Faculty Opinions recommendation of RanGAP mediates GTP hydrolysis without an arginine finger.

GTPaseactivatingproteins (GAPs) down-regulate Ras-like proteins by stimulating their GTP hydrolysis, and a malfunction of this reaction leads to disease formation. In most cases, the molecular mechanism of activation involves stabilization of a catalytic Gln and insertion of a catalytic Arg into the active site by GAP. Rap1 neither possesses a Gln nor does its cognate Rap-GAP employ an Arg. Recently it was proposed that RapGAP provides a catalytic Asn, which substitutes for the Gln found in all other Ras-like proteins (Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R., and Wittinghofer, A. (2004)Nature429, 197–201). Here, RapGAP-mediated activation has been investigated by time-resolved Fourier transform infrared spectroscopy. Although the intrinsic hydrolysis reactions of Rap and Ras are very similar, the GAP-catalyzed reaction shows unique features. RapGAP binding induces a GTP*conformation in which the three phosphate groups are oriented such that they are vibrationally coupled to each other, in contrast to what was seen in the intrinsic and the Ras·RasGAP reactions. However, the charge shift toward β-phosphate observed with RasGAP was also observed for RapGAP. A GDP·Piintermediate accumulates in the GAP-catalyzed reaction, because the release of Piis eight times slower than the cleavage reaction, and significant GTP synthesis from GDP·Piwas observed. Partial steps of the cleavage reaction are correlated with structural changes of protein side groups and backbone. Thus, the Rap·RapGAP catalytic machinery compensates for the absence of acis-Gln by atrans-Asn and for the catalytic Arg by inducing a different GTP conformation that is more prone to be attacked by a water molecule.

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The Ability of GAP1IP4BP To Function as a Rap1 GTPase-Activating Protein (GAP) Requires Its Ras GAP-Related Domain and an Arginine Finger Rather than an Asparagine Thumb

Molecular and Cellular Biology ◽

10.1128/mcb.00427-09 ◽

2009 ◽

Vol 29 (14) ◽

pp. 3929-3940 ◽

Cited By ~ 18

Author(s):

Sabine Kupzig ◽

Dalila Bouyoucef-Cherchalli ◽

Sam Yarwood ◽

Richard Sessions ◽

Peter J. Cullen

Keyword(s):

Binding Site ◽

Molecular Model ◽

Detectable Effect ◽

Gtp Hydrolysis ◽

Amino Terminal ◽

Gtpase Activating Proteins ◽

Close Proximity ◽

Carboxy Terminal ◽

Arginine Finger

ABSTRACT GAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) that includes GAP1m, CAPRI, and RASAL. Composed of a central Ras GAP-related domain (RasGRD), surrounded by amino-terminal C2 domains and a carboxy-terminal PH/Btk domain, these proteins, with the notable exception of GAP1m, possess an unexpected arginine finger-dependent GAP activity on the Ras-related protein Rap1 (S. Kupzig, D. Deaconescu, D. Bouyoucef, S. A. Walker, Q. Liu, C. L. Polte, O. Daumke, T. Ishizaki, P. J. Lockyer, A. Wittinghofer, and P. J. Cullen, J. Biol. Chem. 281:9891-9900, 2006). Here, we have examined the mechanism through which GAP1IP4BP can function as a Rap1 GAP. We show that deletion of domains on either side of the RasGRD, while not affecting Ras GAP activity, do dramatically perturb Rap1 GAP activity. By utilizing GAP1IP4BP/GAP1m chimeras, we establish that although the C2 and PH/Btk domains are required to stabilize the RasGRD, it is this domain which contains the catalytic machinery required for Rap1 GAP activity. Finally, a key residue in Rap1-specific GAPs is a catalytic asparagine, the so-called asparagine thumb. By generating a molecular model describing the predicted Rap1-binding site in the RasGRD of GAP1IP4BP, we show that mutagenesis of individual asparagine or glutamine residues that lie in close proximity to the predicted binding site has no detectable effect on the in vivo Rap1 GAP activity of GAP1IP4BP. In contrast, we present evidence consistent with a model in which the RasGRD of GAP1IP4BP functions to stabilize the switch II region of Rap1, allowing stabilization of the transition state during GTP hydrolysis initiated by the arginine finger.

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Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102699 ◽

1988 ◽

Vol 46 ◽

pp. 122-123

Author(s):

R.A Walker ◽

S. Inoue ◽

E.D. Salmon

Keyword(s):

Dynamic Instability ◽

Microtubule Dynamic ◽

Gtp Hydrolysis ◽

Abrupt Transition ◽

Microtubule Associated Proteins ◽

Asymmetrical Structure ◽

Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

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