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          Institute: MPI für molekulare Physiologie     Collection: Sonstige wissenschaftliche Organisationseinheiten     Display Documents



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ID: 41821.0, MPI für molekulare Physiologie / Sonstige wissenschaftliche Organisationseinheiten
Structural analysis of the mitotic regulator hPin1 in solution - Insights into domain architecture and substrate binding
Authors:Bayer, Elena; Goettsch, Sandra; Mueller, Jonathan W.; Griewel, Bernhard; Guiberman, Elena; Mayr, Lorenz M.; Bayer, Peter
Language:English
Research Context:cell cycle
Date of Publication (YYYY-MM-DD):2003-07-11
Title of Journal:Journal of Biological Chemistry
Journal Abbrev.:J. Biol. Chem.
Volume:278
Issue / Number:28
Start Page:26183
End Page:26193
Review Status:Peer-review
Audience:Experts Only
Intended Educational Use:No
Abstract / Description:The peptidyl-prolyl cis/trans isomerase hPin1 is a phosphorylation-dependent regulatory enzyme whose substrates are proteins involved in regulation of cell cycle, transcription, Alzheimer's disease, and cancer pathogenesis. We have determined the solution structure of the two domain protein hPin1-(1-163) and its separately expressed PPIase domain ( 50-163) ( hPin1(PPIase)) with an root mean square deviation of <0.5 &ANGS; over backbone atoms using NMR. Domain organization of hPin1 differs from that observed in structures solved by x-ray crystallography. Whereas PPIase and WW domain are tightly packed onto each other and share a common binding interface in crystals, our NMR-based data revealed only weak interaction of both domains at their interface in solution. Interaction between the two domains of full-length hPin1 is absent when the protein is dissected into the catalytic and the WW domain. It indicates that the flexible linker, connecting both domains, promotes binding. By evaluation of NOESY spectra we can show that the α1/β1 loop, which was proposed to undergo a large conformational rearrangement in the absence of sulfate and an Ala-Pro peptide, remained in the closed conformation under these conditions. Dissociation constants of 0.4 and 2.0 mM for sulfate and phosphate ions were measured at 12 &DEG;C by fluorescence spectroscopy. Binding of sulfate prevents hPin1 aggregation and changes surface charges across the active center and around the reactive and catalytically essentialCys(113). In the absence of sulfate and/or reducing agent this residue seems to promote aggregation, as observed in hPin1 solutions in vitro.
Free Keywords:PROLYL ISOMERASE PIN1; DEPENDENT PROLINE ISOMERIZATION; PHOSPHOSERINE-BINDING; PHOSPHORYLATION; MITOSIS; P53; RECOGNITION
External Publication Status:published
Document Type:Article
Communicated by:Jürgen Block
Affiliations:MPI für molekulare Physiologie/Sonstige Wissenschaftliche Organisationseinheiten/MPI Nachwuchsgruppe: PD Dr. Peter Bayer
External Affiliations:Interdisciplinary Ctr Magnet Resonance, D-44227 Dortmund, Germany;
Novartis Pharma AG, Lead Discovery Ctr, GSO, CH-4002 Basel, Switzerland;
Identifiers:URL:http://www.jbc.org/cgi/content/abstract/278/28/261... [article abstract with full text links]
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