The dual histidine motif in the active site of Pin1 has a structural rather than catalytic role.
The catalytic domain of the peptidyl-prolyl cis/ trans isomerase Pin1 is a member of the FKBP superfold family. Within its active site are two highly conserved histidine residues, H59 and H157. Despite their sequence conservation in parvulin PPIase domains, the role of these histidine residues remains unclear. Our previous work (Behrsin et al. (2007) J. Mol. Biol. 365, 1143- 1162.) was consistent with a model where one or both histidines had critical roles in a hydrogen bonding network in the active site. Here, we test this model by looking at the effect of mutations to H59 and H157 on Pin1 function, activity, and protein stability. Using a yeast complementation assay, we show that both H59 and H157 can be mutated to non-hydrogen bonding residues and still support viability. Surprisingly, a nonfunctional H59L mutation can be rescued by a mutation of H157, to leucine. This double mutation (H59L/H157L) also had about 5-fold greater isomerase activity than the H59L mutation with a phosphorylated substrate. Structural analyses suggest that rescue of function and activity results from partial rescue of protein stability. Our findings indicate that H59 and H157 are not required for hydrogen bonding within the active site, and in contrast to the active site C113, they do not participate directly in catalysis. Instead, we suggest these histidines play a key role in domain structure or stability.
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Melanie L
ML
Bailey
Brian H
BH
Shilton
Christopher J
CJ
Brandl
David W
DW
Litchfield
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; C000606845:ESS1 protein, S cerevisiae; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase