Figure 6 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Source publication
We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology...
Citations
... The 13 C and 15 N chemical shifts were referenced indirectly. The backbone chemical shifts of MALT1(Casp-IgL 3 ) 338-719 , 1 HN, 15 N, 13 C α , 13 C β and 13 C´ nuclei, have been previously assigned by us (Unnerstale et al. 2016) using the Target Acquisition approach (Isaksson et al. 2013;Jaravine and Orekhov 2006;Jaravine et al. 2008), and can be found in the Biological Magnetic Resonance Data Bank (Ulrich et al. 2008) (http:// www. bmrb. ...
Mucosa-associated lymphoid tissue protein 1 (MALT1) plays a key role in adaptive immune responses by modulating specific intracellular signalling pathways that control the development and proliferation of both T and B cells. Dysfunction of these pathways is coupled to the progress of highly aggressive lymphoma as well as to potential development of an array of different immune disorders. In contrast to other signalling mediators, MALT1 is not only activated through the formation of the CBM complex together with the proteins CARMA1 and Bcl10, but also by acting as a protease that cleaves multiple substrates to promote lymphocyte proliferation and survival via the NF-κB signalling pathway. Herein, we present the partial ¹ H, ¹³ C Ile/Val/Leu-Methyl resonance assignment of the monomeric apo form of the paracaspase-IgL 3 domain of human MALT1. Our results provide a solid ground for future elucidation of both the three-dimensional structure and the dynamics of MALT1, key for adequate development of inhibitors, and a thorough molecular understanding of its function(s).
... This mutation renders the protease inactive as the serine in the catalytic triad (H51-D75-S135) is changed into an alanine with minimal interference on the overall structure (Agback et al. 2020). Applying the target acquisition (TA) methodology (Isaksson et al. 2013;Jaravine et al. 2008), we performed near complete (> 95%) backbone 1 HN, 15 N, 13 C α , 13 CO, 1 H α and sidechain 13 C β chemical shift assignments of the DENV2 S135A NS3pro/NS2B complex. We also assigned the methyl resonances for the side chains of valine, leucine, and isoleucine residues. ...
The serotype II Dengue (DENV 2) virus is the most prevalent of all four known serotypes. Herein, we present nearly complete ¹ H, ¹⁵ N, and ¹³ C backbone and ¹ H, ¹³ C isoleucine, valine, and leucine methyl resonance assignment of the apo S135A catalytically inactive variant of the DENV 2 protease enzyme folded as a tandem formed between the serine protease domain NS3pro and the cofactor NS2B, as well as the secondary structure prediction of this complex based on the assigned chemical shifts using the TALOS-N software. Our results provide a solid ground for future elucidation of the structure and dynamic of the apo NS3pro/NS2B complex, key for adequate development of inhibitors, and a thorough molecular understanding of their function(s).
... The cytoplasmic domain of the Cd3 protein is largely intrinsically disordered and has been characterized by solution NMR spectroscopy [24]. We have investigated the disordered C terminal part, corresponding to residues 153-207 of the human Cd3 protein containing an immunoreceptor tyrosine-based activation motif (ITAM). ...
... ITAMs are composed of two instances of the consensus sequence Y-X-X-[LI] separated by 6-8 residues [25]. We modeled the sequence as it appears in the corresponding BMRB entry (BMRB ID 18889 [24]) having serine and leucine as the first two amino acids instead of tryptophan and serine. We used the deposited chemical shift data for analysis. ...
Ensemble-based structural modeling of flexible protein segments such as intrinsically disordered regions is a complex task often solved by selection of conformers from an initial pool based on their conformity to experimental data. However, the properties of the conformational pool are crucial, as the sampling of the conformational space should be sufficient and, in the optimal case, relatively uniform. In other words, the ideal sampling is both efficient and exhaustive. To achieve this, specialized tools are usually necessary, which might not be maintained in the long term, available on all platforms or flexible enough to be tweaked to individual needs. Here, we present an open-source and extendable pipeline to generate initial protein structure pools for use with selection-based tools to obtain ensemble models of flexible protein segments. Our method is implemented in Python and uses ChimeraX, Scwrl4, Gromacs and neighbor-dependent backbone distributions compiled and published previously by the Dunbrack lab. All these tools and data are publicly available and maintained. Our basic premise is that by using residue-specific, neighbor-dependent Ramachandran distributions, we can enhance the efficient exploration of the relevant region of the conformational space. We have also provided a straightforward way to bias the sampling towards specific conformations for selected residues by combining different conformational distributions. This allows the consideration of a priori known conformational preferences such as in the case of preformed structural elements. The open-source and modular nature of the pipeline allows easy adaptation for specific problems. We tested the pipeline on an intrinsically disordered segment of the protein Cd3ϵ and also a single-alpha helical (SAH) region by generating conformational pools and selecting ensembles matching experimental data using the CoNSEnsX+ server.
... The development of TROSY type [11,19], BEST-TROSY [25,26], and the nonuniform sampling (NUS)-based NMR techniques [27][28][29] has made it possible to enhance the resolution of multidimensional NMR spectra and to study larger IDPs by NMR [30]. Recently, we performed an evaluation of the manual traditional approaches vs. automated, i.e., NUS-based targeted acquisition procedure (TA) [28,[31][32][33] as a mean to decrease the acquisition and analysis time for larger IDPs [30]. It has been demonstrated that in application for IDPs, TA procedure using BEST-TROSY [25] type experiments: 3D HNCO, 3D HNCOCA, 3D HNCA, 3D HNCACO, 3D HNCOCACB, and 3D HNCACB can be preferable from an NMR time-consuming point of view to traditional TROSY type experiments, leading to significantly decreased acquisition time. ...
In recent years, intrinsically disordered proteins (IDPs) and disordered domains have attracted great attention. Many of them contain linear motifs that mediate interactions with other factors during formation of multicomponent protein complexes. NMR spectrometry is a valuable tool for characterizing this type of interactions on both amino acid (aa) and atomic levels. Alphaviruses encode a nonstructural protein nsP3, which drives viral replication complex assembly. nsP3 proteins contain over 200-aa-long hypervariable domains (HVDs), which exhibits no homology between different alphavirus species, are predicted to be intrinsically disordered and appear to be critical for alphavirus adaptation to different cells. Previously, we have shown that nsP3 HVD of chikungunya virus (CHIKV) is completely disordered with low tendency to form secondary structures in free form. In this new study, we used novel NMR approaches to assign the spectra for the nsP3 HVD of Venezuelan equine encephalitis virus (VEEV). The HVDs of CHIKV and VEEV have no homology but are both involved in replication complex assembly and function. We have found that VEEV nsP3 HVD is also mostly disordered but contains a short stable α-helix in its C-terminal fragment, which mediates interaction with the members of cellular Fragile X syndrome protein family. Our NMR data also suggest that VEEV HVD has several regions with tendency to form secondary structures.
... We used the nonuniform sampling (NUS) approach in combination with targeted acquisition (TA) and a new multidimensional decomposition (MDD) signal processing technique. [51][52][53] ...
... The NUS approach provides a solution because it strongly reduces the experimental time and/or allows for higher resolution without the loss of important information, if the obtainable signal-to-noise ratio is sufficient. [59] In addition, a TA and a new MDD signal-processing technique [51][52][53] applied for nonuniform sampled data dramatically reduces the NMR spectroscopy measurement time and simplifies the evaluation of the spectra. In combination with automated methods for signal list generation and resonance assignment (such as FLYA), [60] this method is suitable for the rapid structural screening of small proteins. ...
... Reduction of the NMR spectroscopy measurement time can be achieved by applying TA in combination with the MDD signal-processing technique on NUS data. [51][52][53] Subsequently, a method for automated resonance assignment, FLYA, [60] can be performed by using the program CYANA for automated NOESY crosspeak assignment and structure calculations. [60][61][62][63] The benefit of using this technique is exemplified for the SP-22 small protein. ...
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution‐state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
... NMR experiments were performed at 45 C on a Bruker spectrometer with a Larmor frequency of 800 MHz equipped with a 3-mm triple-resonance TCI cryoProbe. The automated backbone assignment was obtained using a previously reported assignment platform (32), which uses nonuniformly sampled three-dimensional (3D) TROSY-type experiments (HNCO, HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, HN(CA)CO) combined with targeted acquisition (TA) and statistical validation. The details of this TA platform will not be discussed further in this article. ...
... [ 1 H 15 N]-TROSY spectra (Fig. 1) and a set of nonuniformly sampled 3D TROSY-type H-N-C spectra were re-corded for both states. This set formed the basis for 1 H, 13 C, 15 N backbone resonance assignment using TA (32,34), followed by manual inspection. In the dark spectra, 75% of all assignable residues were assigned for the dark-adapted state (Fig. 2). ...
Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although the crystal structures of dark- and light-adapted states have been determined, the molecular mechanisms underlying photoactivation remain elusive. Here, we demonstrate that the conserved tongue region of the PHY domain of a 57-kDa photosensory module of Deinococcus radiodurans phytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.
... Targeted acquisition (TA) (Isaksson et al., 2013;Jaravine and Orekhov, 2006;Jaravine et al., 2008;Unnerstale et al., 2016): Two sets of NMR experiments were recorded. The first was done at 25°C on a 700 MHz Bruker AVANCE III-HD spectrometer equipped with 5 mm CP-TCI probe. ...
... We have previously performed a preliminary backbone assignment of 199-aa-long CHIKV HVD and demonstrated that it is an intrinsically disordered protein domain (IDP) (Meshram et al., 2018). In this work, application of targeted acquisition (TA) (Isaksson et al., 2013;Jaravine and Orekhov, 2006;Jaravine et al., 2008;Unnerstale et al., 2016), extra-large spectroscopy (XLSY) (Pustovalova et al., 2018), and manual verification led to almost complete assignment of the backbone resonances of CHIKV HVD: 97% of 1 H N , 97% of 15 N, 98% 13 C α , 96% of 13 C β , 98% of 13 C O and 73% of 1 H α signals ( Fig. 2 and Fig. S-1, supplementary data). The assignment data unambiguously proved that CHIKV HVD is disordered in solution ( Supplementary Fig. 2) and allowed us to characterize in detail its interactions with the SH3binding domains of CD2AP protein. ...
Alphavirus nsP3 proteins contain long, intrinsically disordered, hypervariable domains, HVD, which serve as hubs for interaction with many cellular proteins. Here, we have deciphered the mechanism and function of HVD interaction with host factors in alphavirus replication. Using NMR spectroscopy, we show that CHIKV HVD contains two SH3 domain-binding sites. Using an innovative chemical shift perturbation signature approach, we demonstrate that CD2AP interaction with HVD is mediated by its SH3-A and SH3-C domains, and this leaves the SH3-B domain available for interaction with other cellular factor(s). This cooperative interaction with two SH3 domains increases binding affinity to CD2AP and possibly induces long-range allosteric effects in HVD. Our data demonstrate that BIN1, CD2AP and SH3KBP1 play redundant roles in initiation of CHIKV replication. Point mutations in both CHIKV HVD binding sites abolish its interaction with all three proteins, CD2AP, BIN1 and SH3KBP1. This results in strong inhibition of viral replication initiation.
... Data were processed using the compressed sensing IRLS algorithm in the mddnmr software (Kazimierczuk and Orekhov 2011;Mayzel et al. 2014). Sequential assignment was partly achieved using the targeted acquisition approach (Jaravine and Orekhov 2006;Jaravine et al. 2008;Isaksson et al. 2013), and complemented by manual inspection of data. Automated assignment was carried out using the FLYA module of CYANA (Schmidt and Guntert 2012). ...
Bacteroides ovatus is a member of the human gut microbiota. The importance of this microbial consortium involves the degradation of complex dietary glycans mainly conferred by glycoside hydrolases. In this study we focus on one such catabolic glycoside hydrolase from B. ovatus. The enzyme, termed BoMan26A, is a β-mannanase that takes part in the hydrolytic degradation of galactomannans. The crystal structure of BoMan26A has previously been determined to reveal a TIM-barrel like fold, but the relation between the protein structure and the mode of substrate processing has not yet been studied. Here we report residue-specific assignments for 95% of the 344 backbone amides of BoMan26A. The assignments form the basis for future studies of the relationship between substrate interactions and protein dynamics. In particular, the potential role of loops adjacent to glycan binding sites is of interest for such studies.
... Another approach is to record projection planes with more or less freedom in selecting projection angles (Eghbalnia and Markley 2017;Murrali et al. 2018;Hiller et al. 2008;Malmodin and Billeter 2006). Data monitoring during acquisition may further save time (Isaksson et al. 2013). Normally, a first step in NMR projects involves obtaining resonance assignments, and many methodological projects are demonstrated on backbone assignments, making use of triple-resonance experiments or their extensions into higher dimensions. ...
A flexible and scalable approach for protein NMR is introduced that builds on rapid data collection via projection spectroscopy and analysis of the spectral input data via joint decomposition. Input data may originate from various types of spectra, depending on the ultimate goal: these may result from experiments based on triple-resonance pulse sequences, or on TOCSY or NOESY sequences, or mixtures thereof. Flexible refers to the free choice of spectra for the joint decompositions depending on the purpose: assignments, structure, dynamics, interactions. Scalable means that the approach is open to the addition of similar or different experiments, e.g. larger proteins may require a wider selection of triple-resonance based experiments. Central to the proposed approach is the mutual support among the different spectra during the spectral analysis: for example, sparser triple-resonance spectra may help decomposing (separating) spin systems in a TOCSY or identifying unique NOEs. In the example presented, backbone plus side chain assignments of ubiquitin were obtained from the combination of either two or three of the following projection experiments: a 4D HCCCONH, a 4D HNCACO and a 3D HNCACB. In all cases, TOCSY data (4D HCCCONH) proved crucial not only for the side chain assignments, but also for the sequential assignment. Even when total recording time was reduced to about 10 h, nearly complete assignments were obtained, with very few missing assignments and even fewer differences to a reference.
Electronic supplementary material
The online version of this article (10.1007/s10858-019-00226-8) contains supplementary material, which is available to authorized users.
... The helical conformation in peptides and proteins can be stabilized in vitro in the presence of TFE due to a lower dielectric constant than water, or through binding to detergent micelles (Isaksson et al., 2013;Shiraki et al., 1995;Sigalov et al., 2006). To assess whether the ITIMs in 3DL1-cyto are capable of forming helices, we examined CON spectral changes of SET-3DL1-cyto in the presence of TFE. ...
KIR3DL1 is an inhibitory killer cell immunoglobulin-like receptor (KIR) that negatively regulates natural killer cell cytotoxicity. The KIR3DL1 cytoplasmic region (3DL1-cyto) is disordered and can be dissected into three segments: (I) H340-V351; (II) M352-D371; and (III) P372-P423. NMR studies indicate that segment II can dynamically adopt a loop-like conformation, and segments I and III can form dynamic helices that may mediate binding to membranes, particularly in the region around the N-terminal (N) immunoreceptor tyrosine-based inhibitory motif (ITIM), consistent with its role in signaling. Furthermore, individual SH2 domains of SHP-2 strongly engage with the unphosphorylated N-ITIM of 3DL1-cyto, while binding of the tandem SHP-2 SH2 domains to the bis-phosphorylated ITIMs results in more extensive conformational changes in segments I and III. The findings enhance our understanding of KIR function and how ITIMs in a target receptor operate in concert to engage the tandem SH2 domains of SHP-2.
