Three-dimensional Structures Of Biological Macromolecules

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Traditional molecular modeling is performed at atomic resolution, which relies on X-ray and NMR experiments to provide structural information. When dealing with biomolecular assemblies of millions of atoms, atomic description of molecular objects becomes very computational inefficient. We developed a method that uses map objects for molecular modeling to efficiently derive structural information from experimental maps, as well as conveniently manipulate map objects, perform conformational search directly using map objects. This development work has been implemented into CHARMM as the EMAP module. This implementation enables CHARMM to manipulate map objects, including map input, output, comparison, docking, etc. 1. Map restrained Self-guided Langevin dynamics simulation study of SARS-CoV-2 spike ectodomain The emergence of SARS-CoV-2 has resulted over 3 million infections and more than 200,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. It has been shown that SARS-CoV-2 S uses angiotensin-converting enzyme 2 (ACE2) to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein that forms homotrimers protruding from the viral surface . S comprises two functional subunits responsible for binding to the host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit). Coronavirus entry into susceptible cells is a complex process that requires the concerted action of receptor-binding and proteolytic processing of the S protein to promote virus-cell fusion. As the coronavirus S glycoprotein is surface-exposed and mediates entry into host cells, it is the main target of neutralizing antibodies (Abs) upon infection and the focus of therapeutic and vaccine design. Conformational transition of SARS-CoV-2 spike is critical for its function to interact with ACE2 and enter host cells. The mechanism of the opening and possibility to mediate this process is the focus of research in development of vaccines. This research benefits from two main aspects, the information extraction from cryo-EM maps and the ability to reproduce the opening process. The map-restrained Self-guided Langevin dynamics method (MapSGLD) developed in this lab is a perfect tool for this type of research. This method accurately extract structure information from cryo-EM maps and achieve large scale conformational search through SGLD to achieve the goal of reproduce conformational evolvement from one state to another. Along the conformational path many opportunities for mediate the process can be discovered and studied. Current, the cryo-EM maps of the open and close state of the SARS-CoV-2 spike ectodomain are available, which provide us unprecedent opportunity to study the mechanism with MapSGLD. It is found that SARS-CoV polyclonal antibodies inhibit SARS-CoV-2 spike-mediated entry into cells. MapSGLD simulation of antibody-spike interaction will provide structure and energetic insight for vaccine development. 2. New Structure and Energy Cycles of Kinesin Dimers Walking on Microtubules Revealed from Molecular Simulations Kinesins are motor proteins that move unidirectionally along microtubules as they hydrolyze ATP. Although the general features of the kinesin walking mechanism are becoming increasingly clear, some key questions remain unanswered, such as how they convert the chemical energy of ATP into mechanical energy and walk processively. In this study, through molecular simulations and free energy calculations, we found that in aqueous solution, kinesin favors an extended form with its microtubule-binding interface (MTBI) motif unfolded, as seen in a recent x-ray structure of kinesin-8. Through the flexible fitting of two newly released cryo-electron microscopy (cryo-EM) maps, we derived atomic structures of the kinesin dimer-microtubule complexes in both two-head-bound and one-head bound states. Free energy calculations showed that kinesin bound to microtubules has a lower free energy than the extended form and that the free energy difference is in the range of the free energy released by ATP hydrolysis. The transition between the extended and compact forms, the structural differences of the leading and trailing heads, and atomic force simulations suggest a completely new mechanism by which kinesin dimers walk on microtubules. A structure cycle and energy cycle are presented to describe kinesin dimers walking on microtubules. Identifying the extended form of kinesin provides a new target for the regulation of kinesins. 3. Investigating the receptor binding and glycan dynamics in the spike protein of SARS-COV-2 The coronavirus (SARS-COV-2) outbreak has become a global pandemic and major threat to human public health. The spike protein in SARS-COV-2 resides on the surface of the virion and mediates entry of the virus into human cells by binding its receptor binding domain (RBD) to the receptor protein (ACE2). In the first part of our project, we investigated the detailed structural mechanism of binding between RBD and ACE2 and its difference with an earlier (SARS-COV) in 2002 using MD simulations. Our analysis showed that RBD of SARS-COV-2 binds its receptor with a higher binding affinity than SARS-COV which is part of the reason for its higher infection rate. We found the hot spots for the interaction between RBD and ACE2 and mutations from the earlier SARS-COV RBD that gifted the spike protein a higher binding affinity in SARS-COV-2. Moreover, numerous mutations have been identified in the RBD of SARS-COV-2 isolated from strains from humans from different parts of the world. We studied the effect of these mutations (until June 2020) as well as other ala-scanning mutations on the binding free energy between RBD and ACE2 using MMPBSA approach. It was found that most of the naturally occurring mutations to RBD either slightly strengthen or have similar binding affinity to ACE2 as the wild-type. Currently, we are working on a more rigorous non-equilibrium free energy perturbation approach (FEP) to evaluate the relative binding affinity of the currently observed mutations (UK variant and South Africa variant). Non-equilibrium FEP is more computationally demanding than MMPBSA, however, it does not suffer from limitations in the MMPBSA such as the choice of implicit solvent. In the second part of this project we studied how the glycan shield of the spike helps the virus to evade the humane immune response by covering the spike protein against any antibody. We performed micro-second long MD simulation of spike protein in two different states corresponding to the receptor binding domain in open or closed states. The role of glycans in shielding of spike were uncovered by a network analysis, in which the high betweenness centrality of glycans at the apex revealed their importance and function in the glycan shield. Furthermore, microdomains of glycans were identified that feature a high degree of intra-communication in these microdomains. An antibody overlap analysis revealed the glycan microdomains as well as individual glycans that inhibit access to the antibody epitopes on the spike protein. The higher number of microdomains in the open state suggested the higher vulnerability of the spike in the open state. Collectively our work presented insights to design antibodies and vaccines against SARS-COV-2.