Prediction of Ras-effector interactions using position energy matrices

MOTIVATION: One of the more challenging problems in biology is to determine the cellular protein interaction network. Progress has been made to predict protein-protein interactions based on structural information, assuming that structural similar proteins interact in a similar way. In a previous publication, we have determined a genome-wide Ras-effector interaction network based on homology models, with a high accuracy of predicting binding and non-binding domains. However, for a prediction on a genome-wide scale, homology modelling is a time-consuming process. Therefore, we here successfully developed a faster method using position energy matrices, where based on different Ras-effector X-ray template structures, all amino acids in the effector binding domain are sequentially mutated to all other amino acid residues and the effect on binding energy is calculated. Those pre-calculated matrices can then be used to score for binding any Ras or effector sequences. RESULTS: Based on position energy matrices, the sequences of putative Ras-binding domains can be scanned quickly to calculate an energy sum value. By calibrating energy sum values using quantitative experimental binding data, thresholds can be defined and thus non-binding domains can be excluded quickly. Sequences which have energy sum values above this threshold are considered to be potential binding domains, and could be further analysed using homology modelling. This prediction method could be applied to other protein families sharing conserved interaction types, in order to determine in a fast way large scale cellular protein interaction networks. Thus, it could have an important impact on future in silico structural genomics approaches, in particular with regard to increasing structural proteomics efforts, aiming to determine all possible domain folds and interaction types. AVAILABILITY: All matrices are deposited in the ADAN database (http://adan-embl.ibmc.umh.es/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Concerted simulations reveal how peroxidase compound III formation results in cellular oscillations

A major problem in mathematical modeling of the dynamics of complex biological systems is the frequent lack of knowledge of kinetic parameters. Here, we apply Brownian dynamics simulations, based on protein three-dimensional structures, to estimate a previously undetermined kinetic parameter, which is then used in biochemical network simulations. The peroxidase-oxidase reaction involves many elementary steps and displays oscillatory dynamics important for immune response. Brownian dynamics simulations were performed for three different peroxidases to estimate the rate constant for one of the elementary steps crucial for oscillations in the peroxidase-oxidase reaction, the association of superoxide with peroxidase. Computed second-order rate constants agree well with available experimental data and permit prediction of rate constants at physiological conditions. The simulations show that electrostatic interactions depress the rate of superoxide association with myeloperoxidase, bringing it into the range necessary for oscillatory behavior in activated neutrophils. Such negative electrostatic steering of enzyme-substrate association presents a novel control mechanism and lies in sharp contrast to the electrostatically-steered fast association of superoxide and Cu/Zn superoxide dismutase, which is also simulated here. The results demonstrate the potential of an integrated and concerted application of structure-based simulations and biochemical network simulations in cellular systems biology.     

Factors determining electrostatic fields in molecular dynamics simulations of the Ras/effector interface

Using molecular dynamics simulations, we explore geometric and physical factors contributing to calculated electrostatic fields at the binding surface of the GTPase Ras with a spectroscopically labeled variant of a downstream effector, the Ras-binding domain of Ral guanine nucleotide dissociation stimulator (RalGDS). A related system (differing by mutation of one amino acid) has been studied in our group using vibrational Stark effect spectroscopy, a technique sensitive to electrostatic fields. Electrostatic fields were computed using the AMBER 2003 force field and averaged over snapshots from molecular dynamics simulation. We investigate geometric factors by exploring how the orientation of the spectroscopic probe changes on Ras-effector binding. In addition, we explore the physical origin of electrostatic fields at our spectroscopic probe by comparing contributions to the field from discrete components of the system, such as explicit solvent, residues on the Ras surface, and residues on the RalGDS surface. These models support our experimental hypothesis that vibrational Stark shifts are caused by Ras binding to its effector and not the structural rearrangements of the effector surface or probe reorientation on Ras-effector binding, for at least some of our experimental probes. These calculations provide physical insight into the origin, magnitude, and importance of electrostatic fields in protein-protein interactions and suggest new experiments to probe the field's role in protein docking.     

A method for measuring binding constants using unpurified in vivo biotinylated ligands

Obtaining accurate kinetics and steady-state binding constants for biomolecular interactions normally requires pure and homogeneous protein preparations. Furthermore, in many cases, one of the ligands must be labeled. Over the past decade, several technologies have been introduced that allow for the measurement of kinetics constants for multiple different interactions in parallel. One such technology is bio-layer interferometry (BLI), which has been used to develop systems that can measure up to 96 biomolecular interactions simultaneously. However, despite the ever-increasing throughput of the tools available for measuring protein–protein interactions, the preparation of pure protein still remains a bottleneck in the process of producing high-quality kinetics data. Here, we show that high-quality binding data can be obtained using soluble lysate fractions containing protein that has been biotinylated in vivo using BirA and then applied to BLI sensors without further purification. Furthermore, we show that BirA ligase does not necessarily need to be co-overexpressed with the protein of interest for biotinylation of the biotin acceptor peptide to occur, suggesting that the activity of endogenous BirA in Escherichia coli is sufficient for producing enough biotinylated protein for a binding experiment.     

Smoothing molecular interactions: the "kinetic buffer" effect of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) widely participate in molecular recognition and signaling processes in cells by interacting with other molecules. Compared with ordered proteins, IDPs usually possess stronger intermolecular interactions in binding. As a result, the interface structure of IDPs in complexes is distinct from that of ordered-protein complexes, and this difference may have essential effect on the response to various perturbations in a cell. In this study, we examined the perturbations of intermolecular interactions and temperature on the coupled folding and binding processes of pKID to KIX domains by performing molecular dynamics simulations. By comparing a series of virtual pKID systems with various degree of disorder, we found that the complex stability and the binding kinetics of the disordered systems were less sensitive to the perturbations than the ordered systems. The origin of the lower response sensitivity of IDPs was attributed to their higher flexibility in the complex interface, which was further supported by an analysis on protein complex structures. On the basis of our simulations and results from the literature, we speculate IDPs may not only interact with their biological partners with high specificity and low affinity but also may be resistant to the perturbations in the environment and transmit signals fast and smooth. We proposed to name it the "kinetic buffer" effect.     

Characterization of domain-peptide interaction interface: prediction of SH3 domain-mediated protein-protein interaction network in yeast by generic structure-based models

Determination of the binding specificity of SH3 domain, a peptide recognition module (PRM), is important to understand their biological functions and reconstruct the SH3-mediated protein-protein interaction network. In the present study, the SH3-peptide interactions for both class I and II SH3 domains were characterized by the intermolecular residue-residue interaction network. We developed generic MIEC-SVM models to infer SH3 domain-peptide recognition specificity that achieved satisfactory prediction accuracy. By investigating the domain-peptide recognition mechanisms at the residue level, we found that the class-I and class-II binding peptides have different binding modes even though they occupy the same binding site of SH3. Furthermore, we predicted the potential binding partners of SH3 domains in the yeast proteome and constructed the SH3-mediated protein-protein interaction network. Comparison with the experimentally determined interactions confirmed the effectiveness of our approach. This study showed that our sophisticated computational approach not only provides a powerful platform to decipher protein recognition code at the molecular level but also allows identification of peptide-mediated protein interactions at a proteomic scale. We believe that such an approach is general to be applicable to other domain-peptide interactions.     

Kinetics of pH-dependent interactions between PD-1 and PD-L1 immune checkpoint proteins from molecular dynamics

Immune checkpoint blockade of signaling pathways such as PD-1/PD-L1 has recently opened up a new avenue for highly efficient immunotherapeutic strategies to treat cancer. Since tumor microenvironments are characterized by lower pH (5.5-7.0), pH-dependent protein-ligand interactions can be exploited as efficient means to regulate drug affinity and specificity for a variety of malignancies. In this article, we investigate the mechanism and kinetics of pH-dependent binding and unbinding processes for the PD-1/PD-L1 checkpoint pair employing classical molecular dynamics simulations. Two representative pH levels corresponding to circumneutral physiological conditions of blood (pH 7.4) and acidic tumor microenvironment (pH 5.5) are considered. Our calculations demonstrate that pH plays a key role in protein-ligand interactions with small pH changes leading to several orders of magnitude increase in binding affinity. By identifying the binding pocket in the PD-1/PD-L1 complex, we show a pivotal role of the His68 protonation state of PD-1in the complex stabilization at low pH. The results on the reaction rate constants are in qualitative agreement with available experimental data. The obtained molecular details are important for further engineering of binding/unbinding kinetics to formulate more efficient immune checkpoint blockade strategies.     

Insights into prion protein function from atomistic simulations

Computer simulations are a powerful tool for studies of biological systems. They have often been used to study prion protein (PrP), a protein responsible for neurodegenerative diseases, which include “mad cow disease” in cattle and Creutzfeldt-Jacob disease in humans. An important aspect of the prion protein is its interaction with copper ion, which is thought to be relevant for PrP’s yet undetermined function and also potentially play a role in prion diseases. For studies of copper attachment to the prion protein, computer simulations have often been used to complement experimental data and to obtain binding structures of Cu-PrP complexes. This paper summarizes the results of recent ab initio calculations of copper-prion protein interactions focusing on the recently discovered concentration-dependent binding modes in the octarepeat region of this protein. In addition to determining the binding structures, computer simulations were also used to make predictions about PrP’s function and the role of copper in prion diseases. The results demonstrate the predictive power and applicability of ab initio simulations for studies of metal-biomolecular complexes.     

Fluorescence approaches for monitoring interactions of Rho GTPases with nucleotides, regulators, and effectors

To understand the manner in which biological macromolecules interact with each other, we need not only structural information, but also details of kinetics and thermodynamics of the processes involved. This is particularly important for key proteins acting in signal transduction such as the small GTPases of the Ras superfamily. The complexity of their roles is constantly increasing since a large number of GTPases have been identified and each of these in turn interacts with a variety of regulatory and signaling proteins such as GAPs, GEFs, and downstream effectors. There are a number of methods that can be used to characterize the specificity, strength, and stoichiometry of such intermolecular interactions, to understand the effect of binding on the protein structure, and, ultimately, to obtain insights into their biological functions. This article discusses the use of fluorescence spectroscopic methods, which allows real-time monitoring of ligand- and protein-protein interactions at submicromolar concentrations, and quantification of the kinetic and equilibrium constants. Fluorescently labeled guanine nucleotides serve as fluorescence reporter groups to investigate the interactions of GTPases of the Rho family (e.g., RhoA, Rac1, and Cdc42). We present examples for quantitative characterization of (i) Rac1 x GDP interaction, (ii) Cdc42-interaction with the GTPase binding domain of the Wiskott Aldrich syndrome protein (three alternative approaches), (iii) accelerated nucleotide exchange reaction of RhoA by the catalytic domains of p190RhoGEF, and (iv) intrinsic and stimulated GTP-hydrolysis reaction by the catalytic domain of p50RhoGAP.     

Kinetic analysis of the effect on Fab binding of identical substitutions in a peptide and its parent protein

Monoclonal antibody 57P, which was raised against tobacco mosaic virus protein, cross-reacts with a peptide corresponding to residues 134-146 of this protein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics the structure in the protein. In this study, we carried out a detailed comparison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the parent protein, and the effect of these substitutions on antibody binding parameters have been measured with a Biacore instrument. Fabs that recognize epitopes located away from the site of mutations were used as indirect probes for the conformational integrity of protein antigens. Their interaction kinetics with all proteins were similar, suggesting that the substitutions had no drastic effect on their conformation. The five substitutions introduced in the peptide and the protein had minor effects on association rate constants (ka) and significant effects on dissociation rate constants (kd) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope was presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation. However, ka values were about 10 times larger and kd values about 5 times larger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed for three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and protein antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to mimic correctly some but not all properties of the protein-Fab 57P interaction and highlight the importance of quantitative analysis of both equilibrium and kinetic binding parameters in the design of synthetic vaccines and drugs.     

Structure-Based Network Analysis of Activation Mechanisms in the ErbB Family of Receptor Tyrosine Kinases: The Regulatory Spine Residues Are Global Mediators of Structural Stability and Allosteric Interactions

The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced “superacceptor” activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD) motif in the catalytic loop and the Asp-Phe-Gly (DFG) motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not limited to the ATP site, and may enhance allosteric cooperativity with the substrate binding region by increasing communication capabilities of mediating residues.     

Display of Ras on filamentous phage through cysteine replacement

Phage display technology has been used in a variety of contexts to understand and manipulate biomolecular interactions between proteins and other biomolecules. In this paper we describe the establishment of a phage display system for elucidation of the interactions between the GTPase Ras and its panel of effectors. It is shown how technical problems associated with phage display of a protein with unpaired cysteines, likely to be caused by the oxidizing environment of the bacterial periplasm into which the protein is directed, can be overcome by cysteine replacement based on functional and structural studies. First, the catalytic domain (residues 1-166) of mammalian H-Ras (Ras) was observed to be displayed on phage in an incorrect conformation not detectable by antibodies recognizing conformational epitopes on Ras. Although truncation of the phage coat protein used as fusion partner (g3p) resulted in minor improvements in the display, Ras was tailored for phage display by cysteine replacement. By replacing the three cysteines at positions 51, 80 and 118 of Ras with the corresponding residues in Saccharomyces cerevisiae RAS1, the resulting fusion-phage is recognized by the conformation-dependent anti-Ras antibodies. Furthermore, display of cysteine-free Ras is demonstrated by GTP-analogue dependent binding to the Ras-binding domain of the Ras-effector Raf1. These data pave the way for analysis of Ras-effector interactions using phage display technology yet demonstrate that phage display of proteins with normally reduced cysteines should be approached with caution.     

Kinetic analysis of the interactions between vaccinia virus complement control protein and human complement proteins C3b and C4b

The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.     

Screening for transient biological interactions as applied to albumin ligands: a new concept for drug discovery

The notion that many biological interactions are based on transient binding (dissociation constants (K(d)) in the range of 10-0.01 mM) is familiar, yet the implications for biological sciences have been realized only recently. An important area of biological sciences is drug design, where the traditional "lock and key" view of binding has prevailed and drug candidates are usually selected on their merits as being tight binders. However, the rationale that transient interactions are of importance for drug discovery is slowly gaining acceptance. These interactions may relate not only to the desired target interaction but also to unwanted interactions creating, for example, toxicity problems. Here we demonstrate, in a high-throughput screening format, affinity selection of weak binders to a model target of albumin by zonal retardation chromatography. It is perceived that this approach can define the "transient drug" as a complement to current drug discovery procedures.     

Backbone Flexibility of CDR3 and Immune Recognition of Antigens

Conformational entropy is an important component of protein–protein interactions; however, there is no reliable method for computing this parameter. We have developed a statistical measure of residual backbone entropy in folded proteins by using the ?–ψ distributions of the 20 amino acids in common secondary structures. The backbone entropy patterns of amino acids within helix, sheet or coil form clusters that recapitulate the branching and hydrogen bonding properties of the side chains in the secondary structure type. The same types of residues in coil and sheet have identical backbone entropies, while helix residues have much smaller conformational entropies. We estimated the backbone entropy change for immunoglobulin complementarity-determining regions (CDRs) from the crystal structures of 34 low-affinity T-cell receptors and 40 high-affinity Fabs as a result of the formation of protein complexes. Surprisingly, we discovered that the computed backbone entropy loss of only the CDR3, but not all CDRs, correlated significantly with the kinetic and affinity constants of the 74 selected complexes. Consequently, we propose a simple algorithm to introduce proline mutations that restrict the conformational flexibility of CDRs and enhance the kinetics and affinity of immunoglobulin interactions. Combining the proline mutations with rationally designed mutants from a previous study led to 2400-fold increase in the affinity of the A6 T-cell receptor for Tax-HLAA2. However, this mutational scheme failed to induce significant binding changes in the already-high-affinity C225–Fab/huEGFR interface. Our results will serve as a roadmap to formulate more effective target functions to design immune complexes with improved biological functions.     

Thermodynamics and kinetics of actin filament nucleation.

We have performed computer simulations and free energy calculations to determine the thermodynamics and kinetics of actin nucleation and thus identify a probable nucleation pathway and critical nucleus size. The binding free energies of structures along the nucleation pathway are found through a combination of electrostatic calculations and estimates of the entropic and surface area contributions. The association kinetics for the formation of each structure are determined through a series of Brownian dynamics simulations. The combination of the binding free energies and the association rate constants determines the dissociation rate constants, allowing for a complete characterization of the nucleation and polymerization kinetics. The results indicate that the trimer is the size of the critical nucleus, and the rate constants produce polymerization plots that agree very well with experimental results over a range of actin monomer concentrations.