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

Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21348-53. doi: 10.1073/pnas.1214431110. Epub 2012 Dec 12.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biocatalysis*
  • Catalytic Domain
  • DNA Mutational Analysis
  • GTPase-Activating Proteins / chemistry
  • GTPase-Activating Proteins / metabolism*
  • Glutamine / metabolism
  • Guanosine Triphosphate / metabolism
  • Humans
  • Hydrolysis
  • Kinetics
  • Mammals / metabolism*
  • Models, Molecular*
  • Spectroscopy, Fourier Transform Infrared
  • rab1 GTP-Binding Proteins / chemistry
  • rab1 GTP-Binding Proteins / metabolism*

Substances

  • GTPase-Activating Proteins
  • TBC1D20 protein, human
  • Glutamine
  • Guanosine Triphosphate
  • Rab1B protein, human
  • rab1 GTP-Binding Proteins

Associated data

  • PDB/4HL4
  • PDB/4HLQ