Yibing Shan

Atomic-Level Characterization of the Structural Dynamics of Proteins

Following Folding Fast Many protein functions involve conformational changes that occur on time-scales between tens of microseconds and milliseconds. This has limited the usefulness of all-atom molecular dynamics simulations, which are performed over shorter time-scales. Shaw et al. (p. 341) now report millisecond-scale, all-atom molecular dynamics simulations in an explicitly represented solvent environment. Simulation of the folding of a WW domain showed a well-defined folding pathway and simulation of the dynamics of bovine pancreatic trypsin inhibitor showed interconversion between distinct conformational states. Millisecond-scale simulations capture biologically relevant structural transitions during protein folding. Molecular dynamics (MD) simulations are widely used to study protein motions at an atomic level of detail, but they have been limited to time scales shorter than those of many biologically critical conformational changes. We examined two fundamental processes in protein dynamics—protein folding and conformational change within the folded state—by means of extremely long all-atom MD simulations conducted on a special-purpose machine. Equilibrium simulations of a WW protein domain captured multiple folding and unfolding events that consistently follow a well-defined folding pathway; separate simulations of the protein’s constituent substructures shed light on possible determinants of this pathway. A 1-millisecond simulation of the folded protein BPTI reveals a small number of structurally distinct conformational states whose reversible interconversion is slower than local relaxations within those states by a factor of more than 1000.

Scalable algorithms for molecular dynamics simulations on commodity clusters

Although molecular dynamics (MD) simulations of biomolecular systems often run for days to months, many events of great scientific interest and pharmaceutical relevance occur on long time scales that remain beyond reach. We present several new algorithms and implementation techniques that significantly accelerate parallel MD simulations compared with current state-of-the-art codes. These include a novel parallel decomposition method and message-passing techniques that reduce communication requirements, as well as novel communication primitives that further reduce communication time. We have also developed numerical techniques that maintain high accuracy while using single precision computation in order to exploit processor-level vector instructions. These methods are embodied in a newly developed MD code called Desmond that achieves unprecedented simulation throughput and parallel scalability on commodity clusters. Our results suggest that Desmonds parallel performance substantially surpasses that of any previously described code. For example, on a standard benchmark, Desmonds performance on a conventional Opteron cluster with 2K processors slightly exceeded the reported performance of IBMs Blue Gene/L machine with 32K processors running its Blue Matter MD code

Pathway and mechanism of drug binding to G-protein-coupled receptors

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 β1- and β2-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.

How Does a Drug Molecule Find Its Target Binding Site

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 sites location, may prove particularly useful in the development of allosteric inhibitors that target previously undiscovered binding sites.

Architecture and Membrane Interactions of the EGF Receptor

Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is crucial to cellular pathways regulating proliferation, migration, and differentiation. Inactive EGFR can exist as both monomers and dimers, suggesting that the mechanism regulating EGFR activity may be subtle. The membrane itself may play a role but creates substantial difficulties for structural studies. Our molecular dynamics simulations of membrane-embedded EGFR suggest that, in ligand-bound dimers, the extracellular domains assume conformations favoring dimerization of the transmembrane helices near their N termini, dimerization of the juxtamembrane segments, and formation of asymmetric (active) kinase dimers. In ligand-free dimers, by holding apart the N termini of the transmembrane helices, the extracellular domains instead favor C-terminal dimerization of the transmembrane helices, juxtamembrane segment dissociation and membrane burial, and formation of symmetric (inactive) kinase dimers. Electrostatic interactions of EGFRs intracellular module with the membrane are critical in maintaining this coupling.

Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor

How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Millisecond-scale molecular dynamics simulations on Anton

Anton is a recently completed special-purpose supercomputer designed for molecular dynamics (MD) simulations of biomolecular systems. The machines specialized hardware dramatically increases the speed of MD calculations, making possible for the first time the simulation of biological molecules at an atomic level of detail for periods on the order of a millisecond-about two orders of magnitude beyond the previous state of the art. Anton is now running simulations on a timescale at which many critically important, but poorly understood phenomena are known to occur, allowing the observation of aspects of protein dynamics that were previously inaccessible to both computational and experimental study. Here, we report Antons performance when executing actual MD simulations whose accuracy has been validated against both existing MD software and experimental observations. We also discuss the manner in which novel algorithms have been coordinated with Antons co-designed, application-specific hardware to achieve these results.

Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3.

The kinase domain of human epidermal growth factor receptor (HER) 3/ErbB3, a member of the EGF receptor (EGFR) family, lacks several residues that are critical for catalysis. Because catalytic activity in EGFR family members is switched on by an allosteric interaction between kinase domains in an asymmetric kinase domain dimer, HER3 might be specialized to serve as an activator of other EGFR family members. We have determined the crystal structure of the HER3 kinase domain and show that it appears to be locked into an inactive conformation that resembles that of EGFR and HER4. Although the crystal structure shows that the HER3 kinase domain binds ATP, we confirm that it is catalytically inactive but can serve as an activator of the EGFR kinase domain. The HER3 kinase domain forms a dimer in the crystal, mediated by hydrophobic contacts between the N-terminal lobes of the kinase domains. This N-lobe dimer closely resembles a dimer formed by inactive HER4 kinase domains in crystal structures determined previously, and molecular dynamics simulations suggest that the HER3 and HER4 N-lobe dimers are stable. The kinase domains of HER3 and HER4 form similar chains in their respective crystal lattices, in which N-lobe dimers are linked together by reciprocal exchange of C-terminal tails. The conservation of this tiling pattern in HER3 and HER4, which is the closest evolutionary homolog of HER3, might represent a general mechanism by which this branch of the HER receptors restricts ligand-independent formation of active heterodimers with other members of the EGFR family.

Gaussian split Ewald: A fast Ewald mesh method for molecular simulation

Gaussian split Ewald (GSE) is a versatile Ewald mesh method that is fast and accurate when used with both real-space and k-space Poisson solvers. While real-space methods are known to be asymptotically superior to k-space methods in terms of both computational cost and parallelization efficiency, k-space methods such as smooth particle-mesh Ewald (SPME) have thus far remained dominant because they have been more efficient than existing real-space methods for simulations of typical systems in the size range of current practical interest. Real-space GSE, however, is approximately a factor of 2 faster than previously described real-space Ewald methods for the level of force accuracy typically required in biomolecular simulations, and is competitive with leading k-space methods even for systems of moderate size. Alternatively, GSE may be combined with a k-space Poisson solver, providing a conveniently tunable k-space method that performs comparably to SPME. The GSE method follows naturally from a uniform framework that we introduce to concisely describe the differences between existing Ewald mesh methods.

Oncogenic Mutations Counteract Intrinsic Disorder in the EGFR Kinase and Promote Receptor Dimerization

The mutation and overexpression of the epidermal growth factor receptor (EGFR) are associated with the development of a variety of cancers, making this prototypical dimerization-activated receptor tyrosine kinase a prominent target of cancer drugs. Using long-timescale molecular dynamics simulations, we find that the N lobe dimerization interface of the wild-type EGFR kinase domain is intrinsically disordered and that it becomes ordered only upon dimerization. Our simulations suggest, moreover, that some cancer-linked mutations distal to the dimerization interface, particularly the widespread L834R mutation (also referred to as L858R), facilitate EGFR dimerization by suppressing this local disorder. Corroborating these findings, our biophysical experiments and kinase enzymatic assays indicate that the L834R mutation causes abnormally high activity primarily by promoting EGFR dimerization rather than by allowing activation without dimerization. We also find that phosphorylation of EGFR kinase domain at Tyr845 may suppress the intrinsic disorder, suggesting a molecular mechanism for autonomous EGFR signaling.