Molecular dynamics simulation of the RNA complex of a double-stranded RNA-binding domain reveals dynamic features of the intermolecular interface and its hydration

The interaction between double-stranded RNA (dsRNA) and the third double-stranded domain (dsRBD) from Drosophila Staufen protein represents a paradigm to understand how the dsRBD protein family, one of the most common RNA-binding protein units, binds dsRNA. The nuclear magnetic resonance (NMR) structure of this complex and the x-ray structure of another family member revealed the stereochemical basis for recognition, but also raised new questions. Although the crystallographic studies revealed a highly ordered interface containing numerous water-mediated contacts, NMR suggested extensive residual motion at the interface. To address how interfacial motion contributes to molecular recognition in the dsRBD-dsRNA system, we conducted a 2-ns molecular dynamics simulation of the complex derived from Staufen protein and of the separate protein and RNA components. The results support the observation that a high degree of conformational flexibility is retained upon complex formation and that this involves interfacial residues that are critical for dsRBD-dsRNA binding. The structural origin of this residual flexibility is revealed by the analysis of the trajectory of motion. Individual basic side chains switch continuously from one RNA polar group to another with a residence time seldom exceeding 100 ps, while retaining favorable interaction with RNA throughout much of the simulation. Short-lived water molecules mediate some of these interactions for a large fraction of the trajectory studied here. This result indicates that water molecules are not statically associated with the interface, but continuously exchange with the bulk solvent on a 1-10-ps time scale. This work provides new insight into dsRBD-dsRNA recognition and builds upon a growing body of evidence, suggesting that short-lived dynamic interactions play important roles in protein-nucleic acid interactions.     

Synthetic peptides mimicking the interleukin-6/gp130 interaction: a two-helix bundle system. Design and conformational studies.

The objective of our study was to mimic in a typical reductionist approach the molecular interactions observed at the interface between the gp130 receptor and interleukin-6 during formation of their complex. A peptide system obtained by reproducing some of the interleukin-6/gp130 molecular interactions into a two-helix bundle structure was investigated. The solution conformational features of this system were determined by CD and NMR techniques. The CD titration experiments demonstrated that the interaction between the designed peptides is specific and based on a well-defined stoichiometry. The NMR data confirmed some of the structural features of the binding mechanism as predicted by the rational design and indicated that under our conditions the recognition specificity and affinity can be explained by the formation of a two-helix bundle. Thus, the data reported herein represent a promising indication on how to develop new peptides able to interfere with formation of the interleukin-6/gp130 complex.     

Molecular dynamics simulation of SRP GTPases: Towards an understanding of the complex formation from equilibrium fluctuations

Signal recognition particle (SRP) and its receptor (SR) play essential role in the SRP‐dependent protein targeting pathway. They interact with one another to precisely regulate the targeting reaction. The mechanism of this interaction consists of at least two discrete conformational states: complex formation and GTPase activation. Although structural studies have provided valuable insights into the understanding of the SRP‐SR interaction, it still remains unclear that how SRP and SR GTPases use their intrinsic conformational flexibilities to exert multiple allosteric regulations on this interaction process. Here, we use computational simulations to present the dynamic behavior of the SRP GTPases at an atomic level to gain further understanding of SRP‐SR interaction. We show that: (i) equilibrium conformational fluctuations contain a cooperative inter‐ and intradomain structural rearrangements that are functionally relevant to complex formation, (ii) a series of residues in different domains are identified to correlate with each other during conformational rearrangements, and (iii) α3 and α4 helices at domain interface actively rearrange their relative conformation to function as a bridge between the N domain and the core region of the G domain. These results, in addition to structural studies, would harness our understanding of the molecular mechanism for SRP and SR interaction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Transient protein interactions studied by NMR spectroscopy: the case of cytochrome C and adrenodoxin

The interaction between yeast iso-1-cytochrome c (C102T) and two forms of bovine adrenodoxin, the wild type and a truncated form comprising residues 4-108, has been investigated using a combination of one- and two-dimensional heteronuclear NMR spectroscopy. Chemical shift perturbations and line broadening of amide resonances in the [(15)N,(1)H]HSQC spectrum for both (15)N-labeled cytochrome c and adrenodoxin in the presence of the unlabeled partner protein indicate the formation of a transient complex, with a K(a) of (4 +/- 1) x 10(4) M(-)(1) and a lifetime of <3 ms. The perturbed residues map over a large surface area for both proteins. For cytochrome c, the dominating effects are located around the exposed heme edge but with other areas also affected upon formation of the complex. In the case of adrenodoxin, effects are seen in both the recognition and core domains, with the largest perturbations in the recognition domain. These results indicate that the complex has a dynamic nature, with delocalized binding of cytochrome c on adrenodoxin. A comparison with other transient complexes of redox proteins places this complex between well-defined complexes such as the cytochrome c-cytochrome c peroxidase complex and entirely dynamic complexes such as the cytochrome b(5)-myoglobin complex.     

Fluorinated cholesterol retains domain-forming activity in sphingomyelin bilayers

Lipid rafts are cholesterol (Chol)-rich microdomains floating in a sea of lipid bilayers. Chol is thought to interact preferentially with sphingolipids such as sphingomyelin (SM) rather than with glycerophospholipids, and this putative SM–Chol interaction is generally recognized as a requirement for raft formation. However, the presence of the specific interaction is still controversial, primarily because of the lack of useful molecular probes for scrutinizing this interaction. Recently, we reported that the dynamic properties of 6-F-Chol in DMPC bilayers are similar to those of unmodified Chol. Hence, in the present study, we first compared the roles of 6-F-Chol and Chol in SM bilayers through detergent insolubility, fluorescence polarization, and ²H NMR experiments. The results demonstrated that 6-F-Chol and Chol behave similarly in SM bilayers, whereas, in SM–DOPC membranes, 6-F-Chol is less effective in domain formation. Then, we analyzed the molecular orientation of 6-F-Chol in SM bilayers using solid-state NMR, and found that the dynamics and orientation of 6-F-Chol in SM bilayers are almost identical to those in DMPC bilayers. This supports the notion of the lack of a putative specific interaction between SM and Chol. Thus, this study demonstrates the utility of 6-F-Chol as a molecular probe for understanding molecular recognition in lipid rafts.     

Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment

Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibodydsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.     

Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.     

Investigating the Dynamic Aspects of Drug-Protein Recognition through a Combination of MD and NMR Analyses: Implications for the Development of Protein-Protein Interaction Inhibitors

In this paper, we investigate the dynamic aspects of the molecular recognition between a small molecule ligand and a flat, exposed protein surface, representing a typical target in the development of protein-protein interaction inhibitors. Specifically, we analyze the complex between the protein Fibroblast Growth Factor 2 (FGF2) and a recently discovered small molecule inhibitor, labeled sm27 for which the binding site and the residues mainly involved in small molecule recognition have been previously characterized. We have approached this problem using microsecond MD simulations and NMR-based characterizations of the dynamics of the apo and holo states of the system. Using direct combination and cross-validation of the results of the two techniques, we select the set of conformational states that best recapitulate the principal dynamic and structural properties of the complex. We then use this information to generate a multi-structure representation of the sm27-FGF2 interaction. We propose this kind of representation and approach as a useful tool in particular for the characterization of systems where the mutual dynamic influence between the interacting partners is expected to play an important role. The results presented can also be used to generate new rules for the rational expansion of the chemical diversity space of FGF2 inhibitors.     

Inhibition of islet amyloid polypeptide fibril formation: a potential role for heteroaromatic interactions

The formation of amyloid fibril is associated with major human diseases, including Alzheimer's disease, prion diseases, and type 2 diabetes. Methods for efficient inhibition of amyloid fibril formation are therefore highly clinically important. A principal approach for the inhibition of amyloid formation is based on the use of modified molecular recognition elements. Here, we demonstrate efficient inhibition of amyloid formation of the type 2 diabetes-related human islet amyloid polypeptide (hIAPP) by a modified aromatic peptide fragment and a small aromatic polyphenol molecule. A molecular recognition assay using peptide array analysis suggested that molecular recognition between hIAPP and its core amyloidogenic module is mediated by aromatic rather than hydrophobic interactions. To study the possible effect of aromatic interactions on inhibition of hIAPP fibril formation, we have used peptide and small molecule inhibitors. The addition of a nonamyloidogenic peptide analogue of the core module NFGAILSS, in which phenylalanine was substituted with tyrosine (NYGAILSS), resulted in substantial inhibition of fibril formation by hIAPP. The inhibition was significantly stronger than the one achieved using a beta-sheet breaker-conjugated peptide NFGAILPP. On the basis of the molecular arrangement of the tyrosine-phenylalanine interaction, we suggest that the inhibition stems from the geometrical constrains of the heteroaromatic benzene-phenol interaction. In line with this notion, we demonstrate remarkable inhibition of hIAPP fibril formation and cytotoxicity toward pancreatic beta-cells by a small polyphenol molecule, the nontoxic phenol red compound. Taken together, our results provide further experimental support for the potential role of aromatic interactions in amyloid formation and establish a novel approach for its inhibition.     

Kinetic studies of RNA-protein interactions using surface plasmon resonance

Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA-protein interactions. Protein-RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA-binding proteins to compete for binding sites, the sequential assembly of ribonucleoprotein complexes, and the ability of bound RNA to move between cellular compartments. Therefore, to attain a complete and biologically relevant understanding of RNA-protein interactions, complex formation must be studied not only in equilibrated reactions, but also as a dynamic process. BIACORE, a surface plasmon resonance-based biosensor technology, allows intermolecular interactions to be measured in real time, and can provide both equilibrium and kinetic information about complex formation. This technology is a powerful tool with which to study the dynamics of RNA-protein interactions. We have used BIACORE extensively to obtain detailed insight into the interaction between RNA and proteins carrying RNA recognition motif domains. Here we discuss the physical principles on which BIACORE is based, and the required instrumentation. We describe how to design well-controlled RNA-protein interaction experiments aimed at yielding high-quality data, and outline the steps required for data analysis. In addition, we present examples to illustrate how kinetic studies have provided us with unique insights into the interaction of the spliceosomal U1A protein and the neuronal HuD protein with their respective RNA targets.     

Mechanisms of amyloid fibril self-assembly and inhibition. Model short peptides as a key research tool

The formation of amyloid fibrils is associated with various human medical disorders of unrelated origin. Recent research indicates that self-assembled amyloid fibrils are also involved in physiological processes in several micro-organisms. Yet, the molecular basis for the recognition and self-assembly processes mediating the formation of such structures from their soluble protein precursors is not fully understood. Short peptide models have provided novel insight into the mechanistic issues of amyloid formation, revealing that very short peptides (as short as a tetrapeptide) contain all the necessary molecular information for forming typical amyloid fibrils. A careful analysis of short peptides has not only facilitated the identification of molecular recognition modules that promote the interaction and self-assembly of fibrils but also revealed that aromatic interactions are important in many cases of amyloid formation. The realization of the role of aromatic moieties in fibril formation is currently being used to develop novel inhibitors that can serve as therapeutic agents to treat amyloid-associated disorders.     

Aromatic-aromatic interactions in the formation of the MDM2-p53 complex

Although the molecular interaction of MDM2 with the transactivation domain of p53 has been thoroughly studied, there is very limited information regarding the steps involved in the recognition mechanism between these proteins. On this basis, we performed four high-temperature molecular dynamics simulations in explicit solvent to gain insight into the interactions involved in the fist contact toward the formation of the complex. We found that the presence of specific intermolecular aromatic pairs at the interface of the complex, around the native-like state of MDM2, is consistent among independent molecular dynamics runs. This observation suggests that aromatic-aromatic interactions are closely related to the first contact between MDM2 and p53. Thus, we propose that aromatic-aromatic interactions are an important, and probably essential, requirement for the formation and stabilization of the MDM2-p53 complex.     

Structural basis of protein–protein interaction studied by NMR

This paper describes efforts of the structural genomics project in the nuclear magnetic resonance (NMR) laboratory at the University of Science and Technology of China. This structural genomics project is biological-functional driven. Targets are mainly selected from two systems: proteins related with regulation of gene expression in humans and other eukaryotes, and proteins existing in the cell junction in humans. The majority of proteins selected from these two systems are related with human health and diseases, and some are potential drug targets. Twenty-five protein structures from Homo sapiens and other eukaryotes have been determined during last 5 years in this laboratory. Nuclear magnetic resonance (NMR) spectroscopy is highly suited to investigate molecular interactions at a close physiological condition and is particularly suited for the study of low-affinity, transient complexes. It can provide information on protein surface interaction, their complex structure, and their dynamic properties during protein recognition. Several examples are given in this paper.     

How FMN binds to anabaena apoflavodoxin: a hydrophobic encounter at an open binding site

Molecular recognition begins when two molecules approach and establish interactions of certain strength. The mechanisms of molecular recognition reactions between biological molecules are not well known, and few systems have been analyzed in detail. We investigate here the reaction between an apoprotein and its physiological cofactor (apoflavodoxin and flavin mononucleotide) that binds reversibly to form a non-covalent complex (flavodoxin) involved in electron transfer reactions. We have analyzed the fast binding reactions between the FMN cofactor (and shorter analogs) and wild type (and nine mutant apoflavodoxins where residues interacting with FMN in the final complex have been replaced). The x-ray structures of two such mutants are reported that show the mutations are well tolerated by the protein. From the calculated microscopic binding rate constants we have performed a Phi analysis of the transition state of complex formation that indicates that the binding starts by interaction of the isoalloxazine-fused rings in FMN with residues of its hydrophobic binding site. In contrast, the phosphate in FMN, known to contribute most to the affinity of the final holoflavodoxin complex, is not bound in the transition state complex. Both the effects of ionic strength and of phosphate concentration on the wild type complex rate constant agree with this scenario. As suggested previously by nmr data, it seems that the isoalloxazine-binding site may be substantially open in solution. Interestingly, although FMN is a charged molecule, electrostatic interactions seem not to play a role in directing the binding, unlike what has been reported for other biological complexes. The binding can thus be best described as a hydrophobic encounter at an open binding site.     

Molecular basis of HIV-1 TAR RNA specific recognition by an acridine tat-antagonist.

We investigated the interaction of a highly potent acridine-based tat-antagonist with the TAR RNA of HIV-1. The wild type TAR RNA and three mutants with U-->C23, G x C-->C x G26-39 or G x C-->A x U26-39 substitutions were used as substrates to study the molecular basis of drug-TAR RNA complex formation. Melting temperature and RNase protection experiments reveal that the G x C26-39 pair is a critical element for specific major groove recognition of TAR at the pyrimidine bulge. The results provide a rational basis for future design of optimized tat/TAR inhibitors.     

根瘤菌NOD因子的感知与信号传导

根瘤菌是一类引起豆科植物结瘤固氮的土壤细菌。根瘤中的类菌体固定空气中的氮气为宿主植物提供充足的氮源。共生体系的建立始于细菌与宿主植物间复杂的信号交换过程。植物产生类黄酮诱导相应的根瘤菌合成分泌结瘤因子 ,后者进而诱导宿主植物根系形态变化以及早期根瘤素基因表达。以下将就宿主植物结瘤因子的特异识别和早期信号传导进行讨论。     

A comparative study of HIV-1 and HTLV-I protease structure and dynamics reveals a conserved residue interaction network

The two retroviruses human T-lymphotropic virus type I (HTLV-I) and human immunodeficiency virus type 1 (HIV-1) are the causative agents of severe and fatal diseases including adult T-cell leukemia and the acquired immune deficiency syndrome (AIDS). Both viruses code for a protease that is essential for replication and therefore represents a key target for drugs interfering with viral infection. The retroviral proteases from HIV-1 and HTLV-I share 31% sequence identity and high structural similarities. Yet, their substrate specificities and inhibition profiles differ substantially. In this study, we performed all-atom molecular dynamics (MD) simulations for both enzymes in their ligand-free states and in complex with model substrates in order to compare their dynamic behaviors and enhance our understanding of the correlation between sequence, structure, and dynamics in this protein family. We found extensive similarities in both local and overall protein dynamics, as well as in the energetics of their interactions with model substrates. Interestingly, those residues that are important for strong ligand binding are frequently not conserved in sequence, thereby offering an explanation for the differences in binding specificity. Moreover, we identified an interaction network of contacts between conserved residues that interconnects secondary structure elements and serves as a scaffold for the protein fold. This interaction network is conformationally stable over time and may provide an explanation for the highly similar dynamic behavior of the two retroviral proteases, even in the light of their rather low overall sequence identity.     

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5- to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication.