Protein engineering and biochemical/biophysical approaches for structural studies of small membrane proteins and their complexes: Application to viroporins

Abstract

We study the structure and function of small viral membrane proteins, which are expressed in the host and function in cellular membranes. Typically, they are multi‐domain multi‐functional proteins aiding virus adaptation and proliferation. Detailed characterization of the structure‐function relationship of these proteins will help to better understand virus‐host interactions and possibly develop protein inhibitors. However, such characterization is challenging due to the proteins’ small size and dynamic nature. Our focus is on viroporins, namely the Vpu, p7 and E proteins from HIV‐1, HCV, and CoV, respectively. Viroporins form ion‐conducting oligomers in cellular membranes leading to increased membrane permeability affecting ion homeostasis in infected cells. In addition, these proteins have soluble domains involved in interactions with host proteins. Currently, the structures of full‐length viroporins are largely uncharacterized and the information about the mechanism of oligomer formation and inter‐domain communication is very limited. Here, we report our effort to produce and solve the structure of stable viroporin oligomers in lipid environment. We use protein engineering to generate a range of fusion constructs aiming to increase the protein/oligomer size and reduce the conformational heterogeneity, thus making the protein amenable for structural determination by electron microscopy (EM) techniques. We produced, expressed in E. coli and purified, several soluble constructs of Vpu, p7 and E proteins fused at their N‐terminals to maltose binding protein (MBP). Thereafter, we interacted these proteins with lyso lipid mixtures of PC and PG, and their oligomerization was observed by negative staining EM and pulse electron paramagnetic resonance (EPR). We further use size‐exclusion chromatography (SEC) to assess the heterogeneity of viroporin oligomers in lyso PC/PG. Indeed, the protein elution exhibits a relatively broad chromatographic profile, indicating the presence of more than one oligomeric form. We further use SEC to test different protein‐to‐lipid molar ratios and the effect of viroporin inhibitor binding, aiming to shift the equilibrium toward stable functional viroporin oligomers. We will also discuss results from EM studies on these stable oligomers and EPR studies on spin‐labeled viroporin assembly. The progress we made in generating protein constructs and assessing protein oligomerization and stability in lipid provides a great promise for the development of successful methodology to determine the structure of full‐length viroporin. This methodology could also be applicable to other small oligomeric membrane proteins.