SOLUTION STRUCTURE OF THE CYTOPLASMIC DOMAIN OF THE HUMAN CD4 GLYCOPROTEIN BY CD AND H-1-NMR SPECTROSCOPY - IMPLICATIONS FOR BIOLOGICAL FUNCTIONS

The human T cell receptor CD4 is a type I integral membrane glycoprote in that is involved in T cell activation and also acts as the primary coreceptor for human immunodeficiency viruses (HIV), Here the structur e of a synthetic 38 amino acid peptide corresponding to the complete c ytoplasmic domain of CD4 (CD4(CYTO)) has been investigated under a var iety of solution conditions using a combination of circular dichroism and homonuclear two-dimensional H-1 nuclear magnetic resonance spectro scopy. In the presence of the membrane mimetic 2,2,2-trifluoroethanol (TFE), a conformational change of CD4(CYTO) from a random coil to an a -helical structure was observed. In keeping with this, CD4(CYTO) has t he potential to associate with membranes as demonstrated by binding st udies of in vitro phosphorylated CD4(CYTO) with microsomal membranes. Both chemical shift and nuclear Overhauser enhancement data in 50% 2,2 ,2-trifluoroethanol solution provide direct experimental evidence for the predominance of a short amphiphatic alpha-helix that is approximat ely 4 turns in length and extends from positions Arg-402 to Lys-417. T he present data provide, for the first time, compelling experimental e vidence that only a fraction of CD4(CYTO) has a propensity for adoptin g secondary structure under conditions that are assumed to exist at or near to the membrane surface and that this a-helical structure is loc ated in the membrane-proximal region of CD4(CYTO). The N-terminal resi dues, that link the alpha-helix to the transmembrane anchor of CD4, an d a substantial C-terminal portion (14-18 residues) of CD4(CYTO) are u nstructured under the solution conditions investigated. Correlation of our structural data with recent studies on the biological activity of CD4(CYTO) indicates that the alpha-helix is of crucial importance for the interaction of CD4 with Nef and Vpu in the process of HIV-mediate d CD4 down-regulation.