Biochemical and structural studies of HIV-1 MA membrane interaction and ubiquitin recognition

This dissertation describes biochemical and structural studies of HIV-1 MA membranes interactions (Chapter 2) and two different conserved ubiquitin binding motifs: the Ubiquitin Interaction Motif (UIM, Chapter 3) and the Npl4 Zinc Finger (NZF, Chapter 4). HIV-1 MA is the N-terminal domain of Gag and is largely responsible for Gag membrane targeting and membrane binding. Both the N-myristoyl group of MA and a highly basic region near the N-terminus are important for MA membrane binding. NMR spectroscopic studies were used to define the sites within MA that change structure upon myristoylation. These studies indicated that the myristoyl group may pack against the N-terminal helices I and II and generally support a myristoyl switch model for MA membrane binding. Biochemical and NMR spectroscopic studies were also performed to map the MA binding site of PtdIns(3,4,5)P3, an important regulator for Gag assembly. The binding site is composed of basic residues that also comprise the membrane binding surface of MA and corresponds well with the sulfate binding site observed in the crystal structure of the MA trimer. This interaction may help target Gag to the plasma membrane for viral assembly. Ubiquitylation is an important posttranslational modification and regulates many important cellular processes. Ubiquitylated substrate proteins are frequently recognized by downstream effector proteins that contain conserved ubiquitin binding motifs. The UIM and NZF motifs are two such recently identified ubiquitin binding motifs. Biochemical analyses demonstrated that both UIM and NZF motifs bind directly and specifically to ubiquitin. They both interact with ubiquitin through a hydrophobic patch surrounding residue 144, highlighting the importance of the 144 surface for ubiquitin function. A crystal structure of the Vps27p UIM revealed that the UIM forms an amphipathic helix. The solution structure of the NZF domain was determined using NMR. The structure is comprised of four antiparallel ?-strands linked by three well ordered loops. The ubiquitin binding site on NZF is also primarily hydrophobic, and chemical shift mapping and sequence conservation analyses demonstrated that a T-Phi; dipeptide sequence in the ubiquitin binding site is highly conserved and forms the major recognition site for ubiquitin.