The HREST-BP simulations were carried out for all systems. than that observed in experimental studies; and specific glycan conformations can maximize or minimize interactions with individual antibodies. More broadly, the enhanced sampling MD simulations described here provide a valuable tool to guide the engineering of specific Env glycoforms for HIV vaccine design. == Introduction == It has been over 30 years since the identification of Human Immunodeficiency Virus (HIV) as the causative agent of Acquire Immunodeficiency Syndrome (AIDS), and despite Levomepromazine marked progress in the development of effective chemotherapeutics, the search for a protective vaccine remains elusive. Recent efforts towards identification Levomepromazine of vaccine candidates have focused in part on the structural biology of the surface molecules of the HIV virion to understand in greater depth the molecular determinants of key epitopes that could serve as targets for protective antibodies13. Significant effort has been placed on elucidation of the three-dimensional (3D) structure of the full HIV Envelope (Env) protein that mediates binding and internalization of the HIV Levomepromazine virion within susceptible target cells, including macrophages and CD4+ T-cells. In addition to studies that have characterized the structure of the Env protein, recent work has also provided crucial structural information regarding the glycan shield that surrounds the Env protein, and represents a barrier to the accessibility of Env-specific antibodies420. Given the supporting evidence of Env protein and glycan Rabbit polyclonal to AGO2 epitopes as targets of broadly-neutralizing antibodies (bnAb), better understanding of the conformational properties of Env glycans could inform vaccine design10,14. To date, there have been over 300 crystallographic structures of the HIV gp120 protein deposited in the Protein Databank21, many of which include the full Env sequence420. While these 3D structural studies of the Env protein have provided important insights into putative epitopes of broadly neutralizing antibodies (bNAbs), they provide little information on the potential dynamic nature of the glycan shield conformations, which could impact induction and antigen recognition of bNAbs. Atomic level molecular dynamics (MD) simulations offer great potential in this area. Indeed, a recent X-ray crystallographic study on the glycan shield was supplemented by MD simulations of the glycosylated Env Levomepromazine performed at room temperature to obtain additional information on the spatial distribution of the oligosaccharides comprising the glycan shield6. While powerful, conformational sampling by MD simulations alone is challenging, a problem that may be overcome by the coupling of MD simulations with enhanced sampling technologies such as Hamiltonian replica exchange (HREX)2232. Recently, novel HREX methods along with improved empirical force fields for polysaccharides have been developed that increase the accuracy of the information from computational methods with respect to the full range of conformations accessible to glycans3340. In addition, the use of glycosidic (GL) cluster analysis allows for uniquely defining the conformations sampled by the glycans with the analysis of 3D volumes sampled by the glycans and antibodies and overlap coefficients (OC) of those volumes41,42, offering insights into the common regions of space sampled by the different moieties. With the goal of obtaining a better understanding of the conformational properties of the glycan shield of the Env, and the accessibility of the underlying proteins, we undertook a HREX-MD33,34study of a glycosylated Env15in which the three gp120/gp41 monomers were differentially glycosylated with high mannose (M5 and M9) glycans (TableS1, supporting information). From this simulation, we obtained a model of substantial glycan conformational heterogeneity, wherein we document the presence of an extensive network of interactions between glycans, with implications for the role of the glycan shield with respect to accessibility of the protein surface to the CD4 receptor, CCR5 and CXCR4 co-receptors, and neutralizing antibodies. Analysis of glycan holes, a phenomenon referring to the omission of specific Env glycans43, provided further insights towards the role for specific glycans in mediating accessibility to adjacent glycans and the underlying protein. In addition, the range of potential interactions between the Env glycans with the CD4 receptor and anti-Env antibodies was determined. These results highlight the potential of MD methods combined with enhanced sampling for probing the structure and dynamics of HIV Env glycoforms in the context of a methodology where the composition of the system can be rigorously defined and studied in molecular detail at biologically relevant temperatures, with the results used in a predictive fashion to facilitate vaccine design. == Results and Discussion == To investigate the conformational heterogeneity of the glycans on the HIV Env, simulation models were generated in which one monomer of the Env trimer (M1) had full glycosylation (23 glycans) and the other two monomers being partially glycosylated with 12 (M2) and 8 (M3) glycans, respectively.