 5B). This
observation strongly suggests that 2RAE corresponds
to the induced form of the repressor. Unlike the
ligand-bound form of SCO3201, both subunits of the
2RAE homodimer are liganded (they are related by
crystallographic symmetry), leading to an a3-a3 dis-
tance that appears to be too large for DNA binding
(41.8

A). Despite this intriguing difference, SCO3201
and 2RAE together constitute a novel category of reg-
ulators whose allosteric mechanisms exploit the special
properties of 3
10
-ajunctions in composite helices [17].
A more detailed analysis of these molecular switches
awaits the structural characterisation of complexes of
the proteins with promoter DNA, as well as the unam-
biguous identification of their natural inducers.
Involvement of SCO3201 in polyamine sensing, pos-
sibly by means of a reverse induction mechanism, may
explain the dramatic effect of overexpression of the
regulator on morphological differentiation and antibi-
otic production in S. coelicolor [14]. A recent study
has demonstrated that Streptomyces are able to detox-
ify and assimilate polyamines very efficiently, enabling
growth even under conditions where these molecules
constitute the sole nitrogen source [37]. Moreover,
polyamines are naturally abundant in the environ-
ments where Streptomyces are typically found [21].In
S. coelicolor,de novo biosynthesis of putrescine and
spermidine has been reported in the late-stationary
phase in rich medium, while cadaverine synthesis
occurred under conditions of iron limitation [38].
These observations strongly suggest that polyamines
play key roles in cell homeostasis, with depletion
likely to act as a nutritional stress signal. As
soon as the polyamine concentration falls below a
Fig. 5. Comparison of SCO3201 to 2RAE, the most similar PDB entry identified by DALI [36]. (A) Superposition of 2RAE (chains related by
crystallographic symmetry in dark blue and purple) onto the ligand-free SCO3201 dimer (chains C and D, grey). The hinge helices (a4) of
2RAE have been highlighted in light blue and pink. The kink in the hinge helix of 2RAE, around residue Gly67, is indicated by arrows. (B)
Detailed view of the composite hinge helix of 2RAE (pink stick model, side-chain atoms beyond C
b
removed except in Pro65) with the rest
of the protein chain in ribbon representation (purple). Hydrogen bonds are shown as dashed lines. Electron density (2mFo-DFc) likely to cor-
respond to an unidentified ligand, contoured at 1.2 r, is shown in blue. The authors of 2RAE interpreted this density as four water mole-
cules at less than 2.2

A from each other (red spheres).
527The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
S. Werten et al. The induction mechanism of SCO3201

certain threshold, genes involved in morphological
development and antibiotic production would need to
be derepressed, triggering differentiation and antibi-
otic production. Overexpression of SCO3201 would
amplify polyamine-dependent repression (and poten-
tially prolong it) as a result of the law of mass action.
Moreover, sequestering of polyamine by the overex-
pressed regulator might protect the former from being
metabolised, allowing maintenance of repression
throughout growth [14]. However, the fact that dele-
tion of SCO3201 has no detectable impact on morpho-
logical differentiation and antibiotic production [14]
suggests that control of the relevant genes involves
additional factors besides SCO3201. Two regulators of
polyamine catabolism have been described in S. coeli-
color: EpuRII (SCO5656), which controls the glnA3
gene encoding a c-glutamylpolyamine synthetase, and
the global regulator of nitrogen metabolism, GlnR,
which has been shown to interact with the glnA2 gene,
encoding a second c-glutamylpolyamine synthetase
[37]. Interestingly, the expression of glnR can only be
induced by cadaverine [37], whereas the expression of
EpuRII can be induced by putrescine, cadaverine and
spermidine [39]. It is therefore conceivable that several
dedicated factors sense different polyamines or groups
of polyamines in the context of an intricate regulatory
network that mediates cross-talk between a variety of
nutritional signals. Apart from sensors of nitrogen
sources [40–43], such a network is likely to include fac-
tors that respond to the presence of inorganic phos-
phate [44–46] and polyphosphates [47–49].
Materials and methods
Protein expression and purification
In all experiments, a SCO3201 deletion mutant was used
that lacks the 20-residue region of low complexity at the
N-terminus of the full-length sequence, which we found
interfered with crystallisation. The construct, corresponding
to amino acids 21–216 of NCBI reference sequence WP_
011028825.1, was expressed and purified essentially as
described earlier [15]. In brief, the His
6
-tagged protein was
overproduced in Escherichia coli strain C41 using a modi-
fied pET-28b vector that instead of the thrombin recogni-
tion sequence encodes a TEV-protease site. Cultures in LB
medium containing 50 lgmL
1
kanamycin were grown
shaking at 37 °C until they reached an OD (600 nm) of
0.8, followed by a further 4 h incubation in the presence of
1m
MIPTG. Upon centrifugation (15 min at 10 000 g,
4°C), cells from 1 L of culture were resuspended in 30 mL
lysis buffer (20 mMTris/HCl pH 7.0, 400 mMNaCl, 20 mM
of imidazole and 1 mMof DTT) and disrupted by means of
sonication for 8 min on ice using a Sonopuls HD 2070
(Bandelin Electronic GmbH, Berlin, Germany). Sonication
was repeated twice, with 2 min of cooling between cycles.
Cell debris was removed by centrifugation for 1 h at
40 000 gand the cleared lysate applied to a Ni
2+
-charged
Poros 20 MC column (Thermo Fisher Scientific GmbH,
Dreieich, Germany). The protein was eluted using lysis buf-
fer containing 250 mMof imidazole. TEV protease was
added directly to the eluate to a final concentration of
0.1 mgmL
1
. The solution was then incubated for 3 days
at 4 °C, leading to complete removal of the His
6
-tag.
SCO3201 was further purified by gel filtration (Superdex
75, GE Healthcare GmbH, Munich, Germany) in a buffer
containing 20 mMTris/HCl pH 8.5, 400 mMNaCl, 50 mM
imidazole and 1 mMDTT. The protein was concentrated to
30 mgmL
1
using a Vivaspin 15 concentrator (Sartorius
Lab Instruments GmbH, G€
ottingen, Germany) with a
10 kDa molecular weight cut-off. The concentrate was
flash-frozen in 50 lL of aliquots using liquid nitrogen and
stored at 80 °C until further use. Total protein yield was
approximately 100 mg from 1 L of culture. The protein
appeared as a single band on Coomassie-stained SDS/
PAGE gels.
X-ray crystallography
Crystals of ligand-free SCO3201 belonging to space group
P2
1
were obtained using the hanging drop vapour diffusion
method at 20 °C, with a reservoir solution containing 12%
ethanol and 0.1 MTris/HCl pH 8.5. For cryoprotection,
crystals were covered in paraffin oil (alkane chain
length ≥20, Carl Roth GmbH, Karlsruhe, Germany) prior
to flash freezing in liquid nitrogen. Diffraction data to a
resolution of 2.7

A were collected at beamline BL14.2,
operated by the Joint Berlin MX-Laboratory at the BESSY
II electron storage ring (Berlin-Adlershof, Germany) [50].
Crystals of the liganded form of SCO3201, which
belonged to space group P2
1
2
1
2
1
, were also obtained via
hanging drop vapour diffusion at 20 °C. Ahead of crystalli-
sation assays, 1 mL protein solution (2 mgmL
1
) was
incubated with 10 lL of paraffin oil. The rationale behind
this approach was that the oil, which was used as a cry-
oprotectant in the determination of the earlier struc-
ture (4CGR), might contain the unknown ligand, in which
case pre-equilibration might increase occupancy and
improve crystal quality. The solution, without the organic
phase, was subsequently concentrated to 50 lL
(40 mgmL
1
) and used for crystallisation. The reservoir
solution in these experiments contained 1 Mof (NH
4
)
H
2
PO
4
and 0.1 Mof Tris/HCl pH 5.0. Again, paraffin oil
was used for cryoprotection. A dataset with a resolution of
1.86

A was collected at beamline P13, operated by EMBL
Hamburg at the PETRA III storage ring (DESY, Ham-
burg, Germany) [51].
528 The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
The induction mechanism of SCO3201 S. Werten et al.

All diffraction data were processed with XDS [52,53]. Both
crystal structures were solved using PHASER [54], in combina-
tion with the CCP4 suite [55]. For the ligand-free form, chain
B of protein data bank (PDB) entry 4CGR was used as the
molecular replacement search model. Searches with chain A
or the complete homodimer (AB) did not yield meaningful
solutions. For the liganded form, the dimer was used as the
search model. The structures were refined using the CCP4
suite [55] and REFMAC5[56,57]. All molecular graphics illus-
trations were produced with PYMOL 0.99rc6 (DeLano Scien-
tific LLC, Palo Alto, CA, USA). Centres of mass and
angles between helices were determined using CALCOM [58]
and QHELIX [59], respectively. Data collection and refinement
statistics can be found in Table 1.
SAXS
SAXS data were collected at beamline BM29 [60] of the
EMBL Outstation at ESRF, Grenoble, using a PILATUS
1Mpixel detector. Measurements covered the momentum
transfer range 0.002–0.60

A
1
(s=4psin(h)/k, where 2his
the scattering angle and kis the X-ray wavelength,
0.992

A). All experiments took place at a temperature of
293 K. A capillary flow cell was used to avoid radiation
damage. Several SCO3201 dilutions (0.19–5.5 mgmL
1
)
were analysed in a buffer containing 20 mMTris/HCl pH
8.5, 400 mMNaCl, 50 mMimidazole and 1 mMDTT. All
data were normalised to the intensity of the transmitted
beam and radially averaged. Scattering of the buffer was
subtracted and the resulting curves were scaled to unity
protein concentration (1 mgmL
1
). For further data analy-
sis, version 3.0.3 of the ATSAS package was used [61]. Theo-
retical scattering patterns based on structural models were
fitted to the experimental data using the program CRYSOL,
which accounts for protein surface hydration [23].Ab initio
models were calculated using the program DAMMIN [24].
Acknowledgements
We would like to thank Martha E. Brennich (EMBL
Grenoble) for SAXS data collection.
Conflict of interest
The authors declare no conflict of interest.
Author contributions
SW, M-JV and WH conceived, initiated and super-
vised this study; PW expressed, purified and crys-
tallised SCO3201; PW and GJP collected X-ray
diffraction data; PW and WH solved and refined the
crystal structures; SW analysed the SAXS data; SW
interpreted the experimental results, analysed the
induction mechanism and produced the artwork; SW
and WH wrote the manuscript.
Peer review
The peer review history for this article is available at
https://publons.com/publon/10.1111/febs.16606.
Data availability statement
Atomic coordinates and X-ray diffraction data for the
crystal structures of ligand-free and induced SCO3201
have been deposited in the Protein Data Bank (PDB)
with accession codes 5EFY and 7PT0. SAXS data and
models have been deposited in the Small-Angle
Table 1. Data collection and structure refinement statistics. Values
in parentheses correspond to the highest resolution shell.
Protein or complex SCO3201
SCO3201/
spermidine
PDB ID 5EFY 7PT0
Data collection
Radiation source,
beamline
BESSY, BL14.2 PETRA III,
EMBL c/o
DESY, P13
Detector RAYONIX MX-225 Pilatus 6M
Wavelength (

A) 0.9184 0.9763
Temperature (K) 100 100
Resolution range (

A) 94.6–2.70 (2.85–2.70) 61.6–1.89
(2.00–1.89)
Unit cell parameters
a,b,c(

A)
a,b,c(°)
51.79, 98.43, 89.61
90, 100.78, 90
55.13, 79.88,
96.87
90, 90, 90
Space group P2
1
P2
1
2
1
2
1
Unique reflections 23 495 (3916) 34 934 (5288)
Redundancy 3.0 (2.9) 6.3 (5.5)
Mean I/r(I) 7.7 (1.4) 20.7 (1.1)
Completeness (%) 96.6 (97.7) 99 (94.1)
R
meas
(%) 6.1 (101.3) 4.0 (148)
Wilson B-factor (

A
2
) 79.1 59.5
CC 1/2 0.998 (0.279) 1 (0.53)
Refinement
R
cryst
/number of
reflections
0.258/22 236 0.204/33 204
R
free
/number of reflections 0.297/1227 0.220/1727
Number of
non-hydrogen
atoms, protein/
water/ligand
5309/23/–3002/87/10
RMSD bond lengths (

A) 0.016 0.010
RMSD bond angles (°) 1.58 1.60
RMSD torsion
angles (°)
4.68 5.29
Ramachandran
parameters
(%), favoured/
allowed/outlier
99.5/0.5/–96.6/2.8/0.6
Average B-factors (

A
2
) 77.6 64.6
529The FEBS Journal 290 (2023) 521–532 Ó2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of
Federation of European Biochemical Societies.
S. Werten et al. The induction mechanism of SCO3201

Scattering Biological Data Bank (SASBDB) with
accession code SASDN75.
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