Characterizing the Interaction of Human CD4 and the HIV-1 accessory Protein VpU using Liquid State NMR

Abstract

Human CD4 is a 433 residue transmembrane protein involved in the body's adaptive immune response. The extracellular domain of CD4 serves as primary receptor of the human immunodeficiency virus (HIV-1) and binds the viral glycoprotein gp120. The cytoplasmic domain of the 81 residue Virus protein U (VpU) plays an important role in downregulation of CD4. It directly binds the cytoplasmic domain of CD4 in the endoplasmic reticulum, which initiates a series of events resulting in CD4 degradation in the proteasome. We characterized the CD4 - VpU interaction by liquid state NMR spectroscopy utilizing paramagnetic relaxation enhancement (PRE). The PRE effect is a distance-dependent enhancement of the spin relaxation in magnetically active nuclei in the vicinity of a paramagnetic centre. PRE leads to line broadening or complete quenching of NMR signals and indicates spatial proximity to the probe. We recorded NMR spectra of the 15N-labelled cytoplasmic domain of VpU in the presence of varying amounts of a single-cysteine-mutant of CD4(372-433), which was labelled with the active PRE-probe, methanethiosulfonate (MTSL). This CD4 polypeptide contains the single transmembrane and the C-terminal cytoplasmic domain of CD4. Experiments were conducted in membrane-mimicking dodecyl phosphocholine (DPC) micelles. Spectra recorded in the presence of paramagnetic MTSL show reduced NMR signal intensities of certain amino acids of 15N-labeled VpU. Additionally, we studied chemical shift perturbations (CSP's) of VpU resonances observed on titrating increasing concentrations of unlabelled CD4. Such chemical shift changes are expected for residues in the binding interface but may also occur at remote sites due to allosteric effects. Our data reveal VpU residues involved in CD4 binding, provide insight into the exchange regime, and yield an estimate of the binding affinity.