NMR Structure Determination of Vpu from HIV-1

Abstract

Virus protein “u” (Vpu) from HIV-1 is a member of the viroporin family of small viral membrane proteins. It consists of a transmembrane (TM) domain and a cytoplasmic domain, which are associated with different biological functions. Vpu transmembrane (TM) enhances the release of newly formed virus particles from the cells as an antagonist for the human immuno restriction factor, BST-2. It is a single alpha-helix that is capable of oligomerization and exhibits ion-channel activity when incorporated into a membrane environment. The wildtype TM conducts monovalent cations, while a mutant in which a histidine is introduced mimics the properties of a proton channel. The cytoplasmic domain causes the removal of CD4 receptor from ER and its subsequent degradation. It is essential to determine the three-dimensional structure of Vpu in order to obtain an understanding of its molecular mechanisms and to develop new classes of anti-viral drugs. A combination of solution and solid-state NMR experiments are used to obtain protein structural information from different constructs of Vpu. Solution experiments gave valuable information toward obtaining an initial vpu structure. Solid-state NMR techniques are employed to study the protein in lipid bilayer, which is a more biologically relevant environment than micelles. For magnetically oriented samples (OS), proteins are incorporated into DMPC/DHPC bicelles. To complement the OS structural data and alleviate resolution problems caused from the highly dynamic regions of the protein, novel MAS experiments are being implemented to study Vpu incorporated into DMPC liposomes. 15N, 13C chemical shift as well as 1H-15N, 1H-13C dipolar couplings can be extracted based on the new experiments and converted to equivalent structural restraints. Structural features of Vpu obtained from the combination of various techniques will be presented.