Crystal structure analysis and solution studies of human Lck SH3; zinc induced homodimerization competes with the binding of proline rich motifs

In cytosolic Src type tyrosine kinases the Src type homology 3 SH3 domain binds to an internal proline rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src type kinase Lck plays an important role during T cell activation and development, since it phosphorylates the T cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near atomic resolution of 1.0 . Unexpectedly, the Lck SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck SH3 domain also forms dimers in solution. The Zn2 dissociation constant from the Lck SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck SH3 homodimerization and binding of regulatory proline rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc binding site are highly conserved among Lck orthologues but not in other Src type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T cell activatio

Crystal Structure of the Human Cytomegalovirus pUL50 pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus Host Protein Interactions

Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1–175 and 50–292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59–87) embraces pUL50 and contributes 1510 Å(2) to the total interface area (1880 Å(2)). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy

Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M

Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM+ HDL contained S1P, whereas ApoM− HDL did not. Moreover, HDL in Apom−/− mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P–human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM+ HDL induced S1P1 receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM− HDL did not. Importantly, lack of S1P in the HDL fraction of Apom−/− mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P1 receptor on endothelial cells, is a vasculoprotective constituent of HDL