Strukturelle Untersuchungen an membranassoziierten Proteinen: NMR-Strukturen des HIV-1 Virus Protein U (39-81) und des humanen CD4 (372-433)

The transmembrane glycoprotein CD4 (cluster determinant 4) plays a prominent role in the adaptive immune response. CD4 is displayed primarily on the surface of T helper cells, but also on subsets of memory and regulatory T lymphocytes. Binding of the lymphocyte specific tyrosine kinase p56LcK to the cytoplasmic domain of CD4 is crucial for antigen receptor-mediated signal transduction. The human immunodeficiency virus (HIV) utilizes CD4 as the main receptor for T-cell invasion. The virus has developed multiple strategies for down-regulation of CD4 in infected cells. The HIV-1 protein U (VpU) is an accessory protein responsible for enhancement of virus particle release and downregulation of CD4. Physical interactions of viral proteins VpU and Nef with the cytoplasmic tail of CD4 initiate a cascade of events leading to degradation of CD4. Phosphorylation of serines 53 and 57 of VpU plays an essential role in CD4 downregulation. Aim of this work was the cloning and production of the interacting fragments of human CD4 and HIV-1 VpU, as well as their structural and dynamical characterisation using NMR-, CD-spectroscopical and molecular biology methods. As the specified interaction of CD4 and VpU takes place close to the membrane of the endoplasmic reticulum, most observations were performed in presence of model membrane systems. To insure in vitro complex binding between CD4 and ligands (e.g. VpU, Nef, p56lck) in direct membrane proximity, CD4 was decided to be membrane-anchored with its transmembrane domain. CD4 is a protein 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. A synthetic gene encoding CD4 amino acid residues 372 to 433 was cloned ; heterologous expression and purification of the CD4 fragment was carried out. The incorporation of the recombinant wild-type CD4tmcyt fragment in lipid membranes and physical interaction with the cytoplasmic domain of VpU (VpUcyt) was demonstrated by centrifugation assays followed by reversed phase chromatographic analysis of the composition of the proteoliposomes. Circular dichroism (CD) spectroscopy of CD4tmcyt in dodecylphosphocholine (DPC) micelles yields about 40% a-helical content. High resolution NMR spectra of uniformly $^{13}$C/$^{15}$ N-labeled CD4tmcyt peptide in membrane simulating DPC micelles proves the possibility of solution NMR studies of this CD4 fragment and its molecular complexes. For easier handling, a cystein-free variant of CD4tmcyt was constructed (CD4mut). The five cysteine residues of the cytoplasmic region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in $\textit{E. coli}$ and purified. Functional binding activity of CD4mut to protein VpUcyt was verified, the Kd of this complex was estimated to be 250 $\mu$M or lower. Close to complete NMR resonance assignment of the $^{1}$H, $^{13}$C, and $^{15}$N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating DPC micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane [...