New NMR Assignment 1 H, 13 C, and 15 N Assignment of the Second PH Domain of Human Pleckstrin (234–350)

Edlich, Christian; Simon, Bernd; Sattler, Michael; Muhle‐Goll, Claudia

doi:10.1007/s10858-005-6053-x

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“…At pH 6.6 many of the backbone amide signals in the loops surrounding the phosphoinositide binding site were no longer observable. Unobservable loop resonances are not uncommon for PH domains, and they can be due to increased solvent exchange with increased pH, as well as conformational exchange of the loop backbone structures on the intermediate NMR timescale (100 ns-0.1 s) [38,39,40]. Because PZA becomes increasingly unstable below pH 7.2, backbone amide assignments of 2D 15 N HSQC spectra of the PH domain were also made at pH 7.2 and 8.0 by analogy to the spectrum at pH 6.6.…”

Section: Resultsmentioning

confidence: 99%

Dynamic interaction between Arf GAP and PH domains of ASAP1 in the regulation of GAP activity

Luo

Jenkins

Randazzo

et al. 2008

Cellular Signalling

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ASAP family Arf GAPs induce the hydrolysis of GTP bound to the Ras superfamily protein Arf1, regulate cell adhesion and migration and have been implicated in carcinogenesis. The ASAP proteins have a core catalytic domain of PH, Arf GAP and Ank repeat domains. The PH domain is necessary for both biological and catalytic functions of ASAP1 and has been proposed to be integrally folded with the Arf GAP domain. Protection studies and analytical ultracentrifugation studies previously reported indicated that the domains are, at least partly, folded together. Here, using NMR spectroscopy and biochemical analysis, we have further tested this hypothesis and characterized the interdomain interaction. A comparison of NMR spectra of three recombinant proteins comprised of either the isolated PH domain of ASAP1, the Arf GAP and ankyrin repeat domain or all three domains indicated that the PH domain did interact with the Arf GAP and Ank repeat domains; however, we found a significant amount of dynamic independence between the PH and Arf GAP domains, consistent with the interactions being transient. In contrast, the Arf GAP and Ank repeat domains form a relatively rigid structure. The PH-Arf GAP domain interaction partially occluded the phosphoinositide binding site in the soluble protein, but binding studies indicated the PIP2 binding site was accessible in ASAP1 bound to a lipid bilayer surface. Phosphoinositide binding altered the conformation of the PH domain, but had little effect on the structure of the Arf GAP domain. Mutations in a loop of the PH domain that contacts the Arf GAP domain affected PIP2 binding and the K m and k cat for converting Arf1•GTP to Arf1•GDP. Based on these results, we generated a homology model of a composite PH/Arf GAP/Ank repeat domain structure. We propose that the PH domain contributes to Arf GAP activity by either binding to or positioning Arf1•GTP that is simultaneously bound to the Arf GAP domain.

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Section: Resultsmentioning

confidence: 99%