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Dynamic binding of PKA regulatory subunit RIα
Justin Gullingsrud, Choel Kim, Susan S. Taylor and J. Andrew McCammon
Recent crystal structures have revealed that regulatory subunit RIα of PKA undergoes a dramatic conformational change upon complex formation with the catalytic subunit. Molecular dynamics studies were initiated to elucidate the contributions of intrinsic conformational flexibility and interactions with the catalytic subunit in formation and stabilization of the complex. Simulations of a single RIα nucleotide-binding domain (NBD), missing cAMP, showed that its C helix spontaneously occupies two distinct conformations: either packed against the nucleotide binding domain as in its cAMP-bound structure, or extended into an intermediate form resembling that of the holoenzyme structure. C helix extension was not seen in a simulation of both RIα NBDs. In a model complex containing both NBDs and the catalytic subunit, well-conserved residues at the interface between the NBDs in the cAMP-bound form were found to stabilize the complex through contacts with the catalytic subunit. The model structure is consistent with available experimental data.
Molecular Dynamics: A Survey of Methods for Simulating the Activity of Proteins
Stewart A. Adcock and J. Andrew McCammon
This review offers an outline of the origin of molecular dynamics simulation for protein systems and how it has developed into a robust and trusted tool. This review then covers more recent advances in theory and an illustrative selection of practical studies in which it played a central role. The range of studies in which MD has played a considerable or pivotal role is immense, and this review cannot do justice to them. Particular emphasis will be placed on the study of dynamic aspects of protein recognition, an area where molecular dynamics has scope to provide broad and far-ranging insights. This review concludes with a brief discussion of the future potential offered to advancement of the biological and biochemical sciences and the remaining issues that must be overcome to allow the full extent of this potential to be realized.
Channel Opening Motion of α7 Nicotinic Acetylcholine Receptor as Suggested by Normal Mode Analysis
Xiaolin Cheng, Benzhuo Lu, Barry Grant, Richard J. Law and J. Andrew McCammon
The gating motion of the human nicotinic acetylcholine receptor (nAChR) α7 was investigated with normal mode analysis (NMA) of two homology models. The first model, referred to as model I, was built from both the Lymnaea stagnalis acetylcholine binding protein (AChBP) and the transmembrane (TM) domain of the Torpedo marmorata nAChR. The second model, referred to as model C, was based solely on the recent electron microscopy structure of the T. marmorata nAChR. Despite structural differences, both models exhibit nearly identical patterns of flexibility and correlated motions. In addition, both models show a similar global twisting motion that may represent channel gating. The similar results obtained for the two models indicate that NMA is most sensitive to the contact topology of the structure rather than its finer detail. The major difference between the low-frequency motions sampled for the two models is that a symmetrical pore-breathing motion, favoring channel opening, is present as the second most dominant motion in model I, whilst largely absent from model C. The absence of this mode in model C can be attributed to its less symmetrical architecture. Finally, as a further goal of the present study, an approximate open channel model, consistent with many experimental findings, has been produced.
Restrained Molecular Dynamics Simulations of HIV-1 Protease: Validating a New Target for Drug Design
Alexander L. Perryman, Jung-Hsin Lin and J. Andrew McCammon
To test the anti-correlated relationship that was recently displayed in conventional Molecular Dynamics (MD) simulations, several different restrained MD simulations on a wild type and on the V82F/I84V drug-resistant mutant of HIV-1 protease were performed. This anticorrelated relationship refers to the observation that compression of the peripheral ear-to-cheek region of HIV protease (i.e., the elbow of the flap to the fulcrum and the cantilever) occurred as the active site flaps were opening, and, conversely, expansion of that ear-to-cheek region occurred as both flaps were closing. An additional examination of this anti-correlated relationship was necessary to determine whether it can be harnessed in a useful manner. Consequently, six different MD experiments were performed that incorporated pair-wise distance restraints in that ear-to-cheek region (i.e., the distance between the α-carbons of Gly40 and Gln61 was restrained to either 7.7 or 10.5 Angstroms, in both monomers). Pushing the backbones of the ear and the cheek regions away from each other slightly did force the flaps that guard the active site to remain closed in both the wild type and the mutant systems--even though there were no ligands in the active sites. Thus, these restrained MD simulations provided evidence that the anti-correlated relationship can be exploited to affect the dynamic behavior of the flaps that guard the active site of HIV-1 protease. These simulations supported our hypothesis of the mechanism governing flap motion, and they are the first step towards validating that peripheral surface as a new target for drug design.
Increased Membrane Affinity of the C1 Domain of Protein Kinase Cδ Compensates for the Lack of Involvement of its C2 Domain in Membrane Recruitment
Jennifer R. Giorgione, Jung-Hsin Lin, J. Andrew McCammon and Alexandra C. Newton
Protein kinase C (PKC) family members are allosterically activated following membrane recruitment by specific membrane-targeting modules. Conventional PKC isozymes are recruited to membranes by two such modules: a C1 domain, which binds diacylglycerol (DAG), and a C2 domain, which is a Ca2+-triggered phospholipid-binding module. In contrast, novel PKC isozymes respond only to DAG, despite the presence of a C2 domain. Here, we address the molecular mechanism of membrane recruitment of the novel isozyme PKCδ. We show that PKCδ and a conventional isozyme, PKCβII, bind membranes with comparable affinities. However, dissection of the contribution of individual domains to this binding revealed that, although the C2 domain is a major determinant in driving the interaction of PKCβII with membranes, the C2 domain of PKCδ does not bind membranes. Instead, the C1B domain is the determinant that drives the interaction of PKCδ with membranes. The C2 domain also does not play any detectable role in the activity or subcellular location of PKCδ in cells; in vivo imaging studies revealed that deletion of the C2 domain does not affect the stimulus-dependent translocation or activity of PKCδ. Thus, the increased affinity of the C1 domain of PKCδ allows this isozyme to respond to DAG alone, whereas conventional PKC isozymes require the coordinated action of Ca2+ binding to the C2 domain and DAG binding to the C1 domain for activation.
Electrostatic Properties of Cowpea Chlorotic Mottle Virus and Cucumber Mosaic Virus Capsids
Robert Konecny, Joanna Trylska, Florence Tama, Deqiang Zhang, Nathan A. Baker, Charles L. Brooks III and J.A. McCammon
Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca2+ can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca2+ ions can induce the capsid contraction and stabilize the native form.
Conformational Transitions in Protein-Protein Association: Binding of Fasciculin-2 to Acetylcholinesterase
Jennifer M. Bui, Zoran Radić, Palmer Taylor and J. Andrew McCammon
The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.
E230Q Mutation of the Catalytic Subunit of cAMP-dependent Protein Kinase Affects Local Structure and the Binding of Peptide Inhibitor
Man-Un Ung, Benzhuo Lu and J.A. McCammon
The active site of the mammalian cAMP-dependent protein kinase catalytic subunit (C-subunit) has a cluster of non-conserved acidic residues -- Glu127, Glu170, Glu203, Glu230 and Asp241 -- that are crucial for substrate recognition and binding. Studies showed that the Glu230 to Gln mutant (E230Q) of the enzyme had physical properties similar to the wild-type enzyme and had decreased affinity for a short peptide substrate, Kemptide. However, recent experiments intended to crystallize ternary complex of the E230Q mutant with MgATP and protein kinase inhibitor (PKI) could only obtain crystals of the apo-enzyme of E230Q mutant. To deduce the possible mechanism that prevented ternary complex formation, we used the relaxed-complex method [J.-H. Lin, A.L. Perryman, J.R. Schames, and J.A. McCammon, J. Amer. Chem. Soc., 2002, vol. 24, pp. 5632-5633] to study PKI binding to the E230Q mutant C-subunit. In the E230Q mutant, we observed local structural changes of the peptide binding site that correlated closely to the reduced PKI affinity. The structural changes occurred in the F-to-G helix loop and appeared to hinder PKI binding. Reduced electrostatic potential repulsion among Asp241 from the helix loop section and the other acidic residues in the peptide binding site appear to be responsible for the structural change.
A Simple Electrostatic Switch Important in the Activation of Type I Protein Kinase A By Cyclic AMP
Dominico Vigil, Jung-Hsin Lin, Christoph A. Sotriffer, Juniper K. Pennypacker, J. Andrew McCammon, Susan S. Taylor
Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RIα R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.
Dependency Map of Proteins in the Small Ribosomal Subunit
Kay Hamacher, Joanna Trylska and J. Andrew McCammon
The assembly of the ribosome has recently become an interesting target for antibiotics in several bacteria. In this work, we extended an analytical procedure to determine native state fluctuations and contact breaking to investigate the protein stability dependence in the 30S small ribosomal subunit of Thermus thermophilus. We determined the causal influence of the presence and absence of proteins in the 30S complex on the binding free energies of other proteins. The predicted dependencies are in overall agreement with the experimentally determined assembly map for another organism, Escherichia coli. We found that the causal influences result from two distinct mechanisms, one is pure internal energy change, the other originates from the entropy change. We discuss the implications on how to target the ribosomal assembly most effectively by suggesting six proteins as targets for mutations or other hindering of their binding. Our results show that by blocking one out of this set of proteins, the association of other proteins is eventually reduced, thus reducing the translation efficiency even more. We could additionally determine the binding dependency of THX -- a peptide not present in the ribosome of E. coli -- and suggest its assembly path.
Coupling hydrophobic, dispersion, and electrostatic contributions in continuum solvent models
J. Dzubiella, J.M.J. Swanson and J.A. McCammon
An implicit solvent model is presented that couples hydrophobic, dispersion, and electrostatic solvation energies by minimizing the system Gibbs free energy with respect to the solvent volume exclusion function. The solvent accessible surface is output of the theory. The method is illustrated with the solvation of simple solutes on different length scales and captures the sensitivity of hydration to the particular form of solute-solvent interactions in agreement with recent computer simulations.
How does Activation Loop Phosphorylation Modulate Catalytic Activity in the cAMP-dependent Protein Kinase: A Theoretical Study
Yuhui Cheng, Yingkai Zhang and J. Andrew McCammon
Phosphorylation mediates the function of many proteins and enzymes. In the catalytic subunit of cAMP-dependent protein kinase, phosphorylation of Thr 197 in the activation loop strongly influences its catalytic activity. In order to provide theoretical understanding about this important regulatory process, classical molecular dynamics simulations and ab initio QM/MM calculations have been carried out on the wild-type PKA-Mg2 ATP-substrate complex and its dephosphorylated mutant, T197A. It was found that pThr 197 not only facilitates the phosphoryl transfer reaction by stabilizing the transition state through electrostatic interactions but also strongly affects its essential protein dynamics as well as the active site conformation.
Characterization of Nonbiological Antimicrobial Polymers in Aqueous Solution and at Water-Lipid Interfaces from All-Atom Molecular Dynamics
Ivaylo Ivanov, Satyavani Vemparala, Vojislava Pophristic, Kenichi Kuroda, William F. DeGrado, J. Andrew McCammon and Michael L. Klein
We have applied molecular dynamics to investigate the structural properties and activity of recently synthesized amphiphilic polymethacrylate derivatives, designed to mimic the antimicrobial activity of natural peptides. The composition, molecular weight, and hydrophobicity (ratio of hydrophobic and cationic units) of these short copolymers can be modulated to achieve structural diversity, which is crucial in controlling the antimicrobial activity. We have carried out all-atom molecular dynamics to systematically investigate the conformations adopted by these copolymers in water and at the water- lipid interface as a function of sequence and the chemical nature of the monomers. For two sequences, we observe partial insertion into the bilayer. Formation of strong interactions between the lipid headgroups and the amine groups of the polymers assists in the initial association with the lipids. However, the primary driving force for the observed partial insertion appears to be the hydrophobic effect. Our results indicate sensitive dependence of the overall shape on the sequence, suggesting that experimentally observed changes in activity can be correlated with particular sequences, providing an avenue for rational design.
Coupling nonpolar and polar solvation free energies in implicit solvent models
J. Dzubiella, J.M.J. Swanson and J.A. McCammon
Recent studies on the solvation of atomistic and nanoscale solutes indicate that a strong coupling exists between the hydrophobic, dispersion, and electrostatic contributions to the solvation free energy, a facet not considered in current implicit solvent models. We suggest a theoretical formalism which accounts for coupling by minimizing the Gibbs free energy of the solvent with respect to a solvent volume exclusion function. The resulting differential equation is similar to the Laplace-Young equation for the geometrical description of capillary interfaces, but is extended to microscopic scales by explicitly considering curvature corrections as well as dispersion and electrostatic contributions. Unlike existing implicit solvent approaches, the solvent accessible surface is an output of our model. The presented formalism is illustrated on spherically or cylindrically symmetrical systems of neutral or charged solutes on different length scales. The results are in agreement with computer simulations and, most importantly, demonstrate that our method captures the strong sensitivity of solvent expulsion and dewetting to the particular form of the solvent-solute interactions.
Potentials of Mean Force for Acetylcholine Unbinding from the α7 Nicotinic Acetylcholine Receptor Ligand-Binding Domain
Deqiang Zhang, Justin Gullingsrud and J. Andrew McCammon
The nicotinic acetylcholine receptor is a prototype ligand-gated ion channel that mediates signal transduction in the neuromuscular junctions and other cholinergic synapses. The molecular basis for the energetics of ligand binding and unbinding is critical to our understanding of the pharmacology of this class of receptors. Here, we used steered molecular dynamics to investigate the unbinding of acetylcholine from the ligand-binding domain of human α7 nicotinic acetylcholine receptor along four different predetermined pathways. Pulling forces were found to correlate well with interactions between acetylcholine and residues in the binding site during the unbinding process. From multiple trajectories along these unbinding pathways, we calculated the potentials of mean force for acetylcholine unbinding. Four available methods based on Jarzynski's equality were used and compared for their efficiencies. The most probable pathway was identified to be along a direction approximately parallel to the membrane. The derived binding energy for acetylcholine was in good agreement with that derived from the experimental binding constant for acetylcholine binding protein, but significantly higher than that for the complete human α7 nicotinic acetylcholine receptor. In addition, it is likely that several intermediate states exist along the unbinding pathways.
Gated Binding of Ligands to HIV-1 Protease: Brownian Dynamics Simulations in a Course-Grained Model
Chia-En Chang, Tongye Shen, Joanna Trylska, Valentina Tozzini and J. Andrew McCammon
The internal motions of proteins may serve as a "gate" in some systems, which controls ligand-protein association. This study applies Brownian dynamics simulations in a coarse-grained model to study the gated association rate constants of HIV-1 proteases and drugs. The computed gated association rate constants of three protease mutants, G48V/V82A/I84V/L90M, G48V and L90M with three drugs, amprenavir, indinavir and saquinavir, yield good agreements with experiments. The work shows that the flap dynamics leads to "slow gating". The simulations suggest that the flap flexibility and the opening frequency of the wild-type, the G48V and L90M mutants are similar, but the flaps of the variant G48V/V82A/I84V/L90M open less frequently, resulting in a lower gated rate constant. The developed methodology is fast and provides an efficient way to predict the gated association rate constants for various protease mutants and ligands.
A Minimal Model for Stabilization of Biomolecules by Hydrocarbon Cross-linking
K. Hamacher, A. Hübsch and J.A. McCammon
Programmed cell death regulating protein motifs play an essential role in the development of an organism, its immune response and disease-related cellular mechanisms. Among those motifs the BH3-domain of the BCL-2 family is found to be of crucial importance. Recent experiments showed how the isolated, otherwise unstructured BH3-peptide can be modified by a hydrocarbon linkage to regain function. We parametrized a reduced, dynamic model for the stability effects of such covalent cross-linking and confirmed that the model reproduces the reinforcement of the structural stability of the BH3 motif by cross-linking. We show that an analytically solvable model for thermostability around the native state is not capable of reproducing the stabilization effect. This points to the crucial importance of the peptide dynamics and the fluctuaions neglected in the analytic model for the cross-linking system to function properly. This conclusion is supported by a through analysis of a simulated Go-model. The resulting model is suitable for rational design of generic cross-linking systems in silicio.
Mapping All-Atom Models onto One-Bead Coarse-Grained Models: General Properties and Applications to a Minimal Polypeptide Model
Valentina Tozzini, Walter Rocchia and J. Andrew McCammon
In the one- and two-bead coarse-grained (CG) models for proteins, the two conformational dihedrals φ and ψ that describe the backbone geometry are no longer present as explicit internal coordinates; thus, the information contained in the Ramachandran plot cannot be used directly. We derive an analytical mapping between these dihedrals and the internal variable describing the backbone conformation in the one- (two-) bead CG models, namely, the pseudo-bond angle and pseudo-dihedral between subsequent Cα's. This is used to derive a new density plot that contains the same information as the Ramachandran plot and can be used with the one- (two-) bead CG models. The use of this mapping is then illustrated with a new one-bead polypeptide model that accounts for transitions between α helices and β sheets.
Elasticity of peptide omega bonds
Tongye Shen, Donald Hamelberg and J. Andrew McCammon
We calculated the changes of the free energy profile of the peptidyl-prolyl torsional angle of the dipeptide valine-proline under pulling forces by simulations. Using a dynamic model built on the equilibrium properties of this system and previously studied dynamic properties of cis-trans isomerization of other dipeptides, we calculated the dynamic viscoelasticity of this degree of freedom. The results show significant differences between how thermal and mechanical forces alter the equilibrium and the dynamics of the isomerization transition. The former does not change the barrier heights but changes the prefactor of the kinetics owing to temperature effects, while the latter changes minima and thus the population. The force that is required to "excite" this degree of freedom is small. Compared to other systems, we found that this degree of freedom becomes already quite rigid at several Hertz, which is a much lower value due to the high barrier of the cis-trans isomerization. We also found that the tensile elastic modulus of densely packed omega bonds is at the order of GPa, which is comparable to that of polymer materials. These results give mechanical properties of polyproline elasticity of a local nature and provide guidance for future experimental designs.
Computing the amino acid specificity of fluctuations in biomolecular systems
K. Hamacher and J.A. McCammon
We developed a new amino acid specific method for the computation of spatial fluctuations of proteins around their native structures. We show the consistency with experimental values and the increased performance in comparison to an established model, based on statistical estimates for a set of test proteins. We apply the new method to HIV-1 protease in its wild-type-form and to a V82F-I84V-mutant that shows resistance to protease-inhibitors. We further show how the method can be successfully used to explain the molecular biophysics of drug resistance of the mutant.
Accelerating Conformational Transitions in Biomolecular Systems
Donald Hamelberg and J. Andrew McCammon
Molecular dynamics simulation is one of the most extensively used biophysical tools available to computational biologists and chemists due to its ability to accurately sample the conformational space of molecular systems. By integrating Newton's equations of motion, this technique evaluates the time-dependent behavior and evolution of a molecular system as it samples its conformational space. Therefore, with an accurate representation of the system's potential energy landscape, the thermodynamic and kinetic properties can be calculated while studying a host of other structural and dynamic phenomena.
The Influence of Macromolecular Crowding on HIV-1 Protease Internal Dynamics
David D.L. Minh, Chia-En Chang, Joanna Trylska, Valentina Tozzini and J. Andrew McCammon
High macromolecular concentrations, or crowded conditions, have been shown to affect a wide variety of molecular processes, including diffusion, association and dissociation, and protein folding and stability. Here, we model the effect of macromolecular crowding on the internal dynamics of a protein, HIV-1 protease, using Brownian dynamics simulations. HIV-1 protease possesses a pair of flaps which are postulated to open in the early stages of its catalytic mechanism. Compared to low concentrations, close-packed concentrations of repulsive crowding agents are found to significantly reduce the fraction of time that the protease flaps are open. Macromolecular crowding is likely to have a major effect on in vivo enzyme activity, and may play an important regulatory role in the viral life cycle.
CIRSE: A solvation energy estimator compatible with flexible protein docking and design applications
David S. Cerutti, Tushar Jain and J. Andrew McCammon
We present the Coordinate Internal Representation of Solvation Energy (CIRSE) for computing the solvation energy of protein configurations in terms of pairwise interactions between their atoms with analytic derivatives. Currently, CIRSE is trained to a Poisson / Surface-Area benchmark, but CIRSE is not meant to fit this benchmark specifically. CIRSE predicts the overall solvation energy of protein structures from 331 NMR ensembles with 0.951 +/- 0.047 correlation and predicts relative solvation energy changes between members of individual ensembles with an accuracy of 15.8 +/- 9.6 kcal/mol. The energy of individual atoms in any of CIRSEs 17 types is predicted with at least 0.98 correlation. We apply the model in energy minimization, rotamer optimization, protein design, and protein docking applications. The CIRSE model shows some propensity to accumulate errors in energy minimization as well as rotamer optimization, but these errors are consistent enough that CIRSE correctly identifies the relative solvation energies of designed sequences as well as putative docked complexes. We analyze the errors accumulated by the CIRSE model during each type of simulation and suggest means of improving the model to be generally useful for all-atom simulations.
Optimization and Computational Evaluation of a Series of Potential Active Site Inhibitors of the V82F/I84V Drug-resistant Mutant of HIV-1 Protease: an Application of the Relaxed Complex Method of Structure-based Drug Design
Alexander L. Perryman, Jung-Hsin Lin and J. Andrew McCammon
The Relaxed Complex method, an approach to structure-based drug design that incorporates the flexibilities of both the ligand and target protein, was applied to the HIV protease system. The control cases used AutoDock3.0.5 to dock a fully-flexible version of the prospective drug JE-2147 (aka SM-319777 or KNI-764) to large ensembles of conformations extracted from conventional, all atom, explicitly-solvated Molecular Dynamics simulations of the wild type and the V82F/I84V drug-resistant mutant of HIV-1 protease. The best set of run parameters from the control cases produced robust results when used against 2,200 different conformations of the wild type HIV-1 protease or against 2,200 conformations of the mutant. The results of the control cases, the published advice from experts, and structural intuition were used to design a new series of 23 potential active site inhibitors. The compounds were evaluated by docking them against 700 different conformations of the V82F/I84V mutant. The results of this first round of lead optimization were quite promising. Approximately one-third of that series performed at least slightly better than the parent compound, and four of those compounds displayed significantly better binding affinities against that drug-resistant mutant (within our computational model).
Computational investigation of pressure profiles in lipid bilayers with embedded proteins
J. Gullingsrud, A. Babakhani and J.A. McCammon
The distribution of surface tension within a lipid bilayer, also referred to as the lateral pressure profile, has been the subject of theoretical scrutiny recently due to its potential to radically alter the function of biomedically important membrane proteins. Experimental measurements of the pressure profile are still hard to come by, leaving first-principles all-atom calculations of the profile as an important investigative tool. We describe and validate an efficient implementation of pressure profile calculations in the molecular dynamics package NAMD, capable of distinguishing between internal, bonded, and nonbonded contributions as well as those of selected atom groups. The new implementation can also be used in conjunction with Ewald summation for long-range electrostatics, improving the accuracy and reproducibility of the calculated profiles. We then describe results of the calculation of a pressure profile for a simple protein-lipid system consisting of melittin embedded in a DMPC bilayer. While the lateral pressure in the protein-lipid system is nearly the same as that of the bilayer alone, partitioning of the lateral pressure by atom type revealed substantial perturbation of the pressure profile and surface tension in an asymmetric manner.
Evaluation and Binding Mode Prediction of Thiopyrone-Based Inhibitors of Anthrax Lethal Factor
Jana A. Lewis, John Mongan, J. Andrew McCammon and Seth M. Cohen
Anthrax lethal factor (LF) is one of three proteins involved in anthrax pathogenesis and lethality. Inactivation of the LF gene in B. anthracis leads to a decrease in virulence by 1000-fold or greater, which suggests that anthrax pathology is highly dependent on LF. Herein, we report an effective inhibitor of anthrax lethal factor based on a heterocyclic chelator scaffold. We also present computational predictions of the binding mode for this inhibitor and evidence that accurate prediction of binding modes requires use of a molecular surface-like boundary between solute and solvent.
Configurational-bias sampling technique for predicting side-chain conformations in proteins
Tushar Jain, David S. Cerutti and J. Andrew McCammon
Prediction of side-chain conformations is an important component of several biological modeling applications. In this work, we have developed and tested an advanced Monte-Carlo sampling strategy for predicting side-chain conformations. Our method is based on a cooperative rearrangement of atoms that belong to a group of neighboring side-chains. This rearrangement is accomplished by deleting groups of atoms from the side-chains in a particular region, and regrowing them with the generation of trial positions that depends both on a rotamer library and a molecular mechanics potential function. This method allows us to incorporate flexibility about the rotamers in the library and explore phase space in a continuous fashion about the primary rotamers. We have tested our algorithm on a set of 76 proteins using the all-atom AMBER99 force-field and electrostatics that are governed by a distance-dependent dielectric function. When the tolerance for correct prediction of the dihedral angles is less than a 20 degree deviation from the native state, our prediction accuracies for χ1 are 83.3%, and for χ1 and χ2 are 65.4%. The accuracies of our predictions are comparable to the best results in the literature that often used Hamiltonians that have been specifically optimized for side-chain packing. We believe that the continuous exploration of phase space enables our method to overcome limitations inherent with using discrete rotamers as trials.
Insight into the role of hydration on protein dynamics
Donald Hamelberg, Tongye Shen and J. Andrew McCammon
The potential energy surface of a protein is rough. This intrinsic energetic roughness affects diffusion, and hence the kinetics. The dynamics of a system undergoing Brownian motion on this surface in an implicit continuum solvent simulation can be tuned via the frictional drag or collision frequency to be comparable to that of experiments or explicit solvent simulations. We show that the kinetic rate constant for a local rotational isomerization in stochastic simulations with continuum solvent and a collision frequency of 2 ps-1 is about 104 times faster than that in explicit water and experiments. A further increase in the collision frequency to 60 ps-1 slows down the dynamics, but does not fully compensate for the lack of explicit water. We also show that the addition of explicit water does not only slow down the dynamics by increasing the frictional drag, but also increases the local energetic roughness of the energy landscape by as much as 1.0 kcal/mol.
Binding of Aminoglycosidic Antibiotics to the Oligonucleotide A-Site Model and 30S Ribosomal Subunit: Poisson-Boltzmann Model, Thermal Denaturation, and Fluorescence Studies
Grace Yang, Joanna Trylska, Yitzhak Tor and J. Andrew McCammon
The binding of paromomycin and similar antibiotics to the oligonucleotide A-site model and the small (30S) ribosomal subunit has been studied using continuum electrostatics methods. Crystallographic information from complexes of paromomycin, tobramycin and geneticin bound to an A-site oligonucleotide, and paromomycin and streptomycin complexed to the 30S subunit was used as a foundation to develop structures of similar antibiotics in the same ribosomal binding site. Relative binding free energies were calculated by combining the electrostatic contribution, which was obtained by solving the Poisson-Boltzmann equation, with a surface area-dependent apolar term, and contributions from conformational changes. These computed results showed good correlation with the experimental data resulting from fluorescence binding assays and thermal denaturation studies, demonstrating the ability of the Poisson-Boltzmann model to provide insight into the electrostatic mechanisms for aminoglycoside binding and direction for designing more effective antibiotics.
Bio3d: An R package for the comparative analysis of protein structures
Barry J. Grant, Ana P.C. Rodrigues, Karim M. ElSawy, J. Andrew McCammon and Leo S.D. Caves
Summary: An automated procedure for the analysis of homologous protein structures has been developed. The method facilitates the characterization of internal conformational differences and inter- conformer relationships and provides a framework for the analysis of protein structural evolution. The method is implemented in bio3d, an R package for the exploratory analysis of structure and sequence data.
Targeted Molecular Dynamics Study of C-Loop Closure and Channel Gating in Nicotinic Receptors
Xiaolin Cheng, Hailong Wang, Barry Grant, Steven M. Sine and J. Andrew McCammon
The initial coupling between ligand binding and channel gating in the human α7 nicotinic acetylcholine receptor (nAChR) has been investigated with targeted molecular dynamics (TMD) simulation. During the simulation, 8 residues at the tip of the C-loop in two alternating subunits were forced to move towards a ligand-bound conformation as captured in the crystallographic structure of acetylcholine binding protein (AChBP) in complex with carbamoylcholine. Comparison of apo- and ligand-bound AChBP structures shows only minor rearrangements distal from the ligand-binding site. In contrast, comparison of apo and TMD simulation structures of the nAChR reveals significant changes towards the bottom of the ligand-binding domain. These structural rearrangements are subsequently translated to the pore domain, leading to a partly open channel within 4 ns of TMD simulation. Furthermore, we confirmed that two highly conserved residue pairs, one located near the ligand-binding pocket (Lys145 and Tyr188), and the other located towards the bottom of the ligand-binding domain (Arg206 and Glu45), are likely to play important roles in coupling agonist binding to channel gating. Overall, our simulations suggest that gating movements of the α7 receptor may involve relatively small structural changes within the ligand-binding domain implying that the gating transition is energy-efficient, and can be easily modulated by agonist binding/unbinding.
In-situ synthesis of an inhibitor of acetylcholinesterase: Configurational selection imposed by steric interactions
Sanjib Senapati, Yuhui Cheng and J. Andrew McCammon
Recently researchers have used acetylcholinesterase (AChE) as a reaction vessel to synthesize its own inhibitors. Thus 1 (syn-TZ2PA6), a femtomolar AChE inhibitor, which is formed in 1:1 mixture with its anti isomer by solution phase reaction from 3 (TZ2) and 4 (PA6), can be synthesized exclusively inside the AChE gorge. Our computational approach based on quantum mechanical/molecular mechanical (QM/MM) calculations, molecular dynamics (MD), and targeted molecular dynamics (TMD) studies answers why 1 is the sole product in the AChE environment. Ab initio QM/MM results show that the reaction in the AChE gorge occurs when 3: azide and 4: acetylene are extended in a parallel orientation. An MD simulation started from the final structure of QM/MM calculations keeps the azide and acetylene's parallel orientation intact for 10 ns simulation time. A TMD simulation applied on an antiparallel azide-acetylene conformation flips the acetylene easily to bring it to parallel to azide. A second set of QM/MM calculations performed on this flipped structure generates a similar minimum-energy path as obtained previously. Even a TMD simulation carried out on a parallel azide-acetylene conformation could not deform their parallel arrangement. All these results thus imply that inside the AChE gorge the azide group of 3 and the acetylene group of 4 always remain parallel, with the consequence that 1 is the only product. The architecture of the gorge plays an important role in this selective formation of 1.
Protein complex formation by acetylcholinesterase and the neurotoxin fasciculin-2 appears to involve an induced-fit mechanism
Jennifer M. Bui and J. Andrew McCammon
Specific, rapid association of protein complexes is essential for all forms of cellular existence. The initial association of two molecules in diffusion-controlled reactions is often influenced by the electrostatic potential. Yet, the detailed binding mechanisms of proteins are highly dependent on the particular system. For the first time, a complete protein complex formation pathway has been delineated using structural information sampled over the course of the transformation reaction. The pathway begins at an encounter complex that is formed by one of the apo forms of neurotoxin fasciculin-2 and its high affinity binding protein, acetylcholinesterase. This is followed by rapid conformational rearrangements into an intermediate complex that subsequently converts to the final complex as observed in crystal structures. Formation of the intermediate complex has also been independently captured in a separate 20-ns molecular dynamics simulation of the encounter complex. Conformational transitions between the apo and liganded states of fasciculin-2 in the presence and absence of acetylcholinesterase are described in terms of their relative free energy profiles that link these two states. The transitions of fascisculin-2 after binding to acetylcholinesterase are significantly faster than in the absence of acetylcholinesterase; the energy barrier between the two conformational states is reduced by half. Conformational rearrangements of fasciculin-2 to the final liganded form not only bring the fasciculin-2/acetylcholinesterase complex to lower energy states, but by controlling transient motions that lead to opening or closing one of the alternative passages to the active site of the enzyme, also maximize the ligand's inhibition of the enzyme.
On the Application of Accelerated Molecular Dynamics to Liquid Water Simulations
César Augusto F. de Oliveira, Donald Hamelberg and J. Andrew McCammon
Our group recently proposed a robust bias potential function that can be used in an efficient all-atom accelerated molecular dynamics (MD) approach to simulate the transition of high energy barriers without any advance knowledge of the potential-energy landscape. The main idea is to modify the potential-energy surface by adding a bias, or boost, potential in regions close to the local minima, such that all transitions rates are increased. By applying the accelerated MD simulation method to liquid water, we observed that this new simulation technique accelerates the molecular motion without losing its microscopic structure and equilibrium properties. Our results showed that the application of a small boost energy on the potential-energy surface significantly reduces the statistical inefficiency of the simulation while keeping all the other calculated properties unchanged. On the other hand, although aggressive acceleration of the dynamics simulation increases the self-diffusion coefficient of water molecules greatly and dramatically reduces the correlation time of the simulation, configurations representative of the true structure of liquid water are poorly sampled. Our results also showed the strength and robustness of this simulation technique, which confirm this approach as a very useful and promising tool to extend the time scale of the all-atom simulations of biological system with explicit solvent models. However, we should keep in mind that there is a compromise between the strength of the boost applied in the simulation and the reproduction of the ensemble average properties.
Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications
Ivaylo Ivanov, Brian R. Chapados, J. Andrew McCammon and John A. Tainer
Proliferating cell nuclear antigen (PCNA) acts as a biologically essential processivity factor that encircles DNA and provides binding sites for polymerase, flap endonuclease-1 (FEN-1) and ligase during DNA replication and repair. We have computationally characterized the interactions of human and Archaeoglobus fulgidus PCNA trimer with double-stranded DNA (ds DNA) using multi-nanosecond classical molecular dynamics simulations. The results reveal the interactions of DNA passing through the PCNA trimeric ring including the contacts formed, overall orientation and motion with respect to the sliding clamp. Notably, we observe pronounced tilting of the axis of dsDNA with respect to the PCNA ring plane reflecting interactions between the DNA phosphodiester backbone and positively charged arginine and lysine residues lining the PCNA inner surface. Covariance matrix analysis revealed a pattern of correlated motions within and between the three equivalent subunits involving the PCNA C-terminal region and linker strand associated with partner protein binding sites. Additionally, principal component analysis identified low frequency global PCNA subunit motions suitable for translocation along duplex DNA. The PCNA motions and interactions with the DNA minor groove, identified here computationally, provide an unexpected basis for PCNA to act in the coordinated handoff of intermediates from polymerase to FEN-1 to ligase during DNA replication and repair.
Order N algorithm for computation of electrostatic interactions in biomolecular systems
Benzhuo Lu, Xiaolin Cheng, Jingfang Huang and J. Andrew McCammon
Poisson-Boltzmann (PB) electrostatics is a well established model in biophysics, however its application to large scale biomolecular processes such as protein-protein encounter is still limited by the efficiency and memory constraints of existing numerical techniques. In this paper, we present an efficient and accurate scheme which incorporates recently developed numerical techniques to enhance our computational ability. In particular, a boundary integral equation (BIE) approach is applied to discretize the linearized PB equation; the resulting integral formulas are well conditioned and are extended to systems with arbitrary numbers of biomolecules. The solution process is accelerated by Krylov subspace methods and a new version of the fast multipole method (FMM). In addition to the electrostatic energy, fast calculations of the forces and torques are made possible by using an interpolation procedure. Numerical experiments show that the implemented algorithm is asymptotically optimal $O(N)$ in both CPU time and required memory, and application to the acetylcholinesterase-fasciculin complex is illustrated.