Structural consequences of phosphorylation of two serine residues in the cytoplasmic domain of HIV-1 VpU.

The human immunodeficiency virus type 1 (HIV-1) protein U (VpU) is an accessory protein responsible for enhancement of viral particle release and down regulation of the T-lymphocyte coreceptor CD4. Direct binding between the cytoplasmic domains of CD4 and VpU as well as phosphorylation of serines 53 and 57 in the cytoplasmic domain of VpU plays a central role in CD4 downregulation. We investigated structural consequences of phosphorylation of the two serines using nuclear magnetic resonance spectroscopy. A uniformly 15N and 13C stable isotope-labeled 45-residue peptide comprising the cytoplasmic domain of VpU (VpUcyt) was recombinantly produced in E .coli. The peptide forms two helices (commonly referred to as helix 2 and 3) in the presence of membrane mimicking dodecylphosphocholine (DPC) micelles, which flank a flexible region containing the two phosphorylation sites. Phosphorylation does not cause any drastic structural changes in the secondary structure of VpUcyt. However, an N-terminal elongation of helix 3 and a slightly reduced helicity at the C-terminus of helix 2 are observed upon phosphorylation based on characteristic changes of 13Calpha and 13Cbeta chemical shifts. Phosphorylation also reduces the local mobility of the protein backbone in the loop region containing the phosphorylation sites according to heteronuclear 1H--15N nuclear Overhauser enhancement (NOE) data.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly ¹⁵N and ¹³C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the ¹H, ¹³C, and ¹⁵N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

NMR structure of the transmembrane and cytoplasmic domains of human CD4 in micelles

The human cluster determinant 4 (CD4) is a type I transmembrane glycoprotein involved in T-cell signalling. It is expressed primarily on the surface of T helper cells but also on subsets of memory and regulatory T lymphocytes (CD4⁺ cells). It serves as a coreceptor in T-cell receptor recognition of MHC II antigen complexes. Besides its cellular functions, CD4 serves as the main receptor for human immunodeficiency virus type I (HIV-1). During T-cell infection, the CD4 extracellular domain is bound by HIV-1 gp120, the viral surface glycoprotein, which triggers a number of conformational changes ultimately resulting in virion entry of the cell. Subsequently, CD4 is downregulated in infected cells by multiple strategies that involve direct interactions of the HIV-1 proteins VpU and Nef with the cytoplasmic part of CD4. In the present work, we describe the NOE-based solution structure of the transmembrane and cytoplasmic domains of the cystein-free variant of CD4 (CD4mut) in dodecylphosphocholine (DPC) micelles. Furthermore, we have characterized micelle-inserted CD4mut by paramagentic relaxation enhancement (PRE) agents and ¹H-¹⁵N heteronuclear NOE data. CD4mut features a stable and well-defined transmembrane helix from M372 to V395 buried in the micellar core and a cytoplasmic helix ranging from A404 to L413. Experimental data suggest the amphipathic cytoplasmic helix to be in close contact with the micellar surface. The role of the amphipathic helix and its interaction with the micellar surface is discussed with respect to the biological function of the full-length CD4 protein.