Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis

Abstract

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, kcatcis and apparent Michaelis constants, KMApp. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide 13C-1H constant time HSQC spectra to determine Kcatcis, Kcattrans, KDcis, and KDtrans for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

Structure and function of the human parvulins Pin1 and Par14/17

“…Based on these measurements a transition state was proposed that is stabilized by an intramolecular hydrogen bond from the amide Pro +1 to the prolyl nitrogen ( Figure 6). This transition state seems to be stabilized and preferentially bound by the enzyme which thereby diminishes the rotational activation barrier (Greenwood et al, 2011;Velazquez and Hamelberg, 2013). As observed in other structural models (Ranganathan et al, 1997;Zhang et al, 2012), the proline ring resides within a hydrophobic pocket formed by the conserved residues Leu 122 , Met 130 and Phe…”

“…In addition, NMR methods have been designed that enable a temperature dependent and tag-free determination of the path-dependent k cat /K M values (Hsu et al, 1990;Justice et al, 1990). Both values can be determined simultaneously by recording exchange spectroscopy experiments with increasing mixing time (Kern et al, 1995;Greenwood et al, 2011) followed by line-shape analysis. All versions of these assays have been used to measure the specificity constants of parvulins, especially for the phosphate-specific Pin1.…”

Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization

“…A previous comprehensive study on the parvulin Pin1 isomerization of an amyloid-based ligand quantified a higher affinity for the cis-Pro containing peptide (43). This finding led to a putative mechanism by which the higher affinity for the less populated conformation would thereby allow balanced access to the isomerase by both conformations.…”

“…As a consequence, some of the substrate molecules will bind and dissociate while in the same conformation, whereas other molecules will bind in one conformation and dissociate in the alternate conformation due to isomerization while in complex with Fpr4. Although it has been possible to determine the conformer-specific affinities in the interaction between Pin1 and a phosphopeptide from the amyloid precursor protein (43), similar analyses with FKBP have not been reported. In the study of Fpr4(280 -392) we were unable to determine conformer specific affinities mainly due to solubility limits of the PPIase domain.…”

“…These K d values are the combination of both the cis-and trans-specific affinities. For Pin1, the affinity toward the cis-conformation is higher than that of the trans-Pro peptide (43). If this condition holds true for Fpr4, then we can place an upper limit on the cis-conformer affinity by fitting the data from Fig.…”