Interaction between the C-terminal domains of N and P proteins of measles virus investigated by NMR

In this paper we investigate the interaction between the C-terminal domains of the measles virus phosphoprotein (XD) and nucleoprotein (N_TAIL) by using nuclear magnetic resonance chemical shift perturbation experiments. Using both N_TAIL constructs and peptides, we show that contrary to the conserved Box2 region (N^489-506), the C-terminal region of N_TAIL (N^513-525) does not directly interact with XD, and yet affects binding to XD. We tentatively propose a model where the C-terminus of N_TAIL would stabilize the N_TAIL-XD complex either via a functional coupling with N^489-506 or by reducing the entropic penalty associated to the binding-coupled-to-folding process.

Structured summary

MINT-7009780, MINT-7009793, MINT-7009808: N-tail (uniprotkb:Q89933) and P (uniprotkb:P03422) bind (MI:0407) by nuclear magnetic resonance (MI:0077)     

Conformational Analysis of the Partially Disordered Measles Virus NTAIL-XD Complex by SDSL EPR Spectroscopy

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N_TAIL) both free and in complex with the X domain (XD, aa 459-507) of the viral phosphoprotein. By comparing measured and modeled temperature-dependent restrictions of the side-chain conformational spaces of 12 SL cysteine-substituted N_TAIL variants, we showed that the 490-500 region of N_TAIL is prestructured in the absence of the partner, and were able to quantitatively estimate, for the first time to our knowledge, the extent of the α-helical sampling of the free form. In addition, we showed that the 505-525 region of N_TAIL conserves a significant degree of freedom even in the bound form. The latter two findings provide a mechanistic explanation for the reported rather high affinity of the N_TAIL-XD binding reaction. Due to the nanosecond timescale of X-band EPR spectroscopy, we were also able to monitor the disordering in the 488-525 region of N_TAIL, in particular the unfolding of the α-helical region when the temperature was increased from 281 K to 310 K.     

Partner-Mediated Polymorphism of an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) recognize their partners through molecular recognition elements (MoREs). The MoRE of the C-terminal intrinsically disordered domain of the measles virus nucleoprotein (N_TAIL) is partly pre-configured as an α-helix in the free form and undergoes α-helical folding upon binding to the X domain (XD) of the viral phosphoprotein. Beyond XD, N_TAIL also binds the major inducible heat shock protein 70 (hsp70). So far, no structural information is available for the N_TAIL/hsp70 complex. Using mutational studies combined with a protein complementation assay based on green fluorescent protein reconstitution, we have investigated both N_TAIL/XD and N_TAIL/hsp70 interactions. Although the same N_TAIL region binds the two partners, the binding mechanisms are different. Hsp70 binding is much more tolerant of MoRE substitutions than XD, and the majority of substitutions lead to an increased N_TAIL/hsp70 interaction strength. Furthermore, while an increased and a decreased α-helicity of the MoRE lead to enhanced and reduced interaction strength with XD, respectively, the impact on hsp70 binding is negligible, suggesting that the MoRE does not adopt an α-helical conformation once bound to hsp70. Here, by showing that the α-helical conformation sampled by the free form of the MoRE does not systematically commit it to adopt an α-helical conformation in the bound form, we provide an example of partner-mediated polymorphism of an IDP and of the relative insensitiveness of the bound structure to the pre-recognition state. The present results therefore contribute to shed light on the molecular mechanisms by which IDPs recognize different partners.     

How Robust Is the Mechanism of Folding-Upon-Binding for an Intrinsically Disordered Protein?

The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (N_TAIL) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of N_TAIL upon binding to XD by measuring the effect on both the folding and binding steps of N_TAIL when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of N_TAIL of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs.     

Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines

Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N_TAIL) were cyanylated at cysteine and their infrared spectra in the C≡N stretching region were recorded both in the absence and in the presence of one of the physiological partners of N_TAIL, namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within N_TAIL, the C≡N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level.     

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K_D) for vitronectin binding to UspA2 was 2.3 × 10^?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a K_D of 7.9 × 10^?8 M. Electron microscopy verified that the active binding domain (UspA2^30–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2^30–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance.     

The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from Methanobrevibacter ruminantium

Methenyltetrahydromethanopterin cyclohydrolase (Mch) is involved in the methanogenesis pathway of archaea as a C₁ unit carrier where N⁵‐formyl‐tetrahydromethanopterin is converted to methenyl‐tetrahydromethanopterin. Mch from Methanobrevibacter ruminantium was cloned, purified, crystallized and its crystal structure solved at 1.37 Å resolution. A biologically active trimer, the enzyme is composed of two domains including an N‐terminal domain of six α‐helices encompassing a series of four β‐sheets and a predominantly anti‐parallel β–sheet at the C‐terminus flanked on one side by α‐helices. Sequence and structural alignments have helped identify residues involved in substrate binding and trimer formation. Proteins 2013; 81:2064–2070. © 2013 Wiley Periodicals, Inc.     

The N-terminal amphipathic region of the Escherichia coli type III secretion system protein EspD is required for membrane insertion and function

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe‐like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N‐terminal segment of EspD, encompassing residues 1–171, contains two amphipathic domains spanning residues 24–41 and 66–83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His₆‐EspD_1–171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His₆‐EspD_1–171 undergoes a pH depended conformational change that increases the α‐helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp₄₇ into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His₆‐EspD_1–171 forms a homodimer that is postulated to promote EspD–EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66–83 gene showed that this protein was secreted but non‐functional.     

Temperature‐induced partially unfolded state of hUBF HMG Box‐5: Conformational and dynamic investigations of the Box‐5 thermal intermediate ensemble

Human upstream binding factor (hUBF) HMG Box‐5 is a highly conserved protein domain, containing 84 amino acids and belonging to the family of the nonspecific DNA‐binding HMG boxes. Its native structure adopts a twisted L shape, which consists of three α‐helices and two hydrophobic cores: the major wing and the minor wing. In this article, we report a reversible three‐state thermal unfolding equilibrium of hUBF HMG Box‐5, which is investigated by differential scanning calorimetry (DSC), circular dichroism spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. DSC data show that Box‐5 unfolds reversibly in two separate stages. Spectroscopic analyses suggest that different structural elements exhibit noncooperative transitions during the unfolding process and that the major form of the Box‐5 thermal intermediate ensemble at 55°C shows partially unfolded characteristics. Compared with previous thermal stability studies of other boxes, it appears that Box‐5 possesses a more stable major wing and two well separated subdomains. NMR chemical shift index and sequential ¹H^N_i‐¹H^N_i+1 NOE analyses indicate that helices 1 and 2 are native‐like in the thermal intermediate ensemble, while helix 3 is partially unfolded. Detailed NMR relaxation dynamics are compared between the native state and the intermediate ensemble. Our results implicate a fluid helix‐turn‐helix folding model of Box‐5, where helices 1 and 2 potentially form the helix 1‐turn‐helix 2 motif in the intermediate, while helix 3 is consolidated only as two hydrophobic cores form to stabilize the native structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Revealing the dimer dissociation and existence of a folded monomer of the mature HIV‐2 protease

Purification and in vitro protein‐folding schemes were developed to produce monodisperse samples of the mature wild‐type HIV‐2 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2_E37K (elevated K_d ～20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a T_m of 60.5 for PR2 as compared with 65.7°C for HIV‐1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the T_m of PR2 increases by 14.8°C in the presence of DRV as compared with 22.4°C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2_T26A) or a deletion of the C‐terminal residues 96–99 (PR2_1–95), drastically increase the K_d (>10⁵‐fold). PR2_T26A and PR2_1–95 consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size‐exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild‐type PR2 and its active‐site mutant PR2_D25N are folded dimers. Addition of twofold excess active‐site inhibitor promotes dimerization of PR2_T26A but not of PR2_1–95, indicating that subunit interactions involving the C‐terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.     

Hyperglucosylated adhesin‐derived peptides as antigenic probes in multiple sclerosis: Structure optimization and immunological evaluation

Peptides mimicking antigenic epitopes targeted by antibodies can be powerful tools to be used as antigen surrogates for the specific diagnosis and treatment of autoimmune diseases. Obtaining structural insights about the nature of peptide–antibody interaction in complex mixtures such as sera is a critical goal. In multiple sclerosis (MS), we previously demonstrated that the N‐linked β‐d ‐glucopyranosyl moieties (N‐Glc) containing epitopes in nontypeable Haemophilus influenzae adhesin C‐terminal portion HMW1(1205–1526) were essential for high‐affinity antibody binding in a subpopulation of MS patients. With the aim of developing peptide probes and assessing their binding properties to antibodies from sera of representative patients, we performed the systematic analysis of synthetic peptides based on HMW1(1347–1354) fragment bearing one or two N‐Glc respectively on Asn‐1349 and/or Asn‐1352. The N‐glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC₅₀ in the range 10^?8–10^?10 M by competitive indirect enzyme‐linked immunosorbent assay (ELISA) in three representative MS patient sera. We selected the di‐N‐glucosylated adhesin peptide Ac‐KAN (Glc)VTLN (Glc)TT‐NH₂ as the shortest sequence able to inhibit high‐avidity interaction with N‐Glc targeting IgM antibodies. Nuclear magnetic resonance (NMR)‐ and circular dichroism (CD)‐based characterization showed that the binding properties of these antigens could not be ascribed to structural differences induced by the presence of up to two N‐glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water.     

A novel post‐ligation thioesterification device enables peptide ligation in the N to C direction: synthetic study of human glycodelin

Human glycodelin consists of 162 amino acid residues and two N‐linked glycans at Asn²⁸ and Asn⁶³. In this study, we synthesized it by a fully convergent strategy using native chemical ligation (NCL) in N to C direction. The four peptide segments corresponding to 1–31, 32–65, 66–105 and 106–162 sequences were synthesized by 9‐fluorenylmethoxycarbonyl based solid‐phase peptide synthesis. At the C‐terminus of the second segment, N‐ethyl‐S‐acetamidomethyl‐cysteine was attached as a post‐ligation thioesterification device. The N‐terminal two segments were condensed by the homocysteine‐mediated NCL at Leu‐Met site, and the product was methylated to convert homocysteine to methionine. After deprotection of acetamidomethyl group on the N‐ethylcysteine residue, the peptide was thioesterified by N‐alkylcysteine‐assisted method. The product was then ligated with the C‐terminal half, which was obtained by the NCL of third and fourth segments, to give the full‐length glycodelin. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway

(3R,5R)‐Clavulanic acid (CA) is a clinically important inhibitor of Class A β‐lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5‐related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P2₁ space groups) of CBG were obtained; in both forms one molecule of acetyl‐CoA (AcCoA) was bound to the N‐terminal GNAT domain, with the C‐terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl‐CoA molecule can bind to CBG. Succinyl‐CoA and myristoyl‐CoA displayed the strongest binding to the “second” CoA binding site, which is likely in the C‐terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N‐terminal GNAT domain plays a structural role whereas the C‐terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl‐CoA occurs preferentially to monomeric rather than dimeric CBG. The N‐terminal AcCoA binding site and the proposed C‐terminal acyl‐CoA binding site of CBG are compared with acyl‐CoA binding sites of other tandem and single domain GNAT proteins. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Targeting the SH3 domain of human osteoclast‐stimulating factor with rationally designed peptoid inhibitors

Human osteoclast‐stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption. The protein contains a modular Src homology 3 (SH3) domain that mediates the intermolecular recognition and interaction of hOSF with its biological partners. Here, we proposed targeting the hOSF SH3 domain to disrupt hOSF–partner interactions for bone disease therapy by using SH3 inhibitors. In the procedure, the primary sequences of three known hOSF‐interacting proteins (c‐Src, SMN and Sam68) were parsed, from which totally 31 octapeptide segments that contain the core SH3‐binding motif PXXP were extracted, and their binding behavior to hOSF SH3 domain was investigated at structural level using a biomolecular modeling protocol. Several SH3‐binding candidates were identified theoretically and then determined to have high or moderate affinity for the domain using fluorescence spectroscopy assays. One potent peptide ⁴²⁵APP ARP VK⁴³² (K_d = 3.2 μM), which corresponds to the residues 425–432 of Sam68 protein, was used as template to derive N substitution of peptides (peptoids). Considering that proline is the only endogenous N‐substituted amino acid that plays a critical role in SH3–peptide binding, the substitution was addressed at the two key proline residues (Pro427 and Pro430) of the template peptide with nine N‐substituted amino acid types. By systematically evaluating the structural and energetic effects of different N‐substituted amino acids presenting at the two proline sites on peptide binding, we rationally designed five peptoid inhibitors and then determined in vitro their binding affinity to hOSF SH3 domain. Consequently, two designed peptoids APP AR( N ‐Clp) VK and APP AR( N ‐Ffa) VK with Pro430 replaced by N‐Clp and N‐Ffa were confirmed to have increased (K_d = 0.87 μM) and comparable (K_d = 2.9 μM) affinities relative to the template, respectively. In addition, we also found that the Pro427 residue plays an essential role in restricting peptide/peptoid conformations to polyproline II (PPII) helix as the basic requirement of SH3 binding so that the residue cannot be modified. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Mapping interactions of gastric inhibitory polypeptide with GIPR N‐terminus using NMR and molecular dynamics simulations

Glucose‐dependent insulinotropic polypeptide (gastric inhibitory polypeptide, or GIP), a 42‐amino acid incretin hormone, modulates insulin secretion in a glucose‐concentration‐dependent manner. Its insulinotropic action is highly dependent on glucose concentration that surmounts the hypoglycemia side effects associated with current therapy. In order to develop a GIP‐based anti‐diabetic therapy, it is essential to establish the 3D structure of the peptide and study its interaction with the GIP receptor (GIPR) in detail. This will give an insight into the GIP‐mediated insulin release process. In this article, we report the solution structure of GIP(1–42, human)NH₂ deduced by NMR and the interaction of the peptide with the N‐terminus of GIPR using molecular modelling methods. The structure of GIP(1–42, human)NH₂ in H₂O has been investigated using 2D‐NMR (DQF‐COSY, TOCSY, NOESY, ¹H‐¹³C HSQC) experiments, and its conformation was built by constrained MD simulations with the NMR data as constraints. The peptide in H₂O exhibits an α‐helical structure between residues Ser8 and Asn39 with some discontinuity at residues Gln29 to Asp35; the helix is bent at Gln29. This bent gives the peptide an ‘L’ shape that becomes more pronounced upon binding to the receptor. The interaction of GIP with the N‐terminus of GIPR was modelled by allowing GIP to interact with the N‐terminus of GIPR under a series of decreasing constraints in a molecular dynamics simulation, culminating with energy minimization without application of any constraints on the system. The canonical ensemble obtained from the simulation was subjected to a detailed energy analysis to identify the peptide–protein interaction patterns at the individual residue level. These interaction energies shed some light on the binding of GIP with the GIPR N‐terminus in a quantitative manner. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.