The Structural Biology Research Of Human Osteoclast Stimulating Factor And Preliminary Crystallography Of LAD From Proteus Vulgaris

(â… ) Osteoclast is the main cell responsible for degradation of bone matrix. Its differentiation and activity requires M-CSF (macrophage colony stimulating factor) and RANKL (receptor activator of nuclear factor kappaB ligand) mediated pathways. Tyrosine protein kinase Src, involved in both M-CSF and RANKL mediated signal networks, is also found to be essential for osteoclast function. Osteoclast stimulating factor (OSF), composed of a short proline rich region, one SH3 domain and ankyrin repeats, is a intracellular protein produced by osteoclasts and shown to indirectly enhance osteoclast formation and activity. A mouse homolog of OSF called SH3P2 can bind proline-rich fragment of Cbl and form an OSF-Cbl-Src triple protein complex, suggesting OSF was involved in Src and Cbl mediated pathways.We cloned, expressed and purified the human OSF protein. Here, we present crystal structures of human OSF in two space groups grown from the same condition. Form I crystal structure is determined by multi-wavelength anomalous diffraction (MAD) at 2.57A resolution in space group P2₁2₁2₁ with cell parameters a=48.06 (?), b=56.86 (?) and c=l70.12 (?).The final R value and Rfree value are 21.0% and 28.4%, respectively. Formâ… crystal structure is determined by molecular replacement at 1.95 (?) resolution in space group P1 with cell parameters a=28.31(?), b=57.94 (?), c=60.48(?),α=61.75°,β=76.53°,γ=90.08°.The final R value and Rfree value are 17.8% and 23.8%, respectively. Models in both crystal forms contain one SH3 domain and four ankyrin repeats, but the relative position of the two domains is different in two forms. It is the first reported protein structure of such a SH3-ANKs domain sequence. The peptide-binding groove of OSF-SH3 domain adopts a conformation different from other SH3 domains and probably needs a conformational switch for binding to its proline rich ligand. This different status of OSF-SH3 is possibly regulated by the C-terminal ankyrin repeats. (â…¡)L-amino acid deaminase(LAD)from Proteus vulgaris is an enzyme that cancatalyze L-type amino acids.Significant conserved motif is found in LAD identifiedas a FAD-binding domain and probably LAD is a L-amino acid oxidase．We cloned,expressed and purified the full length LAD and a N-terminal truncation formâ–³N29LAD.Crystals ofâ–³N29LAD suitable for diffraction were obtained and a 2.9 (?)data set was Collected.Preliminary X-ray diffraction analysis was applied.