Design of immunogens to elicit broadly neutralizing antibodies against HIV targeting the CD4 binding site.

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies.

Multiscale affinity maturation simulations to elicit broadly neutralizing antibodies against HIV.

The design of vaccines against highly mutable pathogens, such as HIV and influenza, requires a detailed understanding of how the adaptive immune system responds to encountering multiple variant antigens (Ags). Here, we describe a multiscale model of B cell receptor (BCR) affinity maturation that employs actual BCR nucleotide sequences and treats BCR/Ag interactions in atomistic detail. We apply the model to simulate the maturation of a broadly neutralizing Ab (bnAb) against HIV. Starting from a germline precursor sequence of the VRC01 anti-HIV Ab, we simulate BCR evolution in response to different vaccination protocols and different Ags, which were previously designed by us. The simulation results provide qualitative guidelines for future vaccine design and reveal unique insights into bnAb evolution against the CD4 binding site of HIV. Our model makes possible direct comparisons of simulated BCR populations with results of deep sequencing data, which will be explored in future applications.

Water dynamics around T0 vs R4 of hemoglobin from local hydrophobicity analysis.

The local hydration around tetrameric hemoglobin (Hb) in its T0 and R4 conformational substates is analyzed based on molecular dynamics simulations. Analysis of the local hydrophobicity (LH) for all residues at the α1ß2 and α2ß1 interfaces, responsible for the quaternary T → R transition, which is encoded in the Monod-Wyman-Changeux model, as well as comparison with earlier computations of the solvent accessible surface area, makes clear that the two quantities measure different aspects of hydration. Local hydrophobicity quantifies the presence and structure of water molecules at the interface, whereas "buried surface" reports on the available space for solvent. For simulations with Hb frozen in its T0 and R4 states, the correlation coefficient between LH and buried surface is 0.36 and 0.44, respectively, but it increases considerably if the 95% confidence interval is used. The LH with Hb frozen and flexible changes little for most residues at the interfaces but is significantly altered for a few select ones: Thr41α, Tyr42α, Tyr140α, Trp37ß, Glu101ß (for T0) and Thr38α, Tyr42α, Tyr140α (for R4). The number of water molecules at the interface is found to increase by ∼25% for T0 → R4, which is consistent with earlier measurements. Since hydration is found to be essential to protein function, it is clear that hydration also plays an essential role in allostery.

ppdx: Automated modeling of protein-protein interaction descriptors for use with machine learning.

This paper describes ppdx, a python workflow tool that combines protein sequence alignment, homology modeling, and structural refinement, to compute a broad array of descriptors for characterizing protein-protein interactions. The descriptors can be used to predict various properties of interest, such as protein-protein binding affinities, or inhibitory concentrations (IC50 ), using approaches that range from simple regression to more complex machine learning models. The software is highly modular. It supports different protocols for generating structures, and 95 descriptors can be currently computed. More protocols and descriptors can be easily added. The implementation is highly parallel and can fully exploit the available cores in a single workstation, or multiple nodes on a supercomputer, allowing many systems to be analyzed simultaneously. As an illustrative application, ppdx is used to parametrize a model that predicts the IC50 of a set of antigens and a class of antibodies directed to the influenza hemagglutinin stalk.

Structural basis for power stroke vs. Brownian ratchet mechanisms of motor proteins.

Two mechanisms have been proposed for the function of motor proteins: The power stroke and the Brownian ratchet. The former refers to generation of a large downhill free energy gradient over which the motor protein moves nearly irreversibly in making a step, whereas the latter refers to biasing or rectifying the diffusive motion of the motor. Both mechanisms require input of free energy, which generally involves the processing of an ATP (adenosine 5'-triphosphate) molecule. Recent advances in experiments that reveal the details of the stepping motion of motor proteins, together with computer simulations of atomistic structures, have provided greater insights into the mechanisms. Here, we compare the various models of the power stroke and the Brownian ratchet that have been proposed. The 2 mechanisms are not mutually exclusive, and various motor proteins employ them to different extents to perform their biological function. As examples, we discuss linear motor proteins Kinesin-1 and myosin-V, and the rotary motor F1-ATPase, all of which involve a power stroke as the essential element of their stepping mechanism.

Insights into the origin of the high energy-conversion efficiency of F1-ATPase.

Our understanding of the rotary-coupling mechanism of F1-ATPase has been greatly enhanced in the last decade by advances in X-ray crystallography, single-molecular imaging, and theoretical models. Recently, Volkán-Kacsó and Marcus [S. Volkán-Kacsó, R. A. Marcus, Proc. Natl. Acad. Sci. U.S.A. 112, 14230 (2015)] presented an insightful thermodynamic model based on the Marcus reaction theory coupled with an elastic structural deformation term to explain the observed γ-rotation angle dependence of the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) exchange rates of F1-ATPase. Although the model is successful in correlating single-molecule data, it is not in agreement with the available theoretical results. We describe a revision of the model, which leads to consistency with the simulation results and other experimental data on the F1-ATPase rotor compliance. Although the free energy liberated on ATP hydrolysis by F1-ATPase is rapidly dissipated as heat and so cannot contribute directly to the rotation, we show how, nevertheless, F1-ATPase functions near the maximum possible efficiency. This surprising result is a consequence of the differential binding of ATP and its hydrolysis products ADP and Pi along a well-defined pathway.

Structure of the EmrE multidrug transporter and its use for inhibitor peptide design.

Small multidrug resistance (SMR) pumps represent a minimal paradigm of proton-coupled membrane transport in bacteria, yet no high-resolution structure of an SMR protein is available. Here, atomic-resolution structures of the Escherichia coli efflux-multidrug resistance E (EmrE) multidrug transporter in ligand-bound form are refined using microsecond molecular dynamics simulations biased using low-resolution data from X-ray crystallography. The structures are compatible with existing mutagenesis data as well as NMR and biochemical experiments, including pKas of the catalytic glutamate residues and the dissociation constant ([Formula: see text]) of the tetraphenylphosphonium+ cation. The refined structures show the arrangement of residue side chains in the EmrE active site occupied by two different ligands and in the absence of a ligand, illustrating how EmrE can adopt structurally diverse active site configurations. The structures also show a stable, well-packed binding interface between the helices H4 of the two monomers, which is believed to be crucial for EmrE dimerization. Guided by the atomic details of this interface, we design proteolysis-resistant stapled peptides that bind to helix H4 of an EmrE monomer. The peptides are expected to interfere with the dimerization and thereby inhibit drug transport. Optimal positions of the peptide staple were determined using free-energy simulations of peptide binding to monomeric EmrE Three of the four top-scoring peptides selected for experimental testing resulted in significant inhibition of proton-driven ethidium efflux in live cells without nonspecific toxicity. The approach described here is expected to be of general use for the design of peptide therapeutics.

Estimation of the breadth of CD4bs targeting HIV antibodies by molecular modeling and machine learning.

HIV is a highly mutable virus for which all attempts to develop a vaccine have been unsuccessful. Nevertheless, few long-infected patients develop antibodies, called broadly neutralizing antibodies (bnAbs), that have a high breadth and can neutralize multiple variants of the virus. This suggests that a universal HIV vaccine should be possible. A measure of the efficacy of a HIV vaccine is the neutralization breadth of the antibodies it generates. The breadth is defined as the fraction of viruses in the Seaman panel that are neutralized by the antibody. Experimentally the neutralization ability is measured as the half maximal inhibitory concentration of the antibody (IC50). To avoid such time-consuming experimental measurements, we developed a computational approach to estimate the IC50 and use it to determine the antibody breadth. Given that no direct method exists for calculating IC50 values, we resort to a combination of atomistic modeling and machine learning. For each antibody/virus complex, an all-atoms model is built using the amino acid sequence and a known structure of a related complex. Then a series of descriptors are derived from the atomistic models, and these are used to train a Multi-Layer Perceptron (an Artificial Neural Network) to predict the value of the IC50 (by regression), or if the antibody binds or not to the virus (by classification). The neural networks are trained by use of experimental IC50 values collected in the CATNAP database. The computed breadths obtained by regression and classification are reported and the importance of having some related information in the data set for obtaining accurate predictions is analyzed. This approach is expected to prove useful for the design of HIV bnAbs, where the computation of the potency must be accompanied by a computation of the breadth, and for evaluating the efficiency of potential vaccination schemes developed through modeling and simulation.

A restrained locally enhanced sampling method (RLES) for finding free energy minima in complex systems.

We present an extension of the locally enhanced sampling method. A restraint potential is introduced to drive the many-replica system to the canonical ensemble corresponding to the physical, single-replica system. Convergence properties are demonstrated using a model rugged two-dimensional potential, for which sampling by conventional equilibrium molecular dynamics is inefficient. Restrained locally enhanced sampling (RLES) is found to explore the space of configurations with an efficiency comparable to that of temperature replica exchange. To demonstrate the potential of RLES for realistic applications, the method is used to fold the 12-residue tryptophan zipper miniprotein in explicit solvent. The RLES algorithm can be incorporated into existing LES implementations with minor code modifications.

Trapping the ATP binding state leads to a detailed understanding of the F1-ATPase mechanism.

The rotary motor enzyme FoF1-ATP synthase uses the proton-motive force across a membrane to synthesize ATP from ADP and Pi (H2PO4(-)) under cellular conditions that favor the hydrolysis reaction by a factor of 2 × 10(5). This remarkable ability to drive a reaction away from equilibrium by harnessing an external force differentiates it from an ordinary enzyme, which increases the rate of reaction without shifting the equilibrium. Hydrolysis takes place in the neighborhood of one conformation of the catalytic moiety F1-ATPase, whose structure is known from crystallography. By use of molecular dynamics simulations we trap a second structure, which is rotated by 40° from the catalytic dwell conformation and represents the state associated with ATP binding, in accord with single-molecule experiments. Using the two structures, we show why Pi is not released immediately after ATP hydrolysis, but only after a subsequent 120° rotation, in agreement with experiment. A concerted conformational change of the α3ß3 crown is shown to induce the 40° rotation of the γ-subunit only when the ßE subunit is empty, whereas with Pi bound, ßE serves as a latch to prevent the rotation of γ. The present results provide a rationalization of how F1-ATPase achieves the coupling between the small changes in the active site of ßDP and the 40° rotation of γ.

A gating mechanism of pentameric ligand-gated ion channels.

Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communication in the nervous system and are involved in fundamental processes such as attention, learning, and memory. They are oligomeric protein assemblies that convert a chemical signal into an ion flux through the postsynaptic membrane, but the molecular mechanism of gating ions has remained elusive. Here, we present atomistic molecular dynamics simulations of the prokaryotic channels from Gloeobacter violaceus (GLIC) and Erwinia chrysanthemi (ELIC), whose crystal structures are thought to represent the active and the resting states of pLGICs, respectively, and of the eukaryotic glutamate-gated chloride channel from Caenorhabditis elegans (GluCl), whose open-channel structure was determined complexed with the positive allosteric modulator ivermectin. Structural observables extracted from the trajectories of GLIC and ELIC are used as progress variables to analyze the time evolution of GluCl, which was simulated in the absence of ivermectin starting from the structure with bound ivermectin. The trajectory of GluCl with ivermectin removed shows a sequence of structural events that couple agonist unbinding from the extracellular domain to ion-pore closing in the transmembrane domain. Based on these results, we propose a structural mechanism for the allosteric communication leading to deactivation/activation of the GluCl channel. This model of gating emphasizes the coupling between the quaternary twisting and the opening/closing of the ion pore and is likely to apply to other members of the pLGIC family.

Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme.

How living systems detect the presence of genotoxic damage embedded in a million-fold excess of undamaged DNA is an unresolved question in biology. Here we have captured and structurally elucidated a base-excision DNA repair enzyme, MutM, at the stage of initial encounter with a damaged nucleobase, 8-oxoguanine (oxoG), nested within a DNA duplex. Three structures of intrahelical oxoG-encounter complexes are compared with sequence-matched structures containing a normal G base in place of an oxoG lesion. Although the protein-DNA interfaces in the matched complexes differ by only two atoms-those that distinguish oxoG from G-their pronounced structural differences indicate that MutM can detect a lesion in DNA even at the earliest stages of encounter. All-atom computer simulations show the pathway by which encounter of the enzyme with the lesion causes extrusion from the DNA duplex, and they elucidate the critical free energy difference between oxoG and G along the extrusion pathway.

Strandwise translocation of a DNA glycosylase on undamaged DNA.

Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.

Enhanced chemical synthesis at soft interfaces: a universal reaction-adsorption mechanism in microcompartments.

A bimolecular synthetic reaction (imine synthesis) was performed compartmentalized in micrometer-diameter emulsion droplets. The apparent equilibrium constant (Keq) and apparent forward rate constant (k1) were both inversely proportional to the droplet radius. The results are explained by a noncatalytic reaction-adsorption model in which reactants adsorb to the droplet interface with relatively low binding energies of a few kBT, react and diffuse back to the bulk. Reaction thermodynamics is therefore modified by compartmentalization at the mesoscale--without confinement on the molecular scale--leading to a universal mechanism for improving unfavorable reactions.

Investigations of α-helix↔ß-sheet transition pathways in a miniprotein using the finite-temperature string method.

A parallel implementation of the finite-temperature string method is described, which takes into account the invariance of coordinates with respect to rigid-body motions. The method is applied to the complex α-helix↔ß-sheet transition in a ß-hairpin miniprotein in implicit solvent, which exhibits much of the complexity of conformational changes in proteins. Two transition paths are considered, one derived from a linear interpolant between the endpoint structures and the other derived from a targeted dynamics simulation. Two methods for computing the conformational free energy (FE) along the string are compared, a restrained method, and a tessellation method introduced by E. Vanden-Eijnden and M. Venturoli [J. Chem. Phys. 130, 194103 (2009)]. It is found that obtaining meaningful free energy profiles using the present atom-based coordinates requires restricting sampling to a vicinity of the converged path, where the hyperplanar approximation to the isocommittor surface is sufficiently accurate. This sampling restriction can be easily achieved using restraints or constraints. The endpoint FE differences computed from the FE profiles are validated by comparison with previous calculations using a path-independent confinement method. The FE profiles are decomposed into the enthalpic and entropic contributions, and it is shown that the entropy difference contribution can be as large as 10 kcal/mol for intermediate regions along the path, compared to 15-20 kcal/mol for the enthalpy contribution. This result demonstrates that enthalpic barriers for transitions are offset by entropic contributions arising from the existence of different paths across a barrier. The possibility of using systematically coarse-grained representations of amino acids, in the spirit of multiple interaction site residue models, is proposed as a means to avoid ad hoc sampling restrictions to narrow transition tubes.

Unsuspected pathway of the allosteric transition in hemoglobin.

Large conformational transitions play an essential role in the function of many proteins, but experiments do not provide the atomic details of the path followed in going from one end structure to the other. For the hemoglobin tetramer, the transition path between the unliganded (T) and tetraoxygenated (R) structures is not known, which limits our understanding of the cooperative mechanism in this classic allosteric system, where both tertiary and quaternary changes are involved. The conjugate peak refinement algorithm is used to compute an unbiased minimum energy path at atomic detail between the two end states. Although the results confirm some of the proposals of Perutz [Perutz MF (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228:726-734], the subunit motions do not follow the textbook description of a simple rotation of one αß-dimer relative to the other. Instead, the path consists of two sequential quaternary rotations, each involving different subdomains and axes. The quaternary transitions are preceded and followed by phases of tertiary structural changes. The results explain the recent photodissociation measurements, which suggest that the quaternary transition has a fast (2 µs) as well as a slow (20 µs) component and provide a testable model for single molecule FRET experiments.

Hemoglobin Bohr effects: atomic origin of the histidine residue contributions.

The Bohr effect in hemoglobin, which refers to the dependence of the oxygen affinity on the pH, plays an important role in its cooperativity and physiological function. The dominant contribution to the Bohr effect arises from the difference in the pKa values of His residues of the unliganded (deoxy) and liganded (carbonmonoxy) structures. Using recent high resolution structures, the residue pKa values corresponding to the two structures are calculated. The method is based on determining the electrostatic interactions between residues in the protein, relative to those of the residue in solution, by use of the linearized finite difference Poisson-Boltzmann equation and Monte Carlo sampling of protonation states. Given that good agreement is obtained with the available experimental values for the contribution of His residues in HbA to the Bohr effect, the calculated results are used to determine the atomic origin of the pKa shift between deoxy and carbonmonoxy HbA. The contributions to the pKa shift calculated by means of the linear response approximation show that the salt bridge involving His146 plays an important role in the alkaline Bohr effect, as suggested by Perutz but that other interactions are significant as well. A corresponding analysis is made for the contribution of His143 to the acid Bohr effect for which there is no proposed explanation. The method used is summarized and the program by which it is implemented is described in the Appendix .

Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1.

A poorly understood aspect of DNA repair proteins is their ability to identify exceedingly rare sites of damage embedded in a large excess of nearly identical undamaged DNA, while catalyzing repair only at the damaged sites. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Trapping and analyzing such DNA-protein complexes is particularly difficult in the case of base extrusion DNA repair proteins because of the complexity of the repair reaction, which involves extrusion of the target base from DNA followed by its insertion into the active site where glycosidic bond cleavage is catalyzed. Here we report the structure of a human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, in which a normal guanine from DNA has been forcibly inserted into the enzyme active site. Although the interactions of the nucleobase with the active site are only subtly different for G versus oxoG, hOGG1 fails to catalyze excision of the normal nucleobase. This study demonstrates that even if hOGG1 mistakenly inserts a normal base into its active site, the enzyme can still reject it on the basis of catalytic incompatibility.

Behind the folding funnel diagram.

This Commentary clarifies the meaning of the funnel diagram, which has been widely cited in papers on protein folding. To aid in the analysis of the funnel diagram, this Commentary reviews historical approaches to understanding the mechanism of protein folding. The primary role of free energy in protein folding is discussed, and it is pointed out that the decrease [corrected] in the configurational entropy as the native state is approached hinders folding, rather than guiding it. Diagrams are introduced that provide a less ambiguous representation of the factors governing the protein folding reaction than the funnel diagram.

A hierarchy of timescales in protein dynamics is linked to enzyme catalysis.

The synergy between structure and dynamics is essential to the function of biological macromolecules. Thermally driven dynamics on different timescales have been experimentally observed or simulated, and a direct link between micro- to milli-second domain motions and enzymatic function has been established. However, very little is understood about the connection of these functionally relevant, collective movements with local atomic fluctuations, which are much faster. Here we show that pico- to nano-second timescale atomic fluctuations in hinge regions of adenylate kinase facilitate the large-scale, slower lid motions that produce a catalytically competent state. The fast, local mobilities differ between a mesophilic and hyperthermophilic adenylate kinase, but are strikingly similar at temperatures at which enzymatic activity and free energy of folding are matched. The connection between different timescales and the corresponding amplitudes of motions in adenylate kinase and their linkage to catalytic function is likely to be a general characteristic of protein energy landscapes.