Charactering The Flexibility/Mobility Of Topological Layers In HIV-1 Gp120 Using Steered Molecular Dynamics

The molecular machinery responsible for cell entry of HIV-1 is the viron-surface-exposed envelope trimer formed by three gp120 and three gp41 glycoproteins via non-covalent interactions,During the process of cell entry,the gp120 subunit is responsible for recognition of and binding to the host cell-surface receptor CD4 and co-receptor CCR5/CXCR4,while gp41 is responsible for the target cell membrane insertion and the fusion between the viral and cell membranes.A large number of structural and biochemical studies have shown that the conformational changes in gp120 and its conformational diversity arising from these changes play a key role in HIV-1 cell entry and evading the host immune surveillance.According to the traditional topology classification,the structure of gp120 can be divided into two domains: the inner domain and the outer domain,where the inner domain involves direct contacts with gp41 that is located in the inside of the Env trimer,and the outer domain is directly exposed to the surface of the trimer.A more refined topological classification approach divides the inner domain into four parts,i.e.,a structurally stable ?-sandwich anchor and the three topological layers(layers 1-3)emanating from the anchor.It has been considered that the changes in the spatial locations of the three topological layers relative to the ?-sandwich anchor and the mutual changes in the spatial locations or orientations of the three topological layers are likely to be the key determinants in determining the conformational variability and diversity of gp120.However,the questions of how big the differences in the conformational flexibility and the ability of spatial reorientation(i.e.,the displacement or shift of an entire structural fragment)are among the three topological layers,what factors cause these differences,and which of the three topological layers contributes most significantly to the conformational diversity of gp120,remain unanswered.In order to answer the above questions,this thesis takes the structural models of gp120-CD4 complex and CD4-free gp120(i.e.,gp120 without CD4)as the research object,for which a series of steered molecular dynamics(SMD)simulations wereperformed to pull separately the layers 1-3,followed by reconstructing the free energy profiles of the pulling processes;in addition,we also calculated the interaction energies between the layer residues and the gp120 structural main body and between the topological layers and CD4 during the pulling processes.In order to evaluate the structural stability of different structural components/regions of gp120 after the pulling procedure of individual layers,we also performed conventional molecular dynamics simulations on the gp120 structures with stretched layers.By comparing the free energy profiles(or free energy curves)produced by pulling the three topological layers,we found that,whether the steered MD simulation was performed on the gp120-CD4 complex or on the CD-free gp120,the free energy(or work)required to force different topological layers to increase the same distance of the center of mass(COM)relative to the COM of the reference group has the order of layer 3 > layer 2 > layer 1;this indicates that the layer 3 has a larger difficulty in experiencing shift/spatial reorientation than layer 2,while the layer 1 has the strongest ability to undergo spatial reorientation.In addition,we observed many and a few local free energy minima on the free energy profiles of pulling the layers 1 and 2,respectively,but almost no local free energy minima on the free energy profile of pulling the layer 3.This indicates that the layer 1 displays the most abundant conformational diversity,followed by the layer 2,but the layer 3 has the most poor conformational diversity.By comparing the free energy profiles of pulling the same topological layer in the presence and absence of CD4,we found that the CD4 binding reduces the free energy required to pull the layer 1 away from gp120 main body while increasing the number of layer 1's naturally accessible conformation substates(i.e.,the number of local minima with the free energy level < 5 kcal/mol);on the contrary,the presence of CD4 increases the free energy that is needed to overcome when pulling the layers 2 and 3,but there is no local minimum with free energy level less than 5kcal/mol that can be detected on the corresponding free energy profiles.The above results indicate that bingding of CD4 to gp120 enhances the ability for the layer 1 to change its conformation and spatial orientation and,hence,increases itsconformational diversity,but weakens the allosteric abilities and the spatial reorientation abilities of the layers 2 and 3.By observing the steered molecular dynamics simulation trajectories and performing the conventional molecular dynamics simulations on the gp120 structures carrying the stretched layer,we found that the layers 1 and 2,especially the layer 1,can change their conformations and spatial orientations as an independent structural unit but bring about only limited influence on the conformation of the remaining structural components of gp120;on the contrary,pulling layer 3 does not cause a large change in its own conformation but destroys the overall conformation of gp120 due to causing the separation of the inner domain from the outer domain.The effect of pulling the layer 2 is mainly reflected in the conformational change and spatial orientation of?2-?3: in the presence of CD4,pulling layer 2 causes the ?2-?3 hairpin to deviate from the ?20-?21 hairpin and hence destroys the bridging sheet but the structural integrity of the ?2-?3 hairpin is preserved;in the absence of CD4,pulling the layer 2 cause the?2-?3 hairpin to fall apart from the main body of gp120 structure accompanying the conformational conversion into a random-coiled loop,indicating that CD4 binding could stabilize the bridging sheet via limiting spatial displacement of the layer 2.In addition,we also note that the steric hindrance effect from CD4 can not only restrict the conformational freedoms of the layers 2 and 3,but also suppress the transition of stretched conformations of individual layers into their respective conformations before pulling.Through calculating the interaction energy between the layer residues and the structural main body during the steered molecular dynamics simulations,we found that Trp69,His66,Val65 on layer 1,Leu122,Val120,Pro118 and Cys119 on layer 2,and layer 3 residues such as Ser256 make important contributions to maintaining the stability of the association between indvidual layers and the main body of gp120 structure.In addition,by calculating the interaction energy between each topological layer and CD4 during the steered dynamics simulation,it was found that the interaction strength between CD4 and layer 1 is almost 0,but that the high-intensity interactionsexist between the layers 2 and 3 and CD4,indicating that besides the spatial hindrance effect,CD4 can also restrict the conformational freedoms of layers 2 and 3 through its favorable interactions with the layers 2 and 3.In the presence of CD4,the enhancement of the reorientation ability of layer 1 is most likely to be due to the changes in the kinetic and thermodynamic behaviors of gp120 arising from the steric hindrance effect of CD4 or the interaction of CD4 with its binding cavity.In summary,among the three topological layers of gp120,the layer 1 has the strongest abilities of conformational change and spatial reorientation,followed by the layer 2,and the layer 3 almost hardly has the ability to undergo conformatonal change and reorientation;consequently,we believe that the conformational change and diversity of gp120 originate mainly from the strong allosteric and reorientation capabilities of the layers 1 and 2.The differences in the interaction strength between different layer residues and structural main body of gp120,the differences in the interaction strength between different topological layers and CD4,and the differences in the steric hindrance effect on individal topological layers exerted by CD4 could collectively regulate and affect the the conformational freedom and spatial reorientation ability of individual layers,ultimately promoting the cell-entry-related conformational changes and the conformational diversity in gp120 structure required for immune evasion.The results presented in this thesis also provide a hint that screening or designing compounds capable of restricting the conformational change of the layer 1 and/or blocking the spatial reorientation of the layer 2(and thus preventing the formation of mature bridging sheet)could be one of the effective strategies for developing the anti-HIV-1 drugs.