title: Molecular basis for an ancient partnership between prolyl isomerase Pin1 and phosphatase inhibitor-2.
abstract: Pin1 is a prolyl isomerase that recognizes phosphorylated Ser/Thr-Pro sites, and phosphatase inhibitor-2 (I-2) is phosphorylated during mitosis at a PSpTP site that is expected to be a Pin1 substrate. However, we previously discovered I-2, but not phospho-I-2, bound to Pin1 as an allosteric modifier of Pin1 substrate specificity [Li, M., et al. (2008) Biochemistry 47, 292]. Here, we use binding assays and NMR spectroscopy to map the interactions on Pin1 and I-2 to elucidate the organization of this complex. Despite having sequences that are ∼50% identical, human, Xenopus, and Drosophila I-2 proteins all exhibited identical, saturable binding to GST-Pin1 with K(0.5) values of 0.3 μM. The (1)H-(15)N heteronuclear single-quantum coherence spectra for both the WW domain and isomerase domain of Pin1 showed distinctive shifts upon addition of I-2. Conversely, as shown by NMR spectroscopy, specific regions of I-2 were affected by addition of Pin1. A single-residue I68A substitution in I-2 weakened binding to Pin1 by half and essentially eliminated binding to the isolated WW domain. On the other hand, truncation of I-2 to residue 152 had a minimal effect on binding to the WW domain but eliminated binding to the isomerase domain. Size exclusion chromatography revealed that wild-type I-2 and Pin1 formed a large (>300 kDa) complex and I-2(I68A) formed a complex of half the size that we propose are a heterotetramer and a heterodimer, respectively. Pin1 and I-2 are conserved among eukaryotes from yeast to humans, and we propose they make up an ancient partnership that provides a means for regulating Pin1 specificity and function.
authors.affiliation: Center for Cell Signaling and Department of Microbiology, University of Virginia School of Medicine, Box 800577-MSB7225, Charlottesville, Virginia 22908, United States.
authors.forename: Furqan
authors.initials: F
authors.lastname: Sami
authors.forename: Caroline
authors.initials: C
authors.lastname: Smet-Nocca
authors.forename: Meera
authors.initials: M
authors.lastname: Khan
authors.forename: Isabelle
authors.initials: I
authors.lastname: Landrieu
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
authors.forename: David L
authors.initials: DL
authors.lastname: Brautigan
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics; D029867:Xenopus Proteins / Q000737:chemistry* / Q000378:metabolism; D000097847:Protein Phosphatase Inhibitory Proteins
chemical_list: D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C000606845:ESS1 protein, S cerevisiae; C000606846:PIN1 protein, human; C000606849:dod protein, Drosophila; C000606851:parvA protein, E coli; C525404:Pin1 protein, Xenopus