(485al) Investigation of Possible Geometrical Constraints On A-1 Helix in b12-Bound HIV gp120 Core through Targeted Molecular Dynamics

The human immunodeficiency virus (HIV) infects its target cells by attaching to specific cell surface receptors, fusing with the cell membrane and injecting the viral capsid into the cell. The HIV envelope glycoproteins (ENV) are responsible for the process of viral entry into the cell. These proteins are attractive targets for both drug development and also vaccine design. Blocking the entry process effectively prevents the virus from infecting new cells and stops it before it establishes a permanent base inside the body. Since envelope glycoproteins are the major immunogenic components of the virus visible to the body's defense systems and are the main targets for neutralizing antibodies, they can be a basis for the hummoral component of a vaccine. Despite major efforts toward designing such a vaccine, development of an effective one has not been successful yet and this has motivated more in-depth investigations into the structure and functional properties of the envelope glycoprotein. These proteins are assembled into spike-like structures (?envelope spikes?) which are mainly composed of trimers of dimers, with each dimer being made up of gp120 and gp41 which are non-covalently attached to each other. gp120 makes up the larger, outward and more visible part of the spike and is available to the immune system, in contrast to gp41 which is mainly hidden by gp120. gp120 mediates viral attachment to cell surface and engages the target receptor and coreceptor. This leads to exposure of gp41 and finally membrane fusion. It has been suggested that during receptor binding, gp120 goes through a large conformational change, which is confirmed by observation of a large negative entropic change in calorimetric measurements. It has also been suggested that its flexible and ?plastic? nature is a major characteristic of gp120 which contributes to its ability to evade the immune system through a mechanism dubbed ?conformational masking?. This highly flexible nature of the molecule and the presence of multiple variable loops on it has prevented the investigators from resolving the native, unliganded structure of HIV gp120. Aside from a crystal structure of the unliganded, presumably native state homologous SIV gp12; the only conformations available up to now for HIV gp120 are those of gp120 ?core? (with some N- and C-terminal deletions and lacking some of the loops) bound to its CD4 receptor (which here we call the activated structure), and that of gp120 bound to a broadly neutralizing antibody, b12 (which we call the b12-bound conformation). It has been suggested that the b12-bound structure, despite having 9 stabilizing mutations, is close to the unliganded gp120. In this work we have used Molecular Dynamics (as implemented in NAMD software package and using the CHARMM force field) as a tool to investigate the conformational flexibility of the apo forms of gp120 and also Targeted Molecular Dynamics to assess the possibility of a conformational transition between the two structures. To this end, we were interested to see whether or not folding of the alpha-1 helix is affected by the mutations, specifically the I109C/Q428C disulphide-bridge forming mutation. The alpha-1 helix is absent from the b12-bound crystal structure, although it is present in all the other available wild-type crystal structures of the molecule. Since the b12-bound state is thought to be thermodynamically closer to the yet-unresolved unliganded state, it is valuable to investigate whether or not the stabilizing mutations might have had any effects on the overall conformation of the b12-bound gp120. We used two protein structures : the original crystal structure of the engineered gp120 in complex with b12 (PDB ID 2NY7) hereafter called 2ny7; and an in silico modified strcuture where the disulphide bridge ?stitching? the B20/B21 strand to alpha-1 was removed and replaced with the wild type mutant, hereafter called DS1F123. It's worth nothing that DS1F123 has been made by the same group which resolved the b12-bound structure, but it didn't yield to crystallographic analysis in complex with b12. We found that when alpha-1 is folded in 2ny7, it stays stable but alignment of the static b12 molecule over the structure produces many overlaps between gp120 and b12 atoms in the B20/B21 region of gp120. In contrast, the same analysis performed on the DS1F123 structure shows that the amount of overlaps stays minimal which can be attributed to the B20/B21 ?slippipng? away from the folded alpha-1 helix due to the absence of the covalent disulphide link (which keeps it bound to alpha-1 in 2NY7), and allowing for a coexistence of bound-b12, folded alpha-1 and B20/B21. This simple analysis suggests that when b12 binds to gp120 in DS1F123 and occludes the F43 pocket where B20/B21 is supposed to sit, movement of the beta strand away from the pocket does not necessitate a movement or probably partial unfolding of alpha-1. In contrast, in 2ny7, binding of b12 and the consequent moving of the B20/B21 away from the pocket imposes a restraint on alpha-1 (due to the disulphide bridge) which can probably result in an unfolding of the helix. Efforts to further strengthen the proposed mechanism using free energy calculations are under way.