Structural and Functional Characterization of Viral Membrane Proteins by Solution NMR Spectroscopy
AbstractViral membrane proteins play an integral role in the viral life cycle and are targets for antiviral drugs and vaccine design. Despite their importance, very little is known about the structure and function of this class of proteins due to several challenges in solving structures. In recent years, many efforts are underway to establish methods to characterize these proteins. Solution NMR spectroscopy offers a unique possibility to study structure, function, dynamics and inhibition of viral transmembrane proteins in a variety of membrane mimetic systems. Here I describe approaches to study transmembrane domains of viral membrane proteins using solution NMR spectroscopy. These methods were used to characterize two viral membrane proteins: p7 channel from hepatitis C virus (HCV) and gp41 fusion protein from human immunodeficiency virus (HIV).
For the p7 channel, conformational dynamics of the channel and modulation of this dynamics by the inhibitor rimantadine was determined. Relaxation dispersion data show that specific residues lining the cavity of the channel tip are subject to large chemical exchange, suggesting significant breathing. Moreover, the regions connecting the narrow and wide regions of the channel, where rimantadine binds, show strong relaxation dispersion. The presence of rimantadine decreases the conformational dynamics in this area. The data provides direct observation of μs-ms dynamics of the p7 channel and supports the molecular wedge mechanism of rimantadine inhibition of the HCV p7 channel.
For the HIV-1 gp41 protein, structure and the biochemical properties of the transmembrane domain were obtained in bicelles that mimic lipid bilayer. The structure shows that the domain forms a well-ordered trimer that protects a highly conserved arginine in the middle of membrane. The trimer is stabilized by an N-terminal coiled coil, and a C-terminal hydrophilic core, which may be structurally coupled to the cytoplasmic tail. Most of the single and double mutations of conserved residues failed to completely disrupt the trimer. However, drastic changes in the hydrophilic core altered trimer stability and antibody sensitivity of the functional envelope spike. Our results show how the gp41 transmembrane domain anchors, stabilizes and modulates the HIV envelope spike and suggest strategies for HIV-1 immunogen design.
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