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.

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant K of 20±4×10  M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Brownian dynamics computer simulations of the diffusional association of electron transport proteins cytochrome c (cyt c) and cytochrome c peroxidase (cyt c per) were performed. A highly detailed and realistic model of the protein structures and their electrostatic interactions was used that was based on an atomic-level spatial description. Several structural features played a role in enhancing and optimizing the electron transfer efficiency of this reaction. Favorable electrostatic interactions facilitated long-lived nonspecific encounters between the proteins that allowed the severe orientational criteria for reaction to be overcome by rotational diffusion during encounters. Thus a "reduction-in-dimensionality" effect operated. The proteins achieved plausible electron transfer orientations in a multitude of electrostatically stable encounter complexes, rather than in a single dominant complex.

The rapid association of barnase and its intracellular inhibitor barstar has been analysed from the effects of mutagenesis and electrostatic screening. A basal association rate constant of 10(5) M(-1) s(-1) is increased to over 5 x 10(9) M(-1) s(-1) by electrostatic forces. The association between the oppositely charged proteins proceeds through the rate-determining formation of an early, weakly specific complex, which is dominated by long-range electrostatic interactions, followed by precise docking to form the high affinity complex. This mode of binding is likely to be used widely in nature to increase association rate constants between molecules and its principles may be used for protein design.

The electrostatic enhancement of the association rate of barnase and barstar is calculated using a transition-state theory like expression and atomic-detail modeling of the protein molecules. This expression predicts that the rate enhancement is simply the average Boltzmann factor in the region of configurational space where association occurs instantaneously in the diffusion-controlled limit. Based on experimental evidence, this "transition state" is defined by configurations in which, relative to the stereospecifically bound complex, the two proteins are shifted apart by approximately 8 A (so a layer of water can be accommodated in the interface) and the two binding surfaces are rotated away by 0 degrees to 3 degrees. The values of the average Boltzmann factor, calculated by solving the Poisson-Boltzmann equation, for the wild-type complex and 16 complexes with single mutations are found to correlate well with experimental results for the electrostatic rate enhancement. The predicted rate enhancement is found to be somewhat insensitive to the precise definition of the transition state, due to the long-range nature of electrostatic interactions. The experimental ionic strength dependence of the rate enhancement is also reasonably reproduced.Copyright 1998 Academic Press Limited.

A protein design strategy was developed to specifically enhance the rate of association (k(on)) between a pair of proteins without affecting the rate of dissociation (k(off)). The method is based on increasing the electrostatic attraction between the proteins by incorporating charged residues in the vicinity of the binding interface. The contribution of mutations towards the rate of association was calculated using a newly developed computer algorithm, which predicted accurately the rate of association of mutant protein complexes relative to the wild type. Using this design strategy, the rate of association and the affinity between TEM1 beta-lactamase and its protein inhibitor BLIP was enhanced 250-fold, while the dissociation rate constant was unchanged. The results emphasize that long range electrostatic forces specifically alter k(on), but do not effect k(off). The design strategy presented here is applicable for increasing rates of association and affinities of protein complexes in general.

The process of protein-protein association starts with their random collision, which may develop into an encounter complex followed by a transition state and final complex formation. Here we aim to experimentally characterize the nature of the transition state of protein-protein association for three different protein-protein interactions; Barnase-Barstar, TEM1-BLIP and IFNalpha2-IFNAR2, and use the data to model the transition state structures. To model the transition state, we determined inter-protein distance-constraints of the activated complex by using double mutant cycles (DMC) assuming that interacting residues are spatially close. Significant DeltaDeltaG(double dagger)(int) values were obtained only between residues on Barnase and Barstar. However, introducing specific mutations that optimize the charge complementarity between BLIP and TEM1 resulted in the introduction of significant DeltaDeltaG(double dagger)(int) values also between residues of these two proteins. While electrostatic interactions make major contributions towards stabilizing the transition state, we show two examples where steric hindrance exerts an effect on the transition state as well. To model the transition-state structures from the experimental DeltaDeltaG(double dagger)(int) values, we introduced a method for structure perturbation, searching for those inter-protein orientations that best support the experimental DeltaDeltaG(double dagger)(int) values. Two types of transition states were found, specific as observed for Barnase-Barstar and the electrostatically optimized TEM1-BLIP mutants, and diffusive as shown for wild-type TEM1-BLIP and IFNalpha2-IFNAR2. The specific transition states are characterized by defined inter-protein orientations, which cannot be modeled for the diffusive transition states. Mutations introduced through rational design can change the transition state from diffusive to specific. Together, these data provide a structural view of the mechanism allowing rates of association to differ by five orders of magnitude between different protein complexes.

The transient complex of bovine myoglobin and cytochrome b(5) has been investigated using a combination of NMR chemical shift mapping, (15)N relaxation data, and protein docking simulations. Chemical shift perturbations observed for cytochrome b(5) amide resonances upon complex formation with either metmyoglobin (Fe(III)) or carbon monoxide-bound myoglobin (Fe(II)) are more than 10-fold smaller than in other transient redox protein complexes. From (15)N relaxation experiments, an increase in the overall correlation time of cytochrome b(5) in the presence of myoglobin is observed, confirming that complex formation is occurring. The chemical shift perturbations of proton and nitrogen amide nuclei as well as heme protons of cytochrome b(5) titrate with increasing myoglobin concentrations, also demonstrating the formation of a weak complex with a K(a) in the inverse millimolar range. The perturbed residues map over a wide surface area of cytochrome b(5), with patches of residues located around the exposed heme 6-propionate as well as at the back of the protein. The nature of the affected residues is mostly negatively charged contrary to perturbed residues in other transient complexes, which are mainly hydrophobic or polar. Protein docking simulations using the NMR data as constraints show several docking geometries both close to and far away from the exposed heme propionates of myoglobin. Overall, the data support the emerging view that this complex consists of a dynamic ensemble of orientations in which each protein constantly diffuses over the surface of the other. The characteristic NMR features may serve as a structural tool for the identification of such dynamic complexes.

The complexes of horse ferrous and ferric cytochrome c with Cd-substituted pea plastocyanin have been characterized by nuclear magnetic resonance, in order to determine the binding sites and to study the effects of complex formation. Reproducible, small chemical shift changes (0.005-0.05 ppm) were observed for protons in both proteins upon formation of a 1:1 complex. The chemical shift changes depended on the ratio of free to bound protein, with a binding constant of 1.0 +/- 0.5 x 10(5) M(-1), indicating that they were caused by complex formation and that free and bound proteins were in fast exchange. Two-dimensional spectra of the complex of ferrocytochrome c and plastocyanin were screened systematically for chemical shift changes. For about 760 protons, or 70% of the assigned protons in the two proteins, the chemical shift in the complex could be established. In plastocyanin and cytochrome c 14% and 17% of the protons, respectively, showed a significant chemical shift change. These protons form two groups. The first consists of a limited number of surface-exposed side-chain protons. These map on the so-called east side of plastocyanin and the front side of cytochrome c. This group of chemical shift changes is interpreted as representing direct effects of binding, and the respective surfaces thus represent the binding sites. The second group includes backbone amide protons and a few aliphatic and aromatic protons in the hydrophobic core of each protein. The chemical shift changes of this group are interpreted as secondary, i.e., caused by very small structural changes which are transmitted deep into the core of the protein. Ferric cytochrome c caused the same chemical shift effects in plastocyanin as the ferrous form; no intermolecular paramagnetic effects were observed. The small size of the chemical shifts and the absence of intermolecular paramagnetic shifts and NOEs suggest that the complex consists of a dynamic ensemble of structures which are in fast exchange, rather than a single static complex. This study shows that small, reproducible chemical shifts can be used effectively to characterize protein complexes in detail.

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.

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.© 2011 The Authors Journal compilation © 2011 FEBS.

Residual dipolar couplings observed in NMR spectra at very high magnetic fields have been measured to a high degree of accuracy for the paramagnetic protein cyanometmyoglobin. Deviations of these measurements from predictions based on available crystallographic and solution structures are largely systematic and well correlated within a given helix of this highly alpha-helical protein. These observations can be explained by invoking collective motion and small displacements of representative helices from their reported average positions in the solid state. Thus, the measurements appear to be capable of providing important insights into slower, collective protein motions, which are likely to be important for function, and which have been difficult to study using established experimental techniques.

Ligand residence times and binding rates have been found to be useful quantities to consider during drug design. The underlying structural and dynamic determinants of these kinetic quantities are difficult to discern. Driven by developments in computational hardware and simulation methodologies, molecular dynamics (MD) studies on full binding and unbinding pathways have emerged recently, showing these structural and dynamic determinants in atomic detail. However, the long timescales related to drug binding and release are still prohibitive to conventional MD simulation. Here we discuss a suite of enhanced sampling methods that have been applied to the study of full ligand binding or unbinding pathways, and reveal the kinetics of drug binding and/or release. We divide these sampling methods into three families (trajectory parallelization, metadynamics, and temperature- based methods), and discuss recent applications of each, as well as their basic theoretical underpinnings including how kinetic information is extracted. We then present an outlook for how the field could evolve, and how the rich variety of sampling methods discussed here can be leveraged in the future for computationally driven drug design.Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Molecular dynamics (MD) and related methods are close to becoming routine computational tools for drug discovery. Their main advantage is in explicitly treating structural flexibility and entropic effects. This allows a more accurate estimate of the thermodynamics and kinetics associated with drug-target recognition and binding, as better algorithms and hardware architectures increase their use. Here, we review the theoretical background of MD and enhanced sampling methods, focusing on free-energy perturbation, metadynamics, steered MD, and other methods most consistently used to study drug-target binding. We discuss unbiased MD simulations that nowadays allow the observation of unsupervised ligand-target binding, assessing how these approaches help optimizing target affinity and drug residence time toward improved drug efficacy. Further issues discussed include allosteric modulation and the role of water molecules in ligand binding and optimization. We conclude by calling for more prospective studies to attest to these methods' utility in discovering novel drug candidates.

In the last three decades, protein and nucleic acid structure determination and comprehension of the mechanisms, leading to their physiological and pathological functions, have become a cornerstone of biomedical sciences. A deep understanding of the principles governing the fates of cells and tissue at the molecular level has been gained over the years, offering a solid basis for the rational design of drugs aimed at the pharmacological treatment of numerous diseases. Historically, affinity indicators ( and IC/EC) have been assumed to be valid indicators of the efficacy of a drug. However, recent studies pointed out that the kinetics of the drug-receptor binding process could be as important or even more important than affinity in determining the drug efficacy. This eventually led to a growing interest in the characterisation and prediction of the rate constants of protein-ligand association and dissociation. For instance, a drug with a longer residence time can kinetically select a given receptor over another, even if the affinity for both receptors is comparable, thus increasing its therapeutic index. Therefore, understanding the molecular features underlying binding and unbinding processes is of central interest towards the rational control of drug binding kinetics. In this review, we report the theoretical framework behind protein-ligand association and highlight the latest advances in the experimental and computational approaches exploited to investigate the binding kinetics.

Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, detailed experimental characterization of the process by which such binding occurs has proven challenging. We conducted relatively long, unguided molecular dynamics simulations in which a ligand (the cancer drug dasatinib or the kinase inhibitor PP1) was initially placed at a random location within a box that also contained a protein (Src kinase) to which that ligand was known to bind. In several of these simulations, the ligand correctly identified its target binding site, forming a complex virtually identical to the crystallographically determined bound structure. The simulated trajectories provide a continuous, atomic-level view of the entire binding process, revealing persistent and noteworthy intermediate conformations and shedding light on the role of water molecules. The technique we employed, which does not assume any prior knowledge of the binding site's location, may prove particularly useful in the development of allosteric inhibitors that target previously undiscovered binding sites.

\n How drugs bind to their receptors—from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or “pose”—has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the\n β\n 1\n - and\n β\n 2\n -adrenergic receptors, initially making contact with a vestibule on each receptor’s extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug–receptor binding and unbinding rates.\n

The interaction between a ligand and a protein involves a multitude of conformational states. To achieve a particular deeply bound pose, the ligand must search across a rough free-energy landscape with many metastable minima. Creating maps of the ligand binding landscape is a great challenge, as binding and release events typically occur on timescales that are beyond the reach of molecular simulation. The WExplore enhanced sampling method is well suited to build these maps because it is designed to broadly explore free-energy landscapes and is capable of simulating ligand release pathways that occur on timescales as long as minutes. WExplore also uses only unbiased trajectory segments, allowing for the construction of Markov state models (MSMs) and conformation space networks that combine the results of multiple simulations. Here, we use WExplore to study two bromodomain-inhibitor systems using multiple docked starting poses (Brd4-MS436 and Baz2B-ICR7) and synthesize our results using a series of MSMs using time-lagged independent component analysis. Ranking the starting poses by exit rate agrees with the crystal structure pose in both cases. We also predict the most stable pose using the equilibrium populations from the MSM but find that the prediction is not robust as a function of MSM parameters. The simulated trajectories are synthesized into network models that visualize the entire binding landscape for each system, and we examine transition paths between deeply bound stable states. We find that, on average, transitions between deeply bound states convert through the unbound state 81% of the time, implying a trial-and-error approach to ligand binding. We conclude with a discussion of the implications of this result for both kinetics-based drug discovery and virtual screening pipelines that incorporate molecular dynamics.Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Plattner, Nuria; Noe, Frank Free Univ Berlin, Dept Math Comp Sci & Bioinformat, D-14195 Berlin, Germany.

This review consists of three major sections. The first and largest section reviews the protein constituents and known properties of the phosphotransferase systems present in well-studied Gram-positive bacteria. These bacteria include species of the following genera: (1) Staphylococcus, (2) Streptococcus, (3) Bacillus, (4) Lactobacillus, (5) Clostridium, (6) Arthrobacter, and (7) Brochothrix. The properties of the different systems are compared. The second major section deals with the regulation of carbohydrate uptake. There are four parts: (1) inhibition by intracellular sugar phosphates in Staphylococcus aureus, (2) PTS-mediated regulation of glycerol uptake in Bacillus subtilis, (3) competition for phospho-HPr in Streptococcus mutans, and (4) the possible involvement of protein kinases in the regulation of sugar uptake via the phosphotransferase system. The third section deals with the phenomenon of inducer expulsion. The first part is concerned with the physiological characterization of the phenomenon; then the consequences of unregulated uptake and expulsion, a futile cycle of energy expenditure, are considered. Finally, the biochemistry of the protein kinase and the protein phosphate phosphatase system, which appears to regulate sugar transport via the phosphotransferase system, is defined. The review, therefore, concentrates on the phosphotransferase system, its functions in carbohydrate transport and phosphorylation, the mechanisms of its regulation, and the mechanism by which it participates in the regulation of other physiological processes in the bacterial cell.

\n Protein–protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermolecular paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to lowly populated states in the fast exchange regime, to characterize the mechanistic details of the transient encounter complex interactions between the N-terminal domain of Enzyme I (EIN) and the histidine-containing phosphocarrier protein (HPr), two major bacterial signaling proteins. Experiments were conducted at an ionic strength of 150 mM NaCl to eliminate any spurious nonspecific associations not relevant under physiological conditions. By monitoring the dependence of the intermolecular transverse PRE (Γ\n 2\n ) rates measured on\n 15\n N-labeled EIN on the concentration of paramagnetically labeled HPr, two distinct types of encounter complex configurations along the association pathway are identified and dissected. The first class, which is in equilibrium with and sterically occluded by the specific complex, probably involves rigid body rotations and small translations near or at the active site. In contrast, the second class of encounter complex configurations can coexist with the specific complex to form a ternary complex ensemble, which may help EIN compete with other HPr binding partners in vivo by increasing the effective local concentration of HPr even when the active site of EIN is occupied.\n

The solution structure of the second protein-protein complex of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system, that between histidine-containing phosphocarrier protein (HPr) and glucose-specific enzyme IIA(Glucose) (IIA(Glc)), has been determined by NMR spectroscopy, including the use of dipolar couplings to provide long-range orientational information and newly developed rigid body minimization and constrained/restrained simulated annealing methods. A protruding convex surface on HPr interacts with a complementary concave depression on IIA(Glc). Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIA(Glc). Formation of the unphosphorylated complex, as well as the phosphorylated transition state, involves little or no change in the protein backbones, but there are conformational rearrangements of the interfacial side chains. Both HPr and IIA(Glc) recognize a variety of structurally diverse proteins. Comparisons with the structures of the enzyme I-HPr and IIA(Glc)-glycerol kinase complexes reveal how similar binding surfaces can be formed with underlying backbone scaffolds that are structurally dissimilar and highlight the role of redundancy and side chain conformational plasticity.

We announce the availability of the Xplor-NIH software package for NMR biomolecular structure determination. This package consists of the pre-existing XPLOR program, along with many NMR-specific extensions developed at the NIH. In addition to many features which have been developed over the last 20 years, the Xplor-NIH package contains an interface with a new programmatic framework written in C++. This interface currently supports the general purpose scripting languages Python and TCL, enabling rapid development of new tools, such as new potential energy terms and new optimization methods. Support for these scripting languages also facilitates interaction with existing external programs for structure analysis, structure manipulation, visualization, and spectral analysis.

Paramagnetic relaxation enhancement (PRE) measurements on (1)H nuclei have the potential to play an important role in NMR structure determination of macromolecules by providing unique long-range (10-35 A) distance information. Recent methodological advances for covalently attaching paramagnetic groups at specific sites on both proteins and nucleic acids have permitted the application of the PRE to various biological macromolecules. However, because artificially introduced paramagnetic groups are exposed to solvent and linked to the macromolecule by several freely rotatable bonds, they are intrinsically flexible. This renders conventional back-calculation of the (1)H-PRE using a single-point representation inaccurate, thereby severely limiting the utility of the (1)H-PRE as a tool for structure refinement. To circumvent these limitations, we have developed a theoretical framework and computational strategy with which to accurately back-calculate (1)H-PREs arising from flexible paramagnetic groups attached to macromolecules. In this scheme, the (1)H-PRE is calculated using a modified Solomon-Bloembergen equation incorporating a "model-free" formalism, based on a multiple-structure representation of the paramagnetic group in simulated annealing calculations. The ensemble approach for (1)H-PRE back-calculation was examined using several SRY/DNA complexes incorporating dT-EDTA-Mn(2+) at three distinct sites in the DNA, permitting a large data set comprising 435 experimental backbone and side-chain (1)H-PREs to be obtained in a straightforward manner from 2D through-bond correlation experiments. Calculations employing complete cross-validation demonstrate that the ensemble representation provides a means to accurately utilize backbone and side-chain (1)H-PRE data arising from a flexible paramagnetic group in structure refinement. The results of (1)H-PRE based refinement, in conjunction with previously obtained NMR restraints, indicate that significant gains in accuracy can be readily obtained. This is particularly significant in the case of macromolecular complexes where intermolecular translational restraints derived from nuclear Overhauser enhancement data may be limited.