Cryo-EM snapshots and protein spin labeling in structural studies of HIV-1 Vpu.

Abstract

We study the structure-function relationship of the HIV-1 encoded Vpu protein. HIV-1 causes HIV/AIDS leading to severe health complications. Understanding the molecular mechanisms of proteins that are key to the virus is important for the design of efficient therapeutics. Vpu forms ion-conducting oligomers in cellular membranes leading to cellular ion disbalance; its monomer is deemed functionally important as well, mostly through Vpu-host proteins interaction. However, the detailed structure of full-length (FL) Vpu in its oligomeric and monomeric states are currently poorly understood. Here, we describe our efforts to identify conditions under which we obtained high-quality cryo-EM data for structural determinations. In our studies, we used a chimera protein of maltose binding protein fused to the N-terminal of FL Vpu (MBP-Vpu). In initial assessments by negative staining EM, we found that MBP-Vpu forms stable oligomers, likely pentamers, in solution. Then, we subjected these pentamers to cryo-EM imaging and obtained high-quality data, which will be used in further structure reconstruction. We will also present cryo-EM data for MBP-Vpu oligomer residing in short-chain lipids, which will be useful to solve the structure of this protein in a lipid environment. Further, we will present results for the expression and purification of the Vpu C-terminal domain as a fusion construct with SUMO protein, and several single cysteine mutants located in the Vpu C-terminal helix 2, a protein region that associates with the membrane surface. These mutants will be spin-labeled and used in cw EPR studies to assess the conformational dynamics of FL Vpu and identify the membrane-contact sites in the Vpu C-terminal region. Overall, we made substantial progress towards characterizing the structure of FL Vpu.