Mutagenesis of the L7 loop connecting beta strands 12 and 13 of calcineurin: evidence for a structural role in activity changes.
Calcineurin is a heterodimer consisting of a catalytic A-subunit and a B-subunit, and is regulated by binding of calmodulin and calcium. The C-terminus of the A-subunit contains an autoinhibitory domain which plays an important role in regulation of calcineurin activity. In this study, we have mutated the L7 loop connecting beta strands 12 and 13 in calcineurin A. These mutants included two chimeric mutants in which a four amino acid stretch in the cognate L7 loops of the related proteins phosphatase-1 (GEFD) or -2A (YRCG) were substituted for the calcineurin sequence DVYN (313-316), a point mutation (L312C), and a truncated mutant in which the YRG sequence replaced residues 313-316. Examination of the activities of these mutants led to the striking finding that truncation of the loop region by one residue resulted in hyperactivation of the calcineurin A-subunit. That the hyperactivation is due to conformational effects on the catalytic core of the enzyme was established since this effect was maintained in truncation mutants (at residues 456 and 388) in which the calmodulin and autoinhibitory domains were deleted. These studies provide evidence that the L7 loop is an important structural element in the conformation of the active site, and may participate in the conformational transitions of calcineurin between a catalytically repressed state and an activated state under the influence of the B-subunit.
Wei
Q
Q
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Lee
E Y
EY
D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004789:Enzyme Activation; D017353:Gene Deletion; D005822:Genetic Vectors; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D017354:Point Mutation; D054645:Protein Phosphatase 1; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D002148:Calmodulin-Binding Proteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010750:Phosphoproteins; D011994:Recombinant Proteins; C008644:nitrophenylphosphate; D008345:Manganese; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D002118:Calcium