Mutation of a highly conserved aspartate residue in subdomain IX abolishes Fer protein-tyrosine kinase activity.
Before the structure of cAMP-dependent protein kinase had been solved, sequence alignments had already suggested that several highly conserved peptide motifs described as kinase subdomains I through XI might play some functional role in catalysis. Crystal structures of several members of the protein kinase superfamily have suggested that the nearly invariant aspartate residue within subdomain IX contributes to the conformational stability of the catalytic loop by forming hydrogen bonds with backbone amides within subdomain VI. However, substitution of this aspartate with alanine or threonine in some protein kinases have indicated that these interactions are not essential for activity. In contrast, we show here that conversion of this aspartate to arginine abolished the catalytic activity of the Fer protein-tyrosine kinase when expressed either in mammalian cells or in bacteria. Structural modeling predicted that the catalytic loop of the FerD743R mutant was disrupted by van der Waal's repulsion between the side chains of the substituted arginine residue in subdomain IX and histidine-683 in subdomain VI. The FerD743R mutant model predicted a shift in the peptide backbone of the catalytic loop, and an outward rotation of histidine-683 and arginine-684 side chains. However, the position and orientation of the presumptive catalytic base, aspartate-685, was not substantially changed. The proposed model explains how substitutions of some, but not all residues could be tolerated at this nearly invariant aspartate in kinase subdomain IX.
Cole
L A
LA
Cancer Research Laboratories, Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Zirngibl
R
R
Craig
A W
AW
Jia
Z
Z
Greer
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry*; D019556:COS Cells; D003198:Computer Simulation; D017931:DNA Primers; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000936:Antigen-Antibody Complex; D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C052602:proto-oncogene protein c-fes-fps; D001224:Aspartic Acid; D011505:Protein-Tyrosine Kinases