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.
Wittlich
Marc
M
Institut für Neurowissenschaften und Biophysik, Biomolekulare NMR, Forschungszentrum Jülich, Jülich,Germany.
Koenig
Bernd W
BW
Willbold
Dieter
D
D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry*; D054298:Human Immunodeficiency Virus Proteins / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010768:Phosphoserine / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000378:metabolism*; D000086082:Viroporin Proteins
D054298:Human Immunodeficiency Virus Proteins; D010768:Phosphoserine; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins