The Disordered Region of the HCV Protein NS5A: Conformational Dynamics,SH3 Binding,and Phosphorylation

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein’s interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191–447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.     

Does Lack of Secondary Structure Imply Intrinsic Disorder in Proteins? A Sequence Analysis

Intrinsically disordered proteins (IDPs)/protein regions (IDPRs) lack unique three-dimensional structure at the level of secondary and/or tertiary structure and are represented as an ensemble of interchanging conformations. To investigate the role of presence/absence of secondary structures in promoting intrinsic disorder in proteins, a comparative sequence analysis of IDPs, IDPRs and proteins with minimal secondary structures (less than 5%) is required. A sequence analysis reveals proteins with minimal secondary structure content have high mean net positive charge, low mean net hydrophobicity and low sequence complexity. Interestingly, analysis of the relative local electrostatic interactions reveal that an increase in the relative repulsive interactions between amino acids separated by three or four residues lead to either loss of secondary structure or intrinsic disorder. IDPRs show increase in both local negative-negative and positive-positive repulsive interactions. While IDPs show a marked increase in the local negative-negative interactions, proteins with minimal secondary structure depict an increase in the local positive-positive interactions. IDPs and IDPRs are enriched in D, E and Q residues, while proteins with minimal secondary structure are depleted of these residues. Proteins with minimal secondary structures have higher content of G and C, while IDPs and IDPRs are depleted of these residues. These results confirm that proteins with minimal secondary structure have a distinctly different propensity for charge, hydrophobicity, specific amino acids and local electrostatic interactions as compared to IDPs/IDPRs. Thus we conclude that lack of secondary structure may be a necessary but not a sufficient condition for intrinsic disorder in proteins.     

Allostery in Orai1 binding to calmodulin revealed from conformational thermodynamics

Here, we study microscopic mechanism of complex formation between Ca²⁺-bound calmodulin (holoCaM) and Orai1 that regulates Ca²⁺-dependent inactivation process in eukaryotic cells. We compute conformational thermodynamic changes in holoCaM with respect to complex of Orai1 bound to C-terminal domain of holoCaM using histograms of dihedral angles of the proteins over trajectories from molecular dynamics simulations. Our analysis shows that the N-terminal domain residues L4, T5, Q41, N42, T44 and E67 of holoCaM get destabilized and disordered due to Orai1 binding to C-terminal domain of calmodulin affect the N-terminal domain residues. Among these residues, polar T44, having maximum destabilization and disorder via backbone fluctuations, shows the largest change in solvent exposure. This suggests that N-terminal domain is allosterically regulated via T44 by the binding of Orai1 to the C-terminal domain.     

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150^Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150^Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150^Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150^Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.     

Molecular dynamics studies on conformational thermodynamics of Orai1–calmodulin complex

Molecular understanding of bio-macromolecular binding is a challenging task due to large sizes of the molecules and presence of variety of interactions. Here, we study the molecular mechanism of calmodulin (CaM) binding to Orai1 that regulates Ca²⁺-dependent inactivation process in eukaryotic cells. Although experimental observations indicate that Orai1 binds to the C-terminal of Ca²⁺-loaded CaM, it is not decisive if N-domain of CaM interacts with Orai1. We address the issue of interaction of different domains of CaM with Orai1 using conformational thermodynamic changes, computed from histograms of dihedral angles over simulated trajectories of CaM, CaM-binding domain of Orai1 and complexes of CaM with Orai1. The changes for all residues of both C and N terminal domains of CaM upon Orai1 binding are compared. Our analysis shows that Orai1binds to both C-terminal and N-terminal domains of CaM, indicating 1:2 stoichiometry. The Orai1 binding to N-terminal domain of CaM is less stable than that to the C-terminal domain. The binding residues are primarily hydrophobic. These observations are in qualitative agreement to the experiments. The conformational thermodynamic changes thus provide a useful computational tool to provide atomic details of interactions in bio-macromolecular binding.     

A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP

Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J(¹⁵N,¹³Cα) couplings. This concept was realized using a unidirectional (H)NCO ¹³C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J(¹⁵N,¹³Cα) couplings were acquired for WIP residues 2–65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16–23 and 45–60, and a helical tendency at residues 28–42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP^2–65 conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein–protein interactions that stabilize the unfolded state. We conclude that J-couplings are a useful measureable in characterizing structural ensembles in IDPs, and that the proposed experiment provides a practical method for accurately performing such measurements, once again emphasizing the power of NMR in studying IDP behavior.     

Molecular determinants for the interaction of human neutrophil alpha defensin 1 with its propeptide

Human neutrophil α-defensins (HNPs) are cationic antimicrobial peptides that are synthesized in vivo as inactive precursors (proHNPs). Activation requires proteolytic excision of their anionic N-terminal inhibitory pro peptide. The pro peptide of proHNP1 also interacts specifically with and inhibits the antimicrobial activity of HNP1 inter-molecularly. In the light of the opposite net charges segregated in proHNP1, functional inhibition of the C-terminal defensin domain by its propeptide is generally thought to be of electrostatic nature. Using a battery of analogs of the propeptide and of proHNP1, we identified residues in the propeptide region important for HNP1 binding and inhibition. Only three anionic residues in the propeptide, Glu¹⁵, Asp²⁰ and Glu²³, were modestly important for interactions with HNP1. By contrast, the hydrophobic residues in the central part of the propeptide, and the conserved hydrophobic motif Val²⁴Val²⁵Val²⁶Leu²⁸ in particular, were critical for HNP1 binding and inhibition. Neutralization of all negative charges in the propeptide only partially activated the bactericidal activity of proHNP1. Our data indicate that hydrophobic forces have a dominant role in mediating the interactions between HNP1 and its propeptide — a finding largely contrasting the commonly held view that the interactions are of an electrostatic nature.     

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca²⁺- and Zn²⁺-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca²⁺-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca²⁺/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152–1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca²⁺/CaM with a dissociation constant of 100–300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a “wrapping around” mode of interaction between RUBpep and Ca²⁺/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.     

Interaction between residues in the Mg2+-binding site regulates BK channel activation

As a unique member of the voltage-gated potassium channel family, a large conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel has a large cytosolic domain that serves as the Ca²⁺ sensor, in addition to a membrane-spanning domain that contains the voltage-sensing (VSD) and pore-gate domains. The conformational changes of the cytosolic domain induced by Ca²⁺ binding and the conformational changes of the VSD induced by membrane voltage changes trigger the opening of the pore-gate domain. Although some structural information of these individual functional domains is available, how the interactions among these domains, especially the noncovalent interactions, control the dynamic gating process of BK channels is still not clear. Previous studies discovered that intracellular Mg²⁺ binds to an interdomain binding site consisting of D99 and N172 from the membrane-spanning domain and E374 and E399 from the cytosolic domain. The bound Mg²⁺ at this narrow interdomain interface activates the BK channel through an electrostatic interaction with a positively charged residue in the VSD. In this study, we investigated the potential interdomain interactions between the Mg²⁺-coordination residues and their effects on channel gating. By introducing different charges to these residues, we discovered a native interdomain interaction between D99 and E374 that can affect BK channel activation. To understand the underlying mechanism of the interdomain interactions between the Mg²⁺-coordination residues, we introduced artificial electrostatic interactions between residues 172 and 399 from two different domains. We found that the interdomain interactions between these two positions not only alter the local conformations near the Mg²⁺-binding site but also change distant conformations including the pore-gate domain, thereby affecting the voltage- and Ca²⁺-dependent activation of the BK channel. These results illustrate the importance of interdomain interactions to the allosteric gating mechanisms of BK channels.     

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D ¹H-¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.     

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1–168) joined to an RNA helicase (residues 180–618) by an 11-amino acid linker (169–179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B₁₈NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173–183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn²⁺ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu¹⁷³ and Pro¹⁷⁴ or replacing Pro¹⁷⁴ with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro¹⁷⁶ to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.     

Repressor--operator interaction in the lac operon. II. Observations at the tyrosines and tryptophans

This paper shows that ¹⁹F-nuelear magnetic resonance spectroscopy on 3-fluoro-tyrosine and 5-fluorotryptophan-substituted wild-type lactose operon repressors from Escherichia coli can be used to examine the interactions with lac operator DNA.A survey of inducer and salt concentration effects on the repressor-operator complex is presented. The data lead us to a scheme for the interactions between the repressor, operator and inducer, in both binary and ternary complexes, that accommodate the results published by others.The complex between the tetrameric repressor and one 36 base-pair operator DNA fragment results in the simultaneous broadening of the resonances from all four N-terminal DNA binding domains. The actual contacts made by these binding domains are similar but probably not identical.The binding of the inducer molecule to the tetrameric repressor results in an allosteric change that can be monitored by the increased intensity of the resonances from individual tyrosine residues in the N-terminal binding domain. This increased N-terminal tyrosine resonance intensity in the complex is transmitted to repressor subunits that have not yet bound an inducer molecule.     

The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a ‘lure and lock’ model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the ‘lure’ step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on ‘top’ of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein–RNA complexes.     

The flexible N-terminal motif of uL11 unique to eukaryotic ribosomes interacts with P-complex and facilitates protein translation

Eukaryotic uL11 contains a conserved MPPKFDP motif at the N-terminus that is not found in archaeal and bacterial homologs. Here, we determined the solution structure of human uL11 by NMR spectroscopy and characterized its backbone dynamics by ¹⁵N–¹H relaxation experiments. We showed that these N-terminal residues are unstructured and flexible. Structural comparison with ribosome-bound uL11 suggests that the linker region between the N-terminal domain and C-terminal domain of human uL11 is intrinsically disordered and only becomes structured when bound to the ribosomes. Mutagenesis studies show that the N-terminal conserved MPPKFDP motif is involved in interacting with the P-complex and its extended protuberant domain of uL10 in vitro. Truncation of the MPPKFDP motif also reduced the poly-phenylalanine synthesis in both hybrid ribosome and yeast mutagenesis studies. In addition, G→A/P substitutions to the conserved GPLG motif of helix-1 reduced poly-phenylalanine synthesis to 9–32% in yeast ribosomes. We propose that the flexible N-terminal residues of uL11, which could extend up to ∼25 Å from the N-terminal domain of uL11, can form transient interactions with the uL10 that help to fetch and fix it into a position ready for recruiting the incoming translation factors and facilitate protein synthesis.     

Mutational Analysis of a CBM Family 5 Chitin-Binding Domain of an Alkaline Chitinase from Bacillus sp. J813

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.     

Modulation of p53 N-terminal transactivation domain 2 conformation ensemble and kinetics by phosphorylation

Abstract

Phosphorylation of protein is critical for various cell processes, which preferentially happens in intrinsically disordered proteins (IDPs). How phosphorylation modulates structural ensemble of disordered peptide remains largely unexplored. Here, using replica exchange molecular dynamics (REMD) and Markov state model (MSM), the conformational distribution and kinetics of p53 N-terminal transactivation domain (TAD) 2 as well as its dual-site phosphorylated form (pSer46, pThr55) were simulated. It reveals that the dual phosphorylation does not change overall size and secondary structure element fraction, while a change in the distribution of hydrogen bonds induces slightly more pre-existing bound helical conformations. MSM analysis indicates that the dual phosphorylation accelerates conformation exchange between disordered and order-like states in target-binding region. It suggests that p53 TAD2 after phosphorylation would be more apt to bind to both the human p62 pleckstrin homology (PH) domain and the yeast tfb1?PH domain through different binding mechanism, where experimentally it exhibits an extended and α-helix conformation, respectively, with increased binding strength in both complexes. Our study implies except binding interface, both conformation ensemble and kinetics should be considered for the effects of phosphorylation on IDPs. Abbreviations IDPs intrinsically disordered proteins

REMD replica exchange molecular dynamics

MSM Markov state model

TAD transactivation domain

PH pleckstrin homology

PRR proline-rich region

DBD DNA-binding domain

TET Tetramerization domain

REG regulatory domain

MD molecular dynamics

PME particle-mesh Ewald

TICA time-lagged independent component analysis

CK Chapman–Kolmogorov

GMRQ generalized matrix Rayleigh quotient

SARW self-avoiding random walk

KID kinase-inducible domain

MFPT mean first passage time

DSSP definition of secondary structure of proteins

RMSD root mean square deviation

R_g radius of gyration

R_ee end to end distance

Communicated by Ramaswamy H. Sarma