Structural Analysis of the Mitotic Regulator hPin1 in Solution

Abstract

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.

The Structure and Dynamics of Tandem WW Domains in a Negative Regulator of Notch Signaling, Suppressor of Deltex

“…The analysis of chemical shift changes thus supports the conclusion that ligand binding induces a conformational change to both domains from an at least partially unfolded state to one that is fully folded and stable. This is in contrast to NMR studies of the YAP65 (38), Prp40 (10), and Pin1 WW domains (44,45) where shifts of the equivalent residues to Trp 17 and Trp 61 are not significantly affected by ligand binding and the domains are stably folded in absence of ligand.…”

“…Chemical shift mapping has previously proved useful in identifying residues directly involved in ligand binding by WW domains (10,37,38,44,45 28 and His 32 residues in WW3 domain display chemical shift changes (Fig. 8B) of a magnitude consistent with the direct involvement of WW3 in ligand binding, whereas the topologically equivalent residues in WW4 show smaller shifts consistent with an indirect effect of Pept1 on the global conformation of WW4.…”

Structure–function–folding relationship in a WW domain

“…In its physiological role, the N-terminal WW domain binds Pro-rich ligands of the consensus sequence (pS͞pT)P, whereas the C-terminal domain catalyzes the Pro cis͞ trans-isomerization at the pS͞pT-P peptide bond. NMR solution studies show that the two domains, which are connected by a flexible solvated linker, interact only weakly before ligand binding (6,7). The structure of the isolated Pin WW domain is virtually superimposable on that of the WW domain in the two-domain hPin1 protein (8).…”

Oxidative stress and its effect on cells functional activity of Alzheimer's disease

“…Pin1 участвует в изомеризации и дефосфорилировании белка tau, сохраняя его функцию и конформацию и тем самым осуществляет регуляцию вхождения клетки в митотический цикл. Снижение его уровня в ядрах нейронов за счёт окислительной деструкции, появление его в цитоплазме и в области NFTs может быть связано с гибелью клеток при АБ [76,114,115].…”