Multiple Ligand-Bound States of a Phosphohexomutase Revealed by Principal Component Analysis of NMR Peak Shifts

Abstract

Enzymes sample multiple conformations during their catalytic cycles. Chemical shifts from Nuclear Magnetic Resonance (NMR) are hypersensitive to conformational changes and ensembles in solution. Phosphomannomutase/phosphoglucomutase (PMM/PGM) is a ubiquitous four-domain enzyme that catalyzes phosphoryl transfer across phosphohexose substrates. We compared states the enzyme visits during its catalytic cycle. Collective responses of Pseudomonas PMM/PGM to phosphosugar substrates and inhibitor were assessed using NMR-detected titrations. Affinities were estimated from binding isotherms obtained by principal component analysis (PCA). Relationships among phosphosugar-enzyme associations emerge from PCA comparisons of the titrations. COordiNated Chemical Shifts bEhavior (CONCISE) analysis provides novel discrimination of three ligand-bound states of PMM/PGM harboring a mutation that suppresses activity. Enzyme phosphorylation and phosphosugar binding appear to drive the open dephosphorylated enzyme to the free phosphorylated state, and on toward ligand-closed states. Domain 4 appears central to collective responses to substrate and inhibitor binding. Hydrogen exchange reveals that binding of a substrate analogue enhances folding stability of the domains to a uniform level, establishing a globally unified structure. CONCISE and PCA of NMR spectra have discovered novel states of a well-studied enzyme and appear ready to discriminate other enzyme and ligand binding states.

The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light–dark cycle

“…Consequently, we used this mathematical approach to identify the pH-dependent changes in folding from the NMR spectra of our pH titrations, using the PCA-based software TREND NMR (34,35). We bypassed the peak picking step and applied the PCA directly to the series of NMR spectra, encouraged by the success of this direct approach in capturing ligandbinding transitions (34,(36)(37)(38)(39). This semiautomatic strategy simplified the complex NMR spectral changes to suggest the 1.…”

Mechanism of Substrate Recognition and Catalysis of the Haloalkanoic Acid Dehalogenase Family Member α-Phosphoglucomutase

“…Specifically, the radiolabel is almost equally partitioned between solvent αG1,6bisP and G6P. This result is in stark contrast to that observed for Pa -αPGM/αPMM in which αG1,6bisP reorientation takes place on the enzyme within the interdomain space (see Figure A). , …”

“…This result is in stark contrast to that observed for Pa-αPGM/αPMM in which αG1,6bisP reorientation takes place on the enzyme within the interdomain space (see Figure 1A). 27,42 In conclusion, our findings show that the reorientation of the αG1,6bisP intermediate that must occur during the catalytic cycling of Ll-αPGM, proceeds via the αG1,6bisP dissociationrebinding pathway represented in Figure 3. The reorientation does not occur within the active site nor does it occur within the space encapsulated by the subunits of the biological homodimer as was first proposed by Nogly et al 24 Substrate Specificity.…”

A novel Fis1 inhibiting peptide reverses diabetic endothelial dysfunction in human resistance arteries

“…18 , 19 Peptide binding affinity was determined by TREND analysis to each spectrum within the titration series. 20, 21 TREND reveals the changes in data by performing principal component analysis, where each concentration point is treated as a unique data point; here, the input is each 1 H, 15 N spectrum with increasing amounts of peptide. After performing principal component analysis, the principal component 1 (PC1) values are normalized to the highest PC1 value resulting in a range from 0 to 1.…”