Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems

Abstract

Cyclophilins catalyze cis ↔ trans isomerization of peptidyl–prolyl bonds, influencing protein folding along with a breadth of other biological functions such as signal transduction. Here, we have determined the microscopic rate constants defining the full enzymatic cycle for three human cyclophilins and a more distantly related thermophilic bacterial cyclophilin when catalyzing interconversion of a biologically representative peptide substrate. The cyclophilins studied here exhibit variability in on-enzyme interconversion as well as an up to 2-fold range in rates of substrate binding and release. However, among the human cyclophilins, the microscopic rate constants appear to have been tuned to maintain remarkably similar isomerization rates without a concurrent conservation of apparent binding affinities. While the structures and active site compositions of the human cyclophilins studied here are highly conserved, we find that the enzymes exhibit significant variability in microsecond to millisecond time scale mobility, suggesting a role for the inherent conformational fluctuations that exist within the cyclophilin family as being functionally relevant in regulating substrate interactions. We have additionally modeled the relaxation dispersion profile given by the commonly employed Carr–Purcell–Meiboom–Gill relaxation dispersion (CPMG-RD) experiment when applied to a reversible enzymatic system such as cyclophilin isomerization and identified a significant limitation in the applicability of this approach for monitoring on-enzyme turnover. Specifically, we show both computationally and experimentally that the CPMG-RD experiment is sensitive to noncatalyzed substrate binding and release in reversible systems even at saturating substrate concentrations unless the on-enzyme interconversion rate is much faster than the substrate release rate.

Networks of Dynamic Allostery Regulate Enzyme Function

“…This variability indicates that, while altering conformational sampling does influence enzymatic function in CypA toward multiple substrates, the specific outcomes are somewhat substrate specific, likely due to differences in the substrate/active-site interactions within each enzyme/substrate complex. While previous correlative measures have hinted at a role for micro- to millisecond dynamics in the catalytic cycle of CypA (Eisenmesser et al, 2005; Fraser et al, 2009; Holliday et al, 2015a), here we have shown a direct connection between the dynamic networks of CypA identified via our RASSMM approach and catalysis of multiple substrates.…”

“…Specifically, R 2 -RD experiments have revealed that nearly 50% of residues within CypA exhibit micro- to millisecond timescale dynamics and that they comprise rates of motion spanning nearly an order of magnitude. More recently, the segmental nature of dynamics in CypA has also been independently confirmed by variable-temperature X-ray crystallography (Keedy et al, 2015) and we have shown that distinct segmental dynamics persist in solution in the presence of saturating concentrations of substrate (Holliday et al, 2015a). Collectively, these studies reveal that significant global communication occurs between non-coherent regions such that mutagenesis or substrate binding affects motions across the protein at sites reporting on distinct structural transitions.…”

“…As shown in Figure 2A, many residues within the binding site exhibit altered R ex values during isomerization relative to apo CypA, as would be expected due to changes in both the local dynamics and the local chemical environment. Because the non-coherent nature of CypA dynamics in both the apo and isomerizing states prevents simultaneous fitting of multiple residues and because FGP-bound CypA dynamics are potentially influenced by both catalysis and the binding/release cycle (Holliday et al, 2015a), we hesitate to directly interpret these R ex changes mechanistically. However, compared with the large number of residues with altered R ex proximal to the substrate, we detect an increase in R ex for only two residues distal to the active site, Val29 and Val6 (Figure 2A).…”

“…The minimal catalytic cycle of PPIases consists of six microscopic rate constants defining the on and off rates for each isomer, as well as the two rate constants defining the on-enzyme interconversion (Figure S6). Lineshape analysis has provided a powerful means to elucidate these rate constants, which are not readily accessible by classic enzymatic assays (De et al, 2012; Greenwood et al, 2011; Holliday et al, 2015a; Kern et al, 1995). Thus, to determine the specific impact that perturbations to the dynamic networks of CypA have on the catalytic cycle, we collected high-resolution 15 N-heteronuclear single quantum coherence (HSQC) spectra of 15 N-FGP in solution with varying concentrations of CypA WT or mutants (representative example shown in Figure 5B).…”

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

“…In NMR studies of CypA, a dynamic continuum has been identified such that the relaxation profiles cannot be globally fit to one or two exchange phenomena and are instead indicative of more localized motions (22). Exchange rates coalesce somewhat during turnover, perhaps suggesting an increase in coordination throughout the protein, but appear to still consist of localized motions that are not fully coherent (23). However, dynamical signals during catalytic turnover could be affected by substrate binding and unbinding, especially if the substrate binding affinity is low, thereby leading to ambiguity in the interpretation of NMR analysis (23).…”