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Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.

ABSTRACT: The Rho and Ras pathways play vital roles in cell growth, division and motility. Cross-talk between the pathways amplifies their roles in cell proliferation and motility and its dysregulation is involved in disease pathogenesis. One important interaction for cross-talk occurs between p120RasGAP (RASA1), a GTPase activating protein (GAP) for Ras, and p190RhoGAP (p190RhoGAP-A, ARHGAP35), a GAP for Rho. The binding of these proteins is primarily mediated by two SH2 domains within p120RasGAP engaging phosphorylated tyrosines of p190RhoGAP, of which the best studied is pTyr-1105. To better understand the interaction between p120RasGAP and p190RhoGAP, we determined the 1.75 Å X-ray crystal structure of the N-terminal SH2 domain of p120RasGAP in the unliganded form, and its 1.6 Å co-crystal structure in complex with a synthesized phosphotyrosine peptide, EEENI(p-Tyr)SVPHDST, corresponding to residues 1100-1112 of p190RhoGAP. We find that the N-terminal SH2 domain of p120RhoGAP has the characteristic SH2 fold encompassing a central beta-sheet flanked by two alpha-helices, and that peptide binding stabilizes specific conformations of the ?E-?F loop and arginine residues R212 and R231. Site-directed mutagenesis and native gel shifts confirm phosphotyrosine binding through the conserved FLVR motif arginine residue R207, and isothermal titration calorimetry finds a dissociation constant of 0.3 ± 0.1 ?M between the phosphopeptide and SH2 domain. These results demonstrate that the major interaction between two important GAP proteins, p120RasGAP and p190RhoGAP, is mediated by a canonical SH2-pTyr interaction.

SUBMITTER: Jaber Chehayeb R

PROVIDER: S-EPMC6938330 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe enzyme dihydropteroate synthase (DHPS) participates in the de novo synthesis of folate cofactors by catalyzing the formation of 7,8-dihydropteroate from condensation of p-aminobenzoic acid with 6-hydroxymethyl-7,8-dihydropteroate pyrophosphate. DHPS is absent from humans, who acquire folates from diet, and has been validated as an antimicrobial therapeutic target by chemical and genetic means. The bacterium Burkholderia cenocepacia is an opportunistic pathogen and an infective agent of cystic fibrosis patients. The organism is highly resistant to antibiotics and there is a recognized need for the identification of new drugs against Burkholderia and related Gram-negative pathogens. Our characterization of the DHPS active site and interactions with the enzyme product are designed to underpin early stage drug discovery.ResultsAn efficient recombinant protein expression system for DHPS from B. cenocepacia (BcDHPS) was prepared, the dimeric enzyme purified in high yield and crystallized. The structure of the apo-enzyme and the complex with the product 7,8-dihydropteroate have been determined to 2.35 Å and 1.95 Å resolution respectively in distinct orthorhombic crystal forms. The latter represents the first crystal structure of the DHPS-pterin product complex, reveals key interactions involved in ligand binding, and reinforces data generated by other structural studies. Comparisons with orthologues identify plasticity near the substrate-binding pocket and in particular a range of loop conformations that contribute to the architecture of the DHPS active site. These structural data provide a foundation for hit discovery. An intriguing observation, an artifact of the analysis, that of a potential sulfenamide bond within the ligand complex structure is mentioned.ConclusionStructural similarities between BcDHPS and orthologues from other Gram-negative species are evident as expected on the basis of a high level of sequence identity. The presence of 7,8-dihydropteroate in the binding site provides details about ligand recognition by the enzyme and the different states of the enzyme allow us to visualize distinct conformational states of loops adjacent to the active site. Improved drugs to combat infections by Burkholderia sp. and related Gram-negative bacteria are sought and our study now provides templates to assist that process and allow us to discuss new ways of inhibiting DHPS.

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Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.

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