Correlation between NS5A Dimerization and Hepatitis C Virus Replication

Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current standard therapy has limited efficacy and serious side effects. Thus, the development of alternate therapies is of tremendous importance. HCV NS5A (nonstructural 5A protein) is a pleiotropic protein with key roles in HCV replication and cellular signaling pathways. Here we demonstrate that NS5A dimerization occurs through Domain I (amino acids 1-240). This interaction is not mediated by nucleic acids because benzonase, RNase, and DNase treatments do not prevent NS5A-NS5A interactions. Importantly, DTT abrogates NS5A-NS5A interactions but does not affect NS5A-cyclophilin A interactions. Other reducing agents such as tris(2-carboxyethyl)phosphine and 2-mercaptoethanol also abrogate NS5A-NS5A interactions, implying that disulfide bridges may play a role in this interaction. Cyclophilin inhibitors, cyclosporine A, and alisporivir and NS5A inhibitor BMS-790052 do not block NS5A dimerization, suggesting that their antiviral effects do not involve the disruption of NS5A-NS5A interactions. Four cysteines, Cys-39, Cys-57, Cys-59, and Cys-80, are critical for dimerization. Interestingly, the four cysteines have been proposed to form a zinc-binding motif. Supporting this notion, NS5A dimerization is greatly facilitated by Zn(2+) but not by Mg(2+) or Mn(2+). Importantly, the four cysteines are vital not only for viral replication but also critical for NS5A binding to RNA, revealing a correlation between NS5A dimerization, RNA binding, and HCV replication. Altogether our data suggest that NS5A-NS5A dimerization and/or multimerization could represent a novel target for the development of HCV therapies.     

Functional interactions between papillomavirus E1 and E2 proteins.

DNA replication of papillomaviruses requires the viral E1 and E2 proteins. These proteins bind cooperatively to the viral origin of replication (ori), which contains binding sites for both proteins, forming an E1-E2-ori complex which is essential for initiation of DNA replication. To map the domains in E2 that are involved in the interaction with E1, we have used chimeric bovine papillomavirus (BPV)/human papillomavirus type 11 (HPV-11) E2 proteins. The results from this study show that both the DNA binding domain and the transactivation domain from BPV E2 independently can interact with BPV E1. However, the roles of these two interactions are different: the interaction between E1 and the activation domain of E2 is necessary and sufficient for cooperativity in binding and for DNA replication; the interaction between E1 and the DNA binding domain of E2 is required only when the binding sites for E1 and E2 are adjacent to each other, and the function of this interaction appears to be to facilitate the interaction between E1 and the transactivation domain of E2. These results indicate that the cooperative binding of E1 and E2 to the BPV ori takes place via a novel two-stage mechanism where one interaction serves as a trigger for the formation of the second, productive, interaction between the two proteins.     

Relation between Mg2+ activation and AMP inhibition of fructose-1,6-diphosphatase from rabbit skeletal muscles

It was shown that AMP, an allosteric inhibitor of fructose-1.6-bisphosphatase, decreases the apparent affinity of the enzyme for the activating cation, Mg2+, which is accompanied by a decrease of the kinetic cooperativity between the Mg2+-binding sites. In its turn, the Mg2+ increase diminishes the enzyme sensitivity to the inhibiting effect of AMP and decreases the cooperativity of the inhibitor binding. The heterotropic interactions between the allosteric inhibitor and activator binding centers are consistent with the predictions of the Monod-Wyman-Changeux model which involves two conformational states of the enzyme (of which one is catalytically inactive) differing in their affinity for the ligands. An increase in pH from 7.4 to 9.0 increases the enzyme affinity for Mg2+ and causes an equilibrium shift towards the catalytically active state of the enzyme.     

Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T(m)) of 48.5 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k(2)/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.     

A model for dynamin self-assembly based on binding between three different protein domains.

Dynamin is a 100-kDa GTPase that assembles into multimeric spirals at the necks of budding clathrin-coated vesicles. We describe three different intramolecular binding interactions that may account for the process of dynamin self-assembly. The first binding interaction is the dimerization of a 100-amino acid segment in the C-terminal half of dynamin. We call this segment the assembly domain, because it appears to be critical for multimerization. The second binding interaction occurs between the assembly domain and the N-terminal GTPase domain. The strength of this interaction is controlled by the nucleotide-bound state of the GTPase domain, as shown with mutations in GTP binding motifs and in vitro binding experiments. The third binding interaction occurs between the assembly domain and a segment that we call the middle domain. This is the segment between the N-terminal GTPase domain and the pleckstrin homology domain. The three different binding interactions suggest a model in which dynamin molecules first dimerize. The dimers are then linked into a chain by a second binding reaction. The third binding interaction might connect adjacent rungs of the spiral.     

Anti-cooperative and cooperative protein-protein interactions between TetR isoforms on synthetic enhancers

Protein-protein interactions play an important role in determining the regulatory output of cis regulatory regions. In this work, we revisit the regulatory output functions recorded for the synthetic enhancers that contain binding sites for TetR. We use our thermodynamic model as an analysis tool to infer that two different types of interactions may take place between the TetR molecules. First, a strong mutually exclusive anti-cooperative interaction precludes the synthetic enhancer from being occupied by more than one AT (the aTc bound TetR isoform) protein, and a second weak cooperative interaction exists between the aTc-free TetR isoform (T). Consequently, this work highlights the power of the synthetic enhancer approach as a tool for studying protein-protein interactions via an experimentally verifiable prediction for the general mode of binding of the TetR repressor.     

Biochemical evidence for interaction between the two nucleotide binding domains of ArsA. Insights from mutants and ATP analogs

ArsA, the peripheral membrane component of the anion-translocating ATPase ArsAB, consists of two nucleotide binding domains (A1 and A2), which are connected by a linker sequence. Previous studies on ArsA have focused on the function of each nucleotide binding domain and the role of the linker, whereas the present study looks at the interactions between the binding domains and their interactions with the linker. It has previously been shown that the A1 domain of ArsA carries out unisite catalysis in the absence of antimonite, while A2 is recruited in multisite catalysis by antimonite in the presence of a functional A1 domain. Multisite catalysis thus seems to result from an interaction between A1 and A2 brought about by antimonite. In the present study, we provide direct biochemical evidence for interaction between the two nucleotide binding domains and show that the linker region acts as a transducer of the conformational changes between them. We find that nucleotide binding to the A2 domain results in a significant, detectable change in the conformation of the A1 domain. Two ATP analogs, FSBA and ATP gamma S, used in this study, were both found to bind preferentially to the A2 domain, and their binding resulted in changing the otherwise compact A1 domain into an open conformation. Point mutations in the A2 domain and the linker region also produced a similar effect on the conformation of A1, thus suggesting that events at A2 are relayed to A1 via the linker. We propose that nucleotide binding to A2 produces a two-tiered conformational change. The significance of these changes in the mechanism of ArsA is discussed.     

Insight into the interactive residues between two domains of human somatic Angiotensin-converting enzyme and Angiotensin II by MM-PBSA calculation and steered molecular dynamics simulation

Angiotensin-converting enzyme (ACE), a membrane-bound zinc metallopeptidase, catalyzes the formation of Angiotensin-II (AngII) and the deactivation of bradykinin in the renin–angiotensin-aldosterone and kallikrein–kinin systems. As a hydrolysis product of ACE, AngII is regarded as an inhibitor and displays stronger competitive inhibition in the C-domain than the N-domain of ACE. However, the AngII binding differences between the two domains and the mechanisms behind AngII dissociation from the C-domain are rarely explored. In this work, molecular docking, Molecular Mechanics/Poisson–Boltzmann Surface Area calculation, and steered molecular dynamics (SMD) are applied to explore the structures and interactions in the binding or unbinding of AngII with the two domains of human somatic ACE. Calculated free energy values suggest that the C-domain–AngII complex is more stable than the N-domain–AngII complex, consistent with available experimental data. SMD simulation results imply that electrostatic interaction is dominant in the dissociation of AngII from the C-domain. Moreover, Gln106, Asp121, Glu123, and Tyr213 may be the key residues in the unbinding pathway of AngII. The simulation results in our work provide insights into the interactions between the two domains of ACE and its natural peptide inhibitor AngII at a molecular level. Moreover, the results provide theoretical clues for the design of new inhibitors.     

Pinpointing the interaction site between semaphorin-3A and its inhibitory peptide

Semaphorin-3A (Sema-3A) is a chemorepellant protein with various biological functions, including kidney development. It interacts with a protein complex consisting of the receptors neuropilin-1 (NRP-1) and plexin-A1. After acute kidney injury, Sema-3A is overexpressed and secreted, leading to a loss of kidney function. The development of peptide inhibitors is a promising approach to modulate the interaction of Sema-3A with its receptor NRP-1. Few interaction points between these binding partners are known. However, an immunoglobulin-like domain-derived peptide of Sema-3A has shown a positive effect on cell proliferation. To specify these interactions between the peptide inhibitor and the Sema-3A–NRP-1 system, the peptides were modified with the photoactivatable amino acids 4-benzoyl-l -phenylalanine or photo-l -leucine by solid-phase peptide synthesis. Activity was tested by an enzyme-linked immunosorbent-based binding assay, and crosslinking experiments were analyzed by Western blot and mass spectrometry, demonstrating a specific binding site of the peptide at Sema-3A. The observed signals for Sema-3A-peptide interaction were found in a defined area of the Sema domain, which was also demonstrated to be involved in NRP-1 binding. The presented data identified the interaction site for further development of therapeutic peptides to treat acute kidney injury by blocking the Sema-3A–NRP-1 interaction.     

Contribution of EGFR and ErbB-3 Heterodimerization to the EGFR Mutation-Induced Gefitinib- and Erlotinib-Resistance in Non-Small-Cell Lung Carcinoma Treatments

EGFR mutation-induced drug resistance has become a major threat to the treatment of non-small-cell lung carcinoma. Essentially, the resistance mechanism involves modifications of the intracellular signaling pathways. In our work, we separately investigated the EGFR and ErbB-3 heterodimerization, regarded as the origin of intracellular signaling pathways. On one hand, we combined the molecular interaction in EGFR heterodimerization with that between the EGFR tyrosine kinase and its inhibitor. For 168 clinical subjects, we characterized their corresponding EGFR mutations using molecular interactions, with three potential dimerization partners (ErbB-2, IGF-1R and c-Met) of EGFR and two of its small molecule inhibitors (gefitinib and erlotinib). Based on molecular dynamics simulations and structural analysis, we modeled these mutant-partner or mutant-inhibitor interactions using binding free energy and its components. As a consequence, the mutant-partner interactions are amplified for mutants L858R and L858R_T790M, compared to the wild type EGFR. Mutant delL747_P753insS represents the largest difference between the mutant-IGF-1R interaction and the mutant-inhibitor interaction, which explains the shorter progression-free survival of an inhibitor to this mutant type. Besides, feature sets including different energy components were constructed, and efficient regression trees were applied to map these features to the progression-free survival of an inhibitor. On the other hand, we comparably examined the interactions between ErbB-3 and its partners (EGFR mutants, IGF-1R, ErbB-2 and c-Met). Compared to others, c-Met shows a remarkably-strong binding with ErbB-3, implying its significant role in regulating ErbB-3 signaling. Moreover, EGFR mutants corresponding to poor clinical outcomes, such as L858R_T790M, possess lower binding affinities with ErbB-3 than c-Met does. This may promote the communication between ErbB-3 and c-Met in these cancer cells. The analysis verified the important contribution of IGF-1R or c-Met in the drug resistance mechanism developed in lung cancer treatments, which may bring many benefits to specialized therapy design and innovative drug discovery.     

Allosteric interactions between the myristate- and ATP-site of the Abl kinase

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors.     

Interactions between amyloidophilic dyes and their relevance to studies of amyloid inhibitors

Amyloid fibrils are filamentous aggregates of peptides and proteins implicated in a range of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. It has been known almost since their discovery that these β-sheet-rich proteinacious assemblies bind a range of specific dyes that, combined with other biophysical techniques, are convenient probes of the process of amyloid fibril formation. Two prominent examples of such dyes are Congo red (CR) and Thioflavin T (ThT). It has been reported that in addition to having a diagnostic role, CR is an inhibitor of the formation of amyloid structures, and these two properties have both been explained in terms of the same specific noncovalent interactions between the fibrils and the dye molecules. In this article, we show by means of quartz-crystal microbalance measurements that the binding of both ThT and CR to amyloid fibrils formed by the peptide whose aggregation is associated with Alzheimer's disease, Aβ(1-42), can be directly observed, and that the presence of CR interferes with the binding of ThT. Light scattering and fluorescence measurements confirm that an interaction exists between these dyes that can interfere with their ability to reflect accurately the quantity of amyloid material present in a given sample. Furthermore, we show that CR does not inhibit the process of amyloid fibril elongation, and therefore demonstrate the ability of the quartz-crystal microbalance method not only to detect and study the binding of small molecules to amyloid fibrils, but also to elucidate the mode of action of potential inhibitors.     

Protein interactions between surface annexin A2 and S100A10 mediate adhesion of breast cancer cells to microvascular endothelial cells

Annexin A2 (AnxA2) and S100A10 are known to form a molecular complex. Using fluorescence-based binding assays, we show that both proteins are localised on the cell surface, in a molecular form that allows mutual interaction. We hypothesized that binding between these proteins could facilitate cell–cell interactions. For cells that express surface S100A10 and surface annexin A2, cell–cell interactions can be blocked by competing with the interaction between these proteins. Thus an annexin A2-S100A10 molecular bridge participates in cell–cell interactions, revealing a hitherto unexplored function of this protein interaction.     

Competition between antigen and anti-idiotypes for rheumatoid factors

Many idiotypic determinants on antibody molecules are thought to be located at the antigen binding site, and therefore the interaction between idiotype (Id) and anti-idiotype (anti-Id) is expected to be inhibited by the antigen. We describe two IgG and one IgM rheumatoid factors whose interactions with their respective anti-Id could only be partially inhibited by very large amounts of antigen, i.e., normal IgG. The anti-Id, however, readily inhibited the binding of their respective rheumatoid factors to IgG. The differences in interaction energies resulted in failure of antigen to readily block the Id-anti-Id interaction, and did not mean that the Id was not at the antigen combining site. The association constants for the Id-anti-Id interactions varied from 1.3 to 14.8 X 10(7) M-1, whereas the strength of the rheumatoid factor antigen bond is on the order of 10(5) M-1 for interaction with monomeric IgG. In addition, the anti-Id were able to remove rheumatoid factors that were bound to solid phase IgG, indicating that anti-Id have the potential for disrupting the immune complexes formed by antigen and antibody.     

An interaction between zyxin and alpha-actinin

Zyxin is an 82-kD protein first identified as a component of adhesion plaques and the termini of stress fibers near where they associate with the cytoplasmic face of the adhesive membrane. We report here that zyxin interacts with the actin cross-linking protein alpha-actinin. Zyxin cosediments with filamentous actin in an alpha-actinin-dependent manner and an association between zyxin and alpha-actinin is observed in solution by analytical gel filtration. The specificity of the interaction between zyxin and alpha-actinin was demonstrated by blot overlay experiments in which 125I-zyxin recognizes most prominently alpha-actinin among a complex mixture of proteins extracted from avian smooth muscle. By these blot overlay binding studies, we determined that zyxin interacts with the NH2-terminal 27-kD domain of alpha-actinin, a region that also contains the actin binding site. Solid phase binding assays were performed to evaluate further the specificity of the binding and to determine the affinity of the zyxin-alpha-actinin interaction. By these approaches we have demonstrated a specific, saturable, moderate-affinity interaction between zyxin and alpha-actinin. Furthermore, double-label immunofluorescence reveals that zyxin and alpha-actinin exhibit extensive overlap in their subcellular distributions in both chicken embryo fibroblasts and pigmented retinal epithelial cells. The significant colocalization of the two proteins is consistent with the possibility that the interaction between zyxin and alpha-actinin has a biologically relevant role in coordinating membrane-cytoskeletal interactions.     

Molecular dynamics (MD) simulations and binding free energy calculation studies between inhibitors and type II dehydroquinase (DHQ2)

Type II dehydroquinase (DHQ2) is the third enzyme of the shikimic acid pathway, and it has been the effective target for tuberculosis (TB). So far, developing multiple potent inhibitors of the DHQ2 of Mycobacterium tuberculosis (DHQ2-Mt) has been considered to be the new therapy to TB. Molecular dynamics simulations followed by molecular mechanics-generalised Born surface area were carried out to calculate the free binding energy and to determine the affinity ability of the four chosen inhibitor molecules, L1, L2, L3 and L4. Energy decomposition analyses show the electrostatic interaction and van der Waals interaction of the ligands to every residue of the DHQ2-Mt. The results suggest that some important residues have different interactions with the four ligands, such as Arg19 and Tyr24. These interactions may have an effect on the ligand binding affinity. The binding affinity of monosubstituted inhibitor is higher than that of disubstituted inhibitor, due to some important interactions with the DHQ2-Mt residues. These computational works will be significant to the theoretical research in the future.     

Modeling the interaction between the N‐terminal domain of the tumor suppressor p53 and azurin

It is known that the half life of the tumor suppressor p53 can be increased by the interaction with the bacterial protein azurin, resulting in an enhanced anti‐tumoral activity. The understanding of the molecular mechanisms on the basis of this phenomenon can open the way to new anti‐cancer strategies. Some experimental works have given evidence of an interaction between p53 and azurin (AZ); however the binding regions of the proteins are still unknown. Recently, fluorescence studies have shown that p53 partakes in the binding with the bacterial protein by its N‐terminal (NT) domain. Here we have used a computational method to get insight into this interacting mode. The model that we propose for the best complex between AZ and p53 has been obtained from a rigid‐body docking, coupled with a molecular dynamics (MD) simulation, a free energy calculation, and validated by mutagenesis analysis. We have found a high degree of geometric fit between the two proteins that are kept together by several hydrophobic interactions and numerous hydrogen bonds. Interestingly, it has emerged that AZ binds essentially to the helices H_I and H_III of the p53 NT domain, which are also interacting regions for the foremost inhibitor of p53, MDM2. Copyright © 2009 John Wiley & Sons, Ltd.