Preliminary X-ray Crystallographic Analysis Of SH3 Domain Of Human AHI1

AHI1 is a common helper provirus integration site for murine leukemias and lymphomas and shown to be closely linked to the c-myb proto-oncogene. Recently researches have showed that AHI1 can affect the production of leukemogen and mutations in AHI1 are a main cause of Joubert Syndrome (JS). Recently research has also showed that AHI1 binds tightly to huntingtin-associated protein 1 (HAP1), which involved in intracellular trafficking and endocytosis of membrane receptors, and forms a stable protein complex in the brain. The interaction between AHI1 and HAP1 is important for the early development of the brain and offer insight into the pathogenesis of JS, but the details of molecular mechanism are unknown. Human AHI1 gene encodes a 1,196 amino-acid protein (AHI1 or Jouberin), which is comprised of an N-terminal coiled-coiled domain, 7 WD40 repeats, an SH3 domain, and several potential SH3 binding sites. Interestingly, the SH3 domain of AHI1 was rather unique since it has the key conserved residues but shares little amino acid sequence identity with the"classical"SH3 domains of Abl, Fyn, and Lck,Further insight could be provided if an atomic structure of AHI1 were available. As an initial effort towards this goal, we have chosen the SH3 domain, a common domain of signaling molecules involved in protein-protein interactions as our research object. In this report, we have cloned and expressed the recombinant human Ahi1 SH3 domain protein using an Escherichia coli expression system. We purified the recombinant protein to an apparent homogenous state and carry out its preliminary characterization and crystallization trials. We also verified the possibility of SH3 domain of AHI1 and HAP1 interaction by GST pull down assay. Recombinant protein was purified in a two-step procedure using affinity and size-exclusion chromatography and the purity of it was about >95%. DLS result show the AHI1 SH3 domain exists predominantly in a single aggregation state in solution. Crystals suitable for diffraction experiments were obtained in 0.2 M Ammonium sulfate, 0.1 M Sodium acetate trihydrate pH 4.6, 30% w/v Polyethylene glycol monomethyl ether 2,000 by a seating-drop vapor diffusion method in 20℃. The crystal diffracted to 2.5 A ? resolution and belonged to the tetragonal space group P41212, with unit-cell parameters a=67.377, b=67.377, c=98.549?,α=β=γ=90°, With one monomer in the asymmetric unit, the Matthews volume was calculated to be 1.94?3 Da-1and the estimated solvent content was 36.69%. GST pull down assay show that AHI1 SH3 domain can not interact with the P-rich region of HAP1 and their interaction maybe mediate by other domain.