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Neutron Crystal Structure of Ras GTPase sets New Paradigm for GTP Hydrolysis
Author(s) -
Knihtila Ryan,
Holzapfel Genevieve,
Weiss Kevin,
Meilleur Flora,
Mattos Carla
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.893.7
Subject(s) - gtpase , gtp' , nucleotide , chemistry , salt bridge , deprotonation , crystallography , active site , stereochemistry , ras superfamily , hydrogen bond , biochemistry , molecule , mutant , enzyme , ion , organic chemistry , gene
Ras GTPase is a prototype for nucleotide‐binding proteins that function as molecular switches in a variety of cellular functions. The switch is modulated by cycling between the inactive GDP bound form and the active GTP bound form, regulated by the opposing actions of guanine nucleotide exchange factors and GTPase activating proteins. Both intrinsic and GAP catalyzed hydrolysis of GTP on Ras have been studied assuming that the phosphate groups of GTP are fully deprotonated at physiological pH, as they are known to be in solution. We present the neutron crystal structure of Ras‐GppNHp, representative of the pre‐catalytic state. The exchangeable hydrogen atoms, replaced by deuterium, are visible in the nuclear density maps, showing a protonated g‐phosphate on GppNHp at pH 7.5. Furthermore, while the nucleophilic water molecule is near several H‐bonding partners, its nuclear density indicates rotational freedom, such that it does not make any strong H‐bond interactions in the active site. Ser17, which coordinates the Mg 2+ appears to be deprotonated. Residues in the conserved NKXD nucleotide‐binding motif do not make significant H‐bond interactions with the nucleotide, except for D119, which makes a well‐known salt bridge with the guanosine base. This work defines for the first time explicit hydrogen positions in the active site of Ras, and given the conserved nature of the nucleotide binding pocket in GTPases, transforms our understanding of GTP hydrolysis catalyzed by Ras and members of the G‐protein superfamily. Work is supported by NSF grant MCB‐1244203.