Conformational Dynamics of Pin1 Regulation of APP processing and Abeta Production
EMSL Project ID
25650
Abstract
The proposed studies fall within the EMSL science theme of "Biological Interactions and Dynamics", specifically in the effort to understand how the "readout", or experimentally measurable outcome of a cellular process, depends on the dynamics of a given step in the process. Peptidyl-prolyl cis/trans isomerization is a particularly effective bridge connecting a kinetically and thermodynamically characterized molecular switch to a measurable cellular output. The proposed experiments build on extensive literature that indicates that aberrant regulation of APP processing and A production is a major cause of Alzheimer's disease, and that phosphorylation of the APP intracellular domain (AICD) at residue T668 plays a central role in this process. Our previous work showed that the phosphorylated pThr668Pro motif in this domain is in equilibrium between distinct cis and trans conformations of the prolyl peptide bond (Ramelot and Nicholson, J Mol Biol, 2001. 307(3): p. 871-84; Ramelot et al., Biochemistry, 2000. 39(10): p. 2714-25). Our recent work shows that cis/trans isomerization of the APP pThr668Pro motif is greatly accelerated by Pin1-catalyzed prolyl isomerization and that Pin1 promotes non-amyloidogenic APP processing and reduces A production (Pastorino et al., Nature, 2006. 440(7083):528-534). We propose to determine the kinetic rates and equilibrium constants for each step in the Pin1 reaction mechanism through NMR lineshape analysis, and to test hypotheses regarding functional motions in Pin1 through relaxation dispersion experiments. We have already obtained lineshapes and relaxation dispersion data for wild-type (wt) Pin1, and now propose to apply the same experimental approaches to characterize a Pin1 mutant (K63A) known to abolish catalysis. This mutant will allow separation of the cis and trans binding processes from catalysis, and will provide important constraints for lineshape fitting of wt-Pin1. Relaxation dispersion NMR experiments on the 15N-Pin1-K63A mutant saturated with substrate will characterize motions on functional timescales. We request a total of 12 days (288 hours) on Mt. St. Helen (500 MHz, 31P-detect probe) for 31P lineshape measurements, and 10.4 days (250 hours) on Ranier (750 MHz, triple resonance probe). These experiments will extend our limited structure-based understanding of Pin1 function, taking it to a new level in which function is explored not by simply examining the proximity and chemistry of atoms within enzyme:substrate or enzyme:inhibitor complexes, but rather by determining correlations between the rates associated with individual steps of the reaction pathway and the rates of backbone motion within the enzyme to elucidate functional motions and to identify critical residues that participate in these motions. The proposed work will assist us in correlating cellular outputs with the effects of "tuning" this switching process through mutation of Pin1, and will potentially advance our understanding of the exquisite choreography of biological processes that are involved in the pathogenicity of Alzheimer's disease.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-06-12
End Date
2009-09-30
Status
Closed
Released Data Link
Team
Principal Investigator