Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail
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Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail. / Gavriljuk, Konstantin; Gazdag, Emerich-Mihai; Itzen, Aymelt; Kötting, Carsten; Goody, Roger S; Gerwert, Klaus.
in: P NATL ACAD SCI USA, Jahrgang 109, Nr. 52, 26.12.2012, S. 21348-53.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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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 -