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44640-G4
NMR Studies of Ligand Flexibility in Protein-Ligand Interactions
This report summarizes progress over the year from 9/01/2006 to 7/31/2007. PRF funds have been used to support my post-doc, whose technical expertise has been essential to our progress. Our project investigates how ligand flexibility changes upon binding a protein receptor. We use solution NMR as our tool to map site-specific dynamics on both protein and ligand.
The protein is human Pin1 (Pin1 hereafter), a peptidyl-prolyl isomerase that targets phospho-Thr/Ser-Pro motifs on other cell signaling proteins important for mitosis. The ligands are phospho-peptides from Pin1 protein substrates. In the last year, we focused mainly on the interaction of Pin1 with EQPLpTPVDL, which represents the substrate phosphatase Cdc25 (Cdc25 hereafter).
To understand changes in ligand flexibility, we must first know the concomitant changes in protein flexibility. We have characterized the changes in protein motion (Pin1) upon binding Cdc25. Pin1 has two domains: (i) a PPIase domain that accelerates cis-trans isomerization of the phospho-Ser/Thr-Pro substrate; (ii) a WW domain that docks Pin1 to its substrate proteins. Using NMR 15N and 2H (deuterium) relaxation, we find the ligand Cdc25 causes changes in functional dynamics in both domains. These findings, which have resulted in two publications acknowledging the ACS-PRF, are as follows:
1. The variable loop of Pin1-WW responsible for substrate docking is intrinsically flexible and rigidifies upon binding Cdc25. Mutating the loop sequence decreases both intrinsic loop flexibility and binding affinity. The results imply that the evolution of the WW specificity loop sequence reflects exploitation of dynamics, as well as chemical moieties, to modulate specificity. These implications extend to other families of docking modules used by modular signaling proteins (such as Pin1).
2. The introduction of Cdc25 substrate causes stiffening of an internal pathway of conserved hydrophobic residues in Pin1. Notably, this path connects two functional sites of Pin1: the domain-domain interface and the catalytic site. This suggests a mechanism for allosteric control within Pin1 that has not been previously observed. Specifically, nearest-neighbor interactions between hydrophobic side chains may propagate dynamics changes from one site to another.
3. Having defined changes in protein motion, we now focus on the ligand dynamics itself. We have carried out natural abundance 13C relaxation measurements of the same Cdc25 peptide in the absence and presence of Pin1. We observe clear evidence of site-specific changes in ligand flexibility upon binding Pin1. In particular, we see enhanced us-ms motions of the phospho-Thr/Pro sequence: the target site on Cdc25 targeted by Pin1. We are following up to distinguish ligand dynamics reflecting catalysis (cis-trans isomerization) versus binding. Additional 2-D exchange experiments provide us estimates of the former. This preliminary data has been presented at the Midwest Protein Folding Conference, 5 May 2007, at Notre Dame. The remainder of the funding period will focus on the ligand motion.
