Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function.
The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide-binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. The Obg/GTP1 proteins share sequence similarity along the putative effector-binding domain. In this study, we examined the functional consequences of altering amino acid residues within this conserved domain, and identified that T193 was critical for CgtA function. The in vitro binding, exchange and GTP hydrolysis of the T192A, T193A and T192AT193A mutant proteins was examined using fluorescent guanine nucleotide analogues (mant-GDP and mant-GTP). Substitution of either T192 and/or T193 for alanine modestly reduced binding to GDP and significantly reduced the binding affinity for GTP. Furthermore, the T193A mutant protein was more severely impaired for binding GTP than the T192A mutant. The T193A mutation appeared to account solely for the impaired GTP binding of the T192AT193A double mutation. This is the first report that demonstrates that a confirmed defect in guanine nucleotide binding and GTP hydrolysis of an Obg-like protein results in the lack of function in vivo.
Lin
B
B
Department of Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA.
Skidmore
J M
JM
Bhatt
A
A
Pfeffer
S M
SM
Pawloski
L
L
Maddock
J R
JR
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D001665:Binding Sites; D016935:Caulobacter crescentus / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D014994:Xylose / Q000378:metabolism
D001426:Bacterial Proteins; D006153:Guanosine Diphosphate; D006147:Guanine; D006160:Guanosine Triphosphate; D014994:Xylose; C108621:CgtA protein, bacteria; D020559:Monomeric GTP-Binding Proteins; D000409:Alanine