Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail

TY - JOUR

T1 - Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail

AU - Gavriljuk, Konstantin

AU - Gazdag, Emerich-Mihai

AU - Itzen, Aymelt

AU - Kötting, Carsten

AU - Goody, Roger S

AU - Gerwert, Klaus

PY - 2012/12/26

Y1 - 2012/12/26

N2 - Rab GTPases, key regulators of vesicular transport, hydrolyze GTP very slowly unless assisted by Rab GTPase-activating proteins (RabGAPs). Dysfunction of RabGAPs is involved in many diseases. By combining X-ray structure analysis and time-resolved FTIR spectroscopy we reveal here the detailed molecular reaction mechanism of a complex between human Rab and RabGAP at the highest possible spatiotemporal resolution and in atomic detail. A glutamine residue of Rab proteins (cis-glutamine) that is essential for intrinsic activity is less important in the GAP-activated reaction. During generation of the RabGAP·Rab:GTP complex, there is a rapid conformational change in which the cis-glutamine is replaced by a glutamine from RabGAP (trans-glutamine); this differs from the RasGAP mechanism, where the cis-glutamine is also important for GAP catalysis. However, as in the case of Ras, a trans-arginine is also recruited to complete the active center during this conformational change. In contrast to the RasGAP mechanism, an accumulation of a state in which phosphate is bound is not observed, and bond breakage is the rate-limiting step. The movement of trans-glutamine and trans-arginine into the catalytic site and bond breakage during hydrolysis are monitored in real time. The combination of X-ray structure analysis and time-resolved FTIR spectroscopy provides detailed insight in the catalysis of human Rab GTPases.

AB - Rab GTPases, key regulators of vesicular transport, hydrolyze GTP very slowly unless assisted by Rab GTPase-activating proteins (RabGAPs). Dysfunction of RabGAPs is involved in many diseases. By combining X-ray structure analysis and time-resolved FTIR spectroscopy we reveal here the detailed molecular reaction mechanism of a complex between human Rab and RabGAP at the highest possible spatiotemporal resolution and in atomic detail. A glutamine residue of Rab proteins (cis-glutamine) that is essential for intrinsic activity is less important in the GAP-activated reaction. During generation of the RabGAP·Rab:GTP complex, there is a rapid conformational change in which the cis-glutamine is replaced by a glutamine from RabGAP (trans-glutamine); this differs from the RasGAP mechanism, where the cis-glutamine is also important for GAP catalysis. However, as in the case of Ras, a trans-arginine is also recruited to complete the active center during this conformational change. In contrast to the RasGAP mechanism, an accumulation of a state in which phosphate is bound is not observed, and bond breakage is the rate-limiting step. The movement of trans-glutamine and trans-arginine into the catalytic site and bond breakage during hydrolysis are monitored in real time. The combination of X-ray structure analysis and time-resolved FTIR spectroscopy provides detailed insight in the catalysis of human Rab GTPases.

KW - Animals

KW - Biocatalysis

KW - Catalytic Domain

KW - DNA Mutational Analysis

KW - GTPase-Activating Proteins

KW - Glutamine

KW - Guanosine Triphosphate

KW - Humans

KW - Hydrolysis

KW - Kinetics

KW - Mammals

KW - Models, Molecular

KW - Spectroscopy, Fourier Transform Infrared

KW - rab1 GTP-Binding Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1073/pnas.1214431110

DO - 10.1073/pnas.1214431110

M3 - SCORING: Journal article

C2 - 23236136

VL - 109

SP - 21348

EP - 21353

JO - P NATL ACAD SCI USA

JF - P NATL ACAD SCI USA

SN - 0027-8424

IS - 52

ER -