Obligatory role in GTP hydrolysis for the amide carbonyl oxygen of the Mg(2+)-coordinating Thr of regulatory GTPases.
When G-protein alpha subunits binds GTP and Mg(2+), they transition from their inactive to their active conformation. This transition is accompanied by completion of the coordination shell of Mg(2+) with electrons from six oxygens: two water molecules, the ss and gamma phosphoryls of GTP, a helix-alpha1 Ser, and a switch I domain (SWI) Thr, and the repositioning of SWI and SWII domains. SWII binds and regulates effector enzymes and facilitates GTP hydrolysis by repositioning the gamma-carbonyl of a Gln. Mutating the Ser generates regulatory GTPases that cannot lock Mg(2+) into its place and are locked in their inactive state with dominant negative properties. Curiously, mutating the Thr appears to reduce GTP hydrolysis. The reason for this difference is not known because it is also not known why removal of the Thr should affect the overall GTPase cycle differently than removal of the Ser. Working with recombinant Gsalpha, we report that mutating its SWI-Thr to either Ala, Glu, Gln, or Asp results not only in diminished GTPase activity but also in spontaneous activation of the SWII domain. Upon close examination of existing alpha subunit crystals, we noted the oxygen of the backbone carbonyl of SWI-Thr and of the gamma-carbonyl of SWII Gln to be roughly equidistant from the oxygen of the hydrolytic H(2)O. Our observations indicate that the Gln and Thr carbonyls play equihierarchical roles in the GTPase process and provide the mechanism that explains why mutating the Thr mimics mutating the Gln and not that of the Ser.
Zurita
Adolfo
A
Laboratory of Neurobiology, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
Zhang
Yinghao
Y
Pedersen
Lee
L
Darden
Tom
T
Birnbaumer
Lutz
L
D000818:Animals; D002413:Cations, Divalent / Q000737:chemistry; D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D002413:Cations, Divalent; D044385:GTP-Binding Protein alpha Subunits; D013912:Threonine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D008274:Magnesium; D010100:Oxygen
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