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 Å 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 ␣1/␤1 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°C by fluorescence spectroscopy. Binding of sulfate prevents hPin1 aggregation and changes surface charges across the active center and around the reactive and catalytically essential Cys 113 . In the absence of sulfate and/or reducing agent this residue seems to promote aggregation, as observed in hPin1 solutions in vitro.

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Many cellular and secreted proteins are chemically modified after their translation is completed. The covalent linkage of a polypeptide chain (modifier) to a substrate protein is a special case of post-translational modification. In the late seventies it was observed that ubiquitin, a small modifier, marks short-lived proteins for degradation by the 26S proteasome. Over the last decade many other ubiquitin-related proteins were discovered and isolated. Attachment of polypeptide chains onto acceptor molecules became a common feature to regulate spatially and timely organized cellular pathways of proteins. This article focuses on the structures of the three modifiers: ubiquitin, RUB and SUMO and the cognate enzymes involved in these modification pathways. We have described the homologies and differences of these proteins and indicate salient topological hallmarks common to modifier-conjugating enzymes. This characterization will help in understanding these regulatory pathways and their similarities and differences in controlling protein fate, from protein degradation signals generated by polyubiquitination to functional modification brought about by RUB and SUMO conjugation.

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