Mechanism of Action of Cyclophilin A Explored by Metadynamics Simulations

Trans/cis prolyl isomerisation is involved in several biological processes, including the development of numerous diseases. In the HIV-1 capsid protein (CA), such a process takes place in the uncoating and recruitment of the virion and is catalyzed by cyclophilin A (CypA). Here, we use metadynamics simulations to investigate the isomerization of CA's model substrate HAGPIA in water and in its target protein CypA. Our results allow us to propose a novel mechanistic hypothesis, which is finally consistent with all of the available molecular biology data

Mapping transiently formed and sparsely populated conformations on a complex energy landscape

Determining the structures, kinetics, thermodynamics and mechanisms that underlie conformational exchange processes in proteins remains extremely difficult. Only in favourable cases is it possible to provide atomic-level descriptions of sparsely populated and transiently formed alternative conformations. Here we benchmark the ability of enhanced-sampling molecular dynamics simulations to determine the free energy landscape of the L99A cavity mutant of T4 lysozyme. We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations. We discover a new tunnel that involves the transient exposure towards the solvent of an internal cavity, and show it to be relevant for ligand escape. Together, our results provide a comprehensive view of the structural landscape of a protein, and point forward to studies of conformational exchange in systems that are less characterized experimentally. DOI: http://dx.doi.org/10.7554/eLife.17505.00

Dynamic design:Manipulation of millisecond timescale motions on the energy landscape of cyclophilin A

Proteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they interconvert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov state modelling (MSM) to explore these ‘excited’ conformational states. Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics, whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R1ρ solution state NMR measurements. By efficiently exploring functionally relevant, but sparsely populated conformations with millisecond lifetimes in silico, our aMD/MSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery

Dynamic design: manipulation of millisecond timescale motions on the energy landscape of cyclophilin A

Proteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they interconvert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov state modelling (MSM) to explore these ‘excited’ conformational states. Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics, whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R1ρ solution state NMR measurements. By efficiently exploring functionally relevant, but sparsely populated conformations with millisecond lifetimes in silico, our aMD/MSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery

Mapping transiently formed and sparsely populated conformations on a complex energy landscape

Abstract Determining the structures, kinetics, thermodynamics and mechanisms that underlie conformational exchange processes in proteins remains extremely difficult. Only in favourable cases is it possible to provide atomic-level descriptions of sparsely populated and transiently formed alternative conformations. Here we benchmark the ability of enhanced-sampling molecular dynamics simulations to determine the free energy landscape of the L99A cavity mutant of T4 lysozyme. We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations. We discover a new tunnel that involves the transient exposure towards the solvent of an internal cavity, and show it to be relevant for ligand escape. Together, our results provide a comprehensive view of the structural landscape of a protein, and point forward to studies of conformational exchange in systems that are less characterized experimentally

Computational Perspective on Intricacies of Interactions, Enzyme Dynamics and Solvent Effects in the Catalytic Action of Cyclophilin A

Cyclophilin A (CypA) is the well-studied member of a group of ubiquitous and evolutionarily conserved families of enzymes called peptidyl–prolyl isomerases (PPIases). These enzymes catalyze the cis-trans isomerization of peptidyl-prolyl bond in many proteins. The distinctive functional path triggered by each isomeric state of peptidyl-prolyl bond renders PPIase-catalyzed isomerization a molecular switching mechanism to be used on physiological demand. PPIase activity has been implicated in protein folding, signal transduction, and ion channel gating as well as pathological condition such as cancer, Alzheimer’s, and microbial infections. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerization by CypA has been the target of intense research. Normal and accelerated molecular dynamic simulations were carried out to understand the catalytic mechanism of CypA in atomistic details. The results reaffirm transition state stabilization as the main factor in the astonishing enhancement in isomerization rate by enzyme. The ensuing intramolecular polarization, as a result of the loss of pseudo double bond character of the peptide bond at the transition state, was shown to contribute only about −1.0 kcal/mol to stabilizing the transition state. This relatively small contribution demonstrates that routinely used fixed charge classical force fields can reasonably describe these types of biological systems. The computational studies also revealed that the undemanding exchange of the free substrate between β- and α-helical regions is lost in the active site of the enzyme, where it is mainly in the β-region. The resultant relative change in conformational entropy favorably contributes to the free energy of stabilizing the transition state by CypA. The isomerization kinetics is strongly coupled to the enzyme motions while the chemical step and enzyme–substrate dynamics are in turn buckled to solvent fluctuations. The chemical step in the active site of the enzyme is therefore not separated from the fluctuations in the solvent. Of special interest is the nature of catalysis in a more realistic crowded environment, for example, the cell. Enzyme motions in such complicated medium are subjected to different viscosities and hydrodynamic properties, which could have implications for allosteric regulation and function

A Kinetic Model of Trp-Cage Folding from Multiple Biased Molecular Dynamics Simulations

Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins. Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For the Trp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap. Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at of 4.4 Å from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the and chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data

Enhanced sampling in molecular dynamics using metadynamics, replica-exchange, and temperature-acceleration

We review a selection of methods for performing enhanced sampling in molecular dynamics simulations. We consider methods based on collective variable biasing and on tempering, and offer both historical and contemporary perspectives. In collective-variable biasing, we first discuss methods stemming from thermodynamic integration that use mean force biasing, including the adaptive biasing force algorithm and temperature acceleration. We then turn to methods that use bias potentials, including umbrella sampling and metadynamics. We next consider parallel tempering and replica-exchange methods. We conclude with a brief presentation of some combination methods. \ua9 2013 by the author; licensee MDPI, Basel, Switzerland