Cholesterol sensing by CD81 is important for hepatitis C virus entry

Machaela Palor,Lenka Stejskal,Piya Mandal,Annasara Lenman,María Pía Alberione,Jared Kirui,Rebecca Moeller,Stefan Ebner,Felix Meissner,Gisa Gerold,Adrian J Shepherd,Joe Grove

doi:10.1074/jbc.ra120.014761

Abstract

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease.

Highlights

Machaela Palor1,‡, Lenka Stejskal1,2,‡, Piya Mandal1, Annasara Lenman3,4, María Pía Alberione4, Jared Kirui4, Rebecca Moeller4, Stefan Ebner5, Felix Meissner5,6, Gisa Gerold3,4,7, Adrian J
The crystal structure of CD81 (Protein Data Bank code 5TCX [26]) reveals an intramembrane cavity bounded by the four transmembrane domains; this contains a single molecule of cholesterol, which is coordinated by hydrogen bonding to the side chains of two residues, Asn18 and Glu219 (Fig. 1A)
We designed a series of mutants to disrupt this interaction: E219A and E219Q, which were previously demonstrated to reduce CD81–cholesterol association [26], and an N18A/E219A double mutation, which should remove all possibility of hydrogen bonding to cholesterol

Summary

Introduction

Machaela Palor1,‡, Lenka Stejskal1,2,‡, Piya Mandal1, Annasara Lenman3,4, María Pía Alberione4, Jared Kirui4 , Rebecca Moeller4, Stefan Ebner5, Felix Meissner5,6 , Gisa Gerold3,4,7 , Adrian J. This suggests the cholesterol-binding pocket of CD81 is important for HCV infection, but viral entry may not be directly dependent on cholesterol association. These data are consistent with specific association between plasma-membrane cholesterol and CD81 and that this interaction can be reduced by mutating residues in the cholesterol-binding pocket of CD81.

Results

Conclusion