Abstract
The human immunodeficiency virus type 1 (HIVโ1) protein U (VpU) is an accessory protein responsible for enhancement of viral particle release and down regulation of the Tโlymphocyte 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 ^15^N and ^13^C stable isotopeโlabeled 45โresidue 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 Nโterminal elongation of helix 3 and a slightly reduced helicity at the Cโterminus of helix 2 are observed upon phosphorylation based on characteristic changes of ^13^C~ฮฑ~ and ^13^C~ฮฒ~ chemical shifts. Phosphorylation also reduces the local mobility of the protein backbone in the loop region containing the phosphorylation sites according to heteronuclear ^1^H๏ฃฟ^15^N nuclear Overhauser enhancement (NOE) data. Copyright ยฉ 2008 European Peptide Society and John Wiley & Sons, Ltd.