SH3 domains are found in proteins associated with receptor tyrosine kinase signal transduction complexes. The solution structure of the SH3 domain of the 85 kd regulatory subunit of phosphatidylinositol 3-kinase is shown to be a compact beta barrel consisting of five beta strands arranged in two beta sheets of three and two strands. The structure is similar to that of chicken brain alpha spectrin but represents a distinct class of SH3 domain, with an insertion between the second and third beta strands that may influence binding specificity. 1H chemical shift changes induced by complex formation with a synthetic peptide derived from the SH3-binding protein dynamin, together with amino acid sequence comparisons, suggest that the ligand-binding site consists of a hydrophobic surface flanked by two charged loops.
"Base on the assignment backbone resonances of PI3-SH3 under native condition, we calculated the secondary structure populations of individual amino acids as a function of protein sequence (Fig. 2). The exceptionally long n-src loop exhibits high helical content, which is consistent with earlier reports (Booker et al. 1993; Koyama et al. 1993a, b; Liang et al. 1996). We next compare our backbone 13 C chemical shifts of PI3-SH3 (Ca, Cb and C 0 ) in native state with those in amyloid state, which were assigned by the use of solid state NMR spectroscopy (Bayro et al. 2010). "
[Show abstract][Hide abstract] ABSTRACT: We report here the near complete assignments of native bovine PI3-SH3 domain, which has been a model system for protein folding, misfolding and amyloid fibril formation. The use of (13)C-detected protonless NMR spectroscopy is instrumental in assigning the spin system of the proline residue at the C-terminus in addition to the missing resonances in proton-based NMR spectra due to rapid solvent exchange. It also helps assign the resonances of all three proline amine nitrogen nuclei, which are underrepresented in the database. Comparison of the backbone (13)C resonances of PI3-SH3 in its native and amyloid fibril states shows that the aggregation of PI3-SH3 is accompanied by major conformational rearrangements.
"The structural organization of the regulatory subunit p85α of the class IA PI3K has been very well studied. Except for the two coiled-coil regions CC1 and CC2, the documented structures of the well-folded SH3 (31), BCR (32), N–SH2 (33) and C–SH2 (34) make p85α a good benchmark for demonstrating the feasibility and efficiency of domain trapping strategies. "
[Show abstract][Hide abstract] ABSTRACT: Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.
Nucleic Acids Research 07/2011; 39(18):e125. DOI:10.1093/nar/gkr548 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in human cancer and represents an attractive target for therapies based on small molecule inhibitors. PI3K isoforms play an essential role in the signal transduction events activated by cell surface receptors including receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs). There are eight known PI3K isoforms in humans, which have been subdivided into three classes (I-III). Therefore PI3Ks show considerable diversity and it remains unclear which kinases in this family should be targeted in cancer. The class I(A) of PI3K comprises the p110alpha, p110beta and p110delta isoforms, which associate with activated RTKs. In human cancer, recent reports have described activating mutations in the PIK3CA gene encoding p110alpha, and inactivating mutations in the phosphatase and tensin homologue (PTEN) gene, a tumour suppressor and antagonist of the PI3K pathway. The PIK3CA mutations described in cancer constitutively activate p110alpha and, when expressed in cells drive oncogenic transformation. Moreover, these mutations cause the constitutive activation of downstream signaling molecules such as Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K) that is commonly observed in cancer cells. In addition to p110alpha, the other isoforms of the PI3K family may also play a role in human cancer, although their individual functions remain to be precisely identified. In this review we will discuss the evidence implicating individual PI3K isoforms in human cancer and their potential as drug targets in this context.
Current Genomics 09/2007; 8(5):271-306. DOI:10.2174/138920207782446160 · 2.34 Impact Factor
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