The courtship of proteins: Understanding the encounter complex

The formation of protein complexes involves an encounter complex, in which proteins show few specific interactions and assume many orientations. Recent kinetic and structural studies have shed light on this elusive state. It is generally dominated by electrostatic interactions, although hydrophobic interactions can play a role. During the encounter phase the proteins remain largely solvated. In extreme cases, the proteins only form an encounter complex, and in many other complexes, the encounter state constitutes a significant amount (5% or more), indicating that the energy difference between encounter and productive complexes is small. Thus, the encounter complex represents an essential part of protein complexes.     

Dynamics in electron transfer protein complexes

Electron transfer proteins transport electrons safely between large redox enzymes. The complexes formed by these proteins are among the most transient. The biological function requires, on the one hand, sufficient specificity of the interaction to allow for rapid and selective electron transfer, and, on the other hand, a fast turnover of the complex. Recent progress in the characterization of the nature of these complexes has demonstrated that the encounter state plays an important role. This state of initial binding is dominated by electrostatic interactions, and consists of an ensemble of orientations. Paramagnetic relaxation enhancement NMR and chemical shift perturbation analysis provide ways for the experimental characterisation of the encounter state. Several studies that have used these techniques have shown that the surface area sample in the encounter state can be limited to the immediate environment of the final, specific complex. The encounter complex can represent a large fraction and, in some small complexes, no specific binding is detected at all. It can be concluded that, in electron transfer protein complexes, a fine balance is sought between the low-specificity encounter state and the high-specificity productive complex to meet the opposing requirements of rapid electron transfer and a high turnover rate.     

Fruitful and Futile Encounters along the Association Reaction between Proteins

The association reaction between pairs of proteins proceeds through an encounter complex that develops into the final complex. Here, we combined Brownian dynamics simulations with experimental studies to analyze the structures of the encounter complexes along the association reaction between TEM1-β-lactamase and its inhibitor, β-lactamase-inhibitor protein. The encounter complex can be considered as an ensemble of short-lived low free-energy states that are stabilized primarily by electrostatic forces and desolvation. For the wild-type, the simulation showed two main encounter regions located outside the physical binding site. One of these regions was located near the experimentally determined transition state. To validate whether these encounters are fruitful or futile, we examined three groups of mutations that altered the encounter. The first group consisted of mutations that increased the experimental rate of association through electrostatic optimization. This resulted in an increase in the size of the encounter region located near the experimentally determined transition state, as well as a decrease in the energy of this region and an increase in the number of successful trajectories (i.e., encounters that develop into complex). A second group of mutations was specifically designed to either increase or decrease the size and energy of the second encounter complex, but either way it did not affect k_on. A third group of mutations consisted of residues that increased k_on without significantly affecting the encounter complexes. These results indicate that the size and energy of the encounter regions are only two of several parameters that lead to fruitful association, and that electrostatic optimization is a major driving force in fast association.     

Protein-protein docking by simulating the process of association subject to biochemical constraints

We present a computational procedure for modeling protein-protein association and predicting the structures of protein-protein complexes. The initial sampling stage is based on an efficient Brownian dynamics algorithm that mimics the physical process of diffusional association. Relevant biochemical data can be directly incorporated as distance constraints at this stage. The docked configurations are then grouped with a hierarchical clustering algorithm into ensembles that represent potential protein-protein encounter complexes. Flexible refinement of selected representative structures is done by molecular dynamics simulation. The protein-protein docking procedure was thoroughly tested on 10 structurally and functionally diverse protein-protein complexes. Starting from X-ray crystal structures of the unbound proteins, in 9 out of 10 cases it yields structures of protein-protein complexes close to those determined experimentally with the percentage of correct contacts >30% and interface backbone RMSD <4 A. Detailed examination of all the docking cases gives insights into important determinants of the performance of the computational approach in modeling protein-protein association and predicting of protein-protein complex structures.     

Temperature differentially affects encounter and docking thermodynamics of antibody--antigen association

Using BIACORE SPR, we have examined the mechanism of temperature effects on the binding kinetics of two closely related antibody Fabs (H10 and H26) which recognize coincident epitopes on hen egg-white lysozyme (HEL), and whose association and dissociation kinetics are best described by the two-step conformational change model which we interpret as molecular encounter and docking. Time-course series data obtained at a series of six temperatures (6, 10, 15, 25, 30 and 37 degrees C) showed that temperature differentially affects the rate constants of the encounter and docking steps. Docking is more temperature-sensitive than the encounter step, and energetically less favorable at higher temperatures. At elevated temperatures, the time required for docking is longer and the apparent increase in off-rate reflects the greater proportion of the molecules failing to dock and remaining in the less stable encounter state. As a consequence, distribution of free energy change between the encounter and docking steps is altered. At physiological temperature (37 degrees C) the docking step of the H26 complex is energetically unfavorable and most complexes essentially do not dock. There is a significant decrease in total free energy change of the H26 complex at higher temperatures. Elevated temperature changes the rate-limiting step of H26--HEL association from the encounter to the docking step, but not that of H10--HEL. Our results indicate that the mechanism by which elevated temperature reduces the affinities of antigen--antibody complexes is to decrease the net docking rate, and/or stability of the docked complex; at higher temperatures, a smaller proportion of the complexes actually anneal to a more stable docked state. This mechanism may have broad applicability to other receptor--ligand complexes.     

Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation.     

New insights into the mechanism of protein-protein association

Association of a protein complex follows a two-step mechanism, with the first step being the formation of an encounter complex that evolves into the final complex. Here, we analyze recent experimental data of the association of TEM1-beta-lactamase with BLIP using theoretical calculations and simulation. We show that the calculated Debye-Hückel energy of interaction for a pair of proteins during association resembles an energy funnel, with the final complex at the minima. All attraction is lost at inter-protein distances of 20 A, or rotation angles of >60 degrees from the orientation of the final complex. For faster-associating protein complexes, the energy funnel deepens and its volume increases. Mutations with the largest impact on association (hotspots for association) have the largest effect on the size and depth of the energy funnel. Analyzing existing evidence, we suggest that the transition state along the association pathway is the formation of the final complex from the encounter complex. Consequently, pairs of proteins forming an encounter complex will tend to dissociate more readily than to evolve into the final complex. Increasing directional diffusion by increasing favorable electrostatic attraction results in a faster forming and slower dissociating encounter complex. The possible applicability of electrostatic calculations for protein-protein docking is discussed.     

On the protein–protein diffusional encounter complex

When two proteins diffuse together to form a bound complex, an intermediate is formed at the end‐point of diffusional association which is called the encounter complex. Its characteristics are important in determining association rates, yet its structure cannot be directly observed experimentally. Here, we address the problem of how to construct the ensemble of three‐dimensional structures which constitute the protein–protein diffusional encounter complex using available experimental data describing the dependence of protein association rates on mutation and on solvent ionic strength and viscosity. The magnitude of the association rates is fitted well using a variety of definitions of encounter complexes in which the two proteins are located at up to about 17 Å root‐mean‐squared distance from their relative arrangement in the bound complex. Analysis of the ionic strength dependence of bimolecular association rates shows that this is determined to a greater extent by the (protein charge) – (salt ion) separation distance than by the protein–protein charge separation distance. Consequently, ionic strength dependence of association rates provides little information about the geometry of the encounter complex. On the other hand, experimental data on electrostatic rate enhancement, mutation and viscosity dependence suggest a model of the encounter complex in which the two proteins form a subset of the contacts present in the bound complex and are significantly desolvated. Copyright © 1999 John Wiley & Sons, Ltd.     

Potential Regulatory Role of Competitive Encounter Complexes in Paralogous Phosphotransferase Systems

There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS^sugar) and one for nitrogen regulation (PTS^Ntr), that utilize proteins enzyme I^sugar (EI^sugar) and HPr, and enzyme I^Ntr (EI^Ntr) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein–protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EI^Ntr:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI^Ntr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI^Ntr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EI^Ntr. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes.     

A mechanistic model of the cysteine synthase complex

Plants and bacteria assimilate and incorporate inorganic sulfur into organic compounds such as the amino acid cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase (CS) complex. The CS complex is composed of the enzymes serine acetyl transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL). Although it is experimentally known that formation of the CS complex influences cysteine production, the exact biological function of the CS complex, the mechanism of reciprocal regulation of the constituent enzymes and the structure of the complex are still poorly understood. Here, we used docking techniques to construct a model of the CS complex from mitochondrial Arabidopsis thaliana. The three-dimensional structures of the enzymes were modeled by comparative techniques. The C-termini of SAT, missing in the template structures but crucial for CS formation, were modeled de novo. Diffusional encounter complexes of SAT and OAS-TL were generated by rigid-body Brownian dynamics simulation. By incorporating experimental constraints during Brownian dynamics simulation, we identified complexes consistent with experiments. Selected encounter complexes were refined by molecular dynamics simulation to generate structures of bound complexes. We found that although a stoichiometric ratio of six OAS-TL dimers to one SAT hexamer in the CS complex is geometrically possible, binding energy calculations suggest that, consistent with experiments, a ratio of only two OAS-TL dimers to one SAT hexamer is more likely. Computational mutagenesis of residues in OAS-TL that are experimentally significant for CS formation hindered the association of the enzymes due to a less-favorable electrostatic binding free energy. Since the enzymes from A. thaliana were expressed in Escherichia coli, the cross-species binding of SAT and OAS-TL from E. coli and A. thaliana was explored. The results showed that reduced cysteine production might be due to a cross-binding of A. thaliana OAS-TL with E. coli SAT. The proposed models of the enzymes and their complexes provide mechanistic insights into CS complexation.     

Electrostatically accelerated coupled binding and folding of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are now recognized to be prevalent in biology, and many potential functional benefits have been discussed. However, the frequent requirement of peptide folding in specific interactions of IDPs could impose a kinetic bottleneck, which could be overcome only by efficient folding upon encounter. Intriguingly, existing kinetic data suggest that specific binding of IDPs is generally no slower than that of globular proteins. Here, we exploited the cell cycle regulator p27(Kip1) (p27) as a model system to understand how IDPs might achieve efficient folding upon encounter for facile recognition. Combining experiments and coarse-grained modeling, we demonstrate that long-range electrostatic interactions between enriched charges on p27 and near its binding site on cyclin A not only enhance the encounter rate (i.e., electrostatic steering) but also promote folding-competent topologies in the encounter complexes, allowing rapid subsequent formation of short-range native interactions en route to the specific complex. In contrast, nonspecific hydrophobic interactions, while hardly affecting the encounter rate, can significantly reduce the efficiency of folding upon encounter and lead to slower binding kinetics. Further analysis of charge distributions in a set of known IDP complexes reveals that, although IDP binding sites tend to be more hydrophobic compared to the rest of the target surface, their vicinities are frequently enriched with charges to complement those on IDPs. This observation suggests that electrostatically accelerated encounter and induced folding might represent a prevalent mechanism for promoting facile IDP recognition.     

The kinetic basis for dual recognition in colicin endonuclease-immunity protein complexes

The antibacterial activity of E colicin endonucleases (DNases) is counteracted by the binding of immunity proteins; the affinities of cognate and non-cognate complexes differing by up to ten orders of magnitude. Here, we address the mechanism of complex formation using a combination of protein engineering, pre-steady-state kinetics and isothermal titration calorimetry, in order to understand the underlying basis for specificity. Contrary to previous work, we show that a pre-equilibrium mechanism does not explain the binding kinetics. Instead, the data are best explained by a modified induced-fit mechanism where cognate and non-cognate complexes alike form a non-specific, conformationally dynamic encounter complex, most likely centred on conserved interactions at the interface. The dynamics appear to be an intrinsic property of the encounter complex where the proteins move relative to one another, thereby sampling different conformations rather than being "induced" by binding. This allows optimal alignment of interface specificity sites, without producing energetically costly conformational changes, essential for high-affinity binding. Importantly, specificity is achieved without slowing the rate of association, an important requirement for rapid inhibition of the colicin in the producing bacterial cell. A rigid-body rotation model is also consistent with the observation that specificity contacts in colicin-immunity protein complexes can involve different regions of the interface. Such a kinetic discrimination mechanism explains the ability of DNase-specific immunity proteins to display dual recognition specificity, wherein they are broadly cross-reactive yet are highly specific, achieving femtomolar binding affinities in complexes with their cognate DNases.     

On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase

We have carried out a series of studies on the binding of a substrate mimic to the enzyme lactate dehydrogenase (LDH) using advanced kinetic approaches, which begin to provide a molecular picture of the dynamics of ligand binding for this protein. Binding proceeds via a binding-competent subpopulation of the nonligated form of the protein (the LDH/NADH binary complex) to form a protein-ligand encounter complex. The work here describes the collapse of the encounter complex to form the catalytically competent Michaelis complex. Isotope-edited static Fourier transform infrared studies on the bound oxamate protein complex reveal two kinds of oxamate environments: 1), a major populated structure wherein all significant hydrogen-bonding patterns are formed at the active site between protein and bound ligand necessary for the catalytically productive Michaelis complex and 2), a minor structure in a configuration of the active site that is unfavorable to carry out catalyzed chemistry. This latter structure likely simulates a dead-end complex in the reaction mixture. Temperature jump isotope-edited transient infrared studies on the binding of oxamate with LDH/NADH suggest that the evolution of the encounter complex between LDH/NADH and oxamate collapses via a branched reaction pathway to form the major and minor bound species. The production of the catalytically competent protein-substrate complex has strong similarities to kinetic pathways found in two-state protein folding processes. Once the encounter complex is formed between LDH/NADH and substrate, the ternary protein-ligand complex appears to “fold” to form a compact productive complex in an all or nothing like fashion with all the important molecular interactions coming together at the same time.     

Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including approximately 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.     

IL-2 complex treatment can protect naive mice from bacterial and viral infection

IL-2 complexes have substantial effects on the cellular immune system, and this approach is being explored for therapeutic application in infection and cancer. However, the impact of such treatments on subsequent encounter with pathogens has not been investigated. In this study, we report that naive mice treated with a short course of IL-2 complexes show enhanced protection from newly encountered bacterial and viral infections. IL-2 complex treatment expands both the NK and CD8 memory cell pool, including a recently described population of preexisting memory-phenotype T cells responsive to previously unencountered foreign Ags. Surprisingly, prolonged IL-2 complex treatment decreased CD8 T cell function and protective immunity. These data reveal the impact of cytokine complex treatment on the primary response to infection.     

Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy

Many biomolecular interactions proceed via a short-lived encounter state, consisting of multiple, lowly-populated species invisible to most experimental techniques. Recent development of paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) spectroscopy has allowed to directly visualize such transient intermediates in a number of protein-protein and protein-DNA complexes. Here we present an analysis of the recently published PRE NMR data for a protein complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). First, we describe a simple, general method to map out the spatial and temporal distributions of binding geometries constituting the Cc-CcP encounter state. We show that the spatiotemporal mapping provides a reliable estimate of the experimental coverage and, at higher coverage levels, allows to delineate the conformational space sampled by the minor species. To further refine the encounter state, we performed PRE-based ensemble simulations. The generated solutions reproduce well the experimental data and lie within the allowed regions of the encounter maps, confirming the validity of the mapping approach. The refined encounter ensembles are distributed predominantly in a region encompassing the dominant form of the complex, providing experimental proof for the results of classical theoretical simulations.     

Electron transfers amongst cytochrome f, plastocyanin and photosystem I: kinetics and mechanisms

The review covers the theory and practice of the determination of kinetic constants for the electron transfer reactions in chloroplast thylakoid membranes between plastocyanin and cytochrome f in cytochrome bf complexes, and between plastocyanin and the reaction centre of photosystem I. Effects of ionic strength and pH are featured. The contribution of mutant studies is included. It is concluded that nearly all data from in vitro experiments can be interpreted with a reaction scheme in which an encounter complex between donor and acceptor is formed by long-range electrostatic attraction, followed by rearrangement during which metal centres become close enough for rapid intra-complex electron transfer. In vivo experiments so far cast doubt on this particular sequence, but their interpretation is not straightforward. Means of modelling the bimolecular complex between cytochrome f and plastocyanin are outlined, and two likely structures are illustrated. The complex formed by plastocyanin and photosystem I in higher plants involves the PsaF subunit, but its structure has not been fully determined.     

Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo

DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.