Structure and dynamics of Pin1 during catalysis by NMR

Wladimir Labeikovsky

Elan Eisenmesser

Daryl Bosco

Dorothee Kern

Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham MA 02454, USA

^{To whom correspondence should be directed.}ude.siednarb@nrekd (Dorothee Kern)

^{D.A.B. present address: Serono International Inc., Rockland, MA}

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Abstract

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the us-ms timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidyl-prolyl isomerase Pin1 catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer’s disease. ^{N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of substrate, suggesting that this area is already “primed” for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intra-molecular NOEs. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.}

Keywords: protein dynamics, NMR spectroscopy, peptidylprolyl isomerase, catalysis

Abstract

Footnotes

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