Life takes place through the concerted flow of numerous biological processes. At the molecular level, this involves highly specific and transient protein-protein and protein-ligand interactions. The specificity and function of a given protein is determined by its unique three-dimensional structure and by motions of groups of atoms within this scaffold. We are interested in observing changes in atomic level structure and dynamics induced by perturbations, such as ligand binding or phosphorylation, that are associated with these transient interactions. Such information provides insights into unanswered questions regarding the origins of binding energy and the mechanisms by which protein function is regulated. These questions are critical in practical endeavors such as drug design and protein engineering.
One of our lab's main foci is Pin1. Pin1 is a peptidyl-prolyl isomerase (PPIase) which catalyzes the cis-trans isomerization in many proteins, including the amyloid precursor protein (APP) from which the pathogenic amyloid-beta peptide (Aβ) is proteolytically derived. The accumulation of Aβ peptides in the brain, leading to the formation of neuritic plaques, is one of the major hallmarks of Alzheimer's Disease (AD). Uncatalyzed cis-trans isomerization can be thought of as a molecular switch where a peptide bond alternates slowly between two conformations (cis and trans) and a large energy barrier between them prohibits rapid "switching" between the states. Pin1 lowers this energy barrier and allows for fast "switching" between cis and trans conformations. Pin1 has been shown to bind APP, to catalyze isomerization of the peptide bond at a conserved phosphorylated Thr668-Pro669 motif and to protect against amyloidogenic processing of APP. Additionally, Pin1 has been shown to be down-regulated or inhibited in AD neurons. Taken together, these results indicate that Pin1-catalyzed acceleration of the APP cis-trans isomerization rate represents a molecular timer that regulates APP processing and Aβ production.
An advantageous approach in elucidating the structural and functional interactions between Pin1 and APP is to utilize Pin1's individual domains, WW and PPIase, to examine the enzyme-substrate interactions using NMR spectroscopy and isothermal titration calorimetry (ITC). We examine both the binding of substrate to the WW domain of Pin1and the binding and catalysis of substrate to/by the PPIase domain. In both cases, a smaller form of APP (a 21-residue phosphopeptide of APP, G659-Q679, referred to herein as pT20) is utilized as the substrate for Pin1; pT20 has been shown to display structural and dynamic features indistinguishable from the native intracellular C-terminal domain of APP (AICD). Further, we take advantage of isotopic labeling of either the substrate or the Pin1 domains to observe the same binding or catalytic event from two perspectives. The substrate perspective allows the distinct cis and trans isomers to be tracked separately, yielding isomer-specific interactions, while the protein perspective reveals residue-specific changes, suggesting structure-based mechanisms for binding/catalysis.
We have performed NMR titration experiments and subsequent lineshape analysis on pT20 and the individual Pin1 domains. More than 50% of the residues in the WW domain show substantial chemical shift changes upon substrate binding, suggesting a global response (within the domain) to binding. 15N-labeled substrate also displays substantial chemical shift changes upon binding to WW. From lineshape analysis of the titration data, we extracted off-rates for substrate binding, and binding constants were determined from ITC. Similar experiments were performed on the PPIase domain, which senses changes due to substrate binding and catalysis. The overall goal of these experiments is to comprehensively determine the microscopic rates for each step in the Pin1-catalyzed pT20 isomerization mechanism. Binding constants determined from independent ITC experiments confirm these rates. Finally, results from both ITC and NMR titration experiments provide thermodynamic information on the free energy landscape of the Pin1-pT20 interaction.
Yet, even in routine HSQC (Mulder et al., 1996) spectra, the supreme sensitivity of NMR line shapes to the kinetics and equilibria of individual reaction steps in a complex multi-state scheme is apparent. For complex systems in which multiple states are at equilibrium, NMR lineshape analysis provides a powerful tool with which to determine the microscopic kinetic rates and equilibrium constants connecting the different states in the reaction scheme, complementing techniques such as CPMG and ZZ exchange. The detailed analysis of NMR line shapes is a well-established but underutilized tool for examining protein interactions and motions that occur within the microsecond to millisecond timescale (Gunther and Schaffhausen, 2002; Kern et al., 1995; Kovrigin and Loria, 2006). The processes of ligand binding and release, conformational exchange, and enzymatic turnover can all occur on this timescale (Mittermaier and Kay, 2009a). While classically applied to 1D NMR spectra, lineshape analysis is easily applied to 2D spectra by peak slice extraction. NMR lineshape broadening and changes in chemical shift in both dimensions of a series of HSQC spectra can yield information about kinetics, populations, and unobservable chemical shifts on a residue-specific basis (Gunther and Schaffhausen, 2002). Because lineshape analysis uses data taken at multiple concentrations of interacting proteins, it is especially applicable to complex models with three, four, or more states (Gunther and Schaffhausen, 2002). Software has been developed to fit lineshape data to multiple, arbitrarily complex models, allowing easy comparisons between models (http://lineshapekin.net/, http://lineshapekin.net/). Simultaneously fitting data from multiple peaks can allow a global understanding of complex physical processes to emerge. In addition to ligand binding and protein dynamics (Craven et al., 1996; Johnson et al., 1998), lineshape analysis is applicable to the study of catalytic mechanisms and enzyme function, such as that of PPIases (Kern et al., 1995). The advantages of NMR lineshape analysis over standard biochemical isomerase assays lie in the ability to use the natural substrate sequence and the convenience of performing experiments at chemical equilibrium.