ESR Resolves the C Terminus Structure of the Ligand-free Human Glutathione S-Transferase A1-1

Abstract

Nitroxide-and Cu 2þ-based electron spin resonance (ESR) are combined to provide insight into the conformational states of the functionally important a-helix of the human glutathione S-transferase A1. Distance measurements on various spinlabeled dimeric human glutathione S-transferase A1-1 all result in bimodal distance distributions, indicating that the C-terminus exists in two distinct conformations in solution, one of which closely matches that found in the crystal structure of the ligandbound enzyme. These measurements permit the generation of a model of the unliganded conformation. Room temperature ESR indicates that the second conformation has high mobility, potentially enabling the enzyme's high degree of substrate promiscuity. This model is then validated using computational modeling and further Cu 2þ-based ESR distance measurements. Cu 2þ-based ESR also provides evidence that the secondary structure of the second conformation is of helical nature. Addition of S-hexyl glutathione results in a shift in relative populations, favoring the state that is similar to the previously known structure of the ligand-bound enzyme.

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site‐Specific Elucidation of Hidden Dynamics in Enzymes

“…Upon ligand binding the α9 helix appears to rigidify and adopt the active conformation. These interpretations are consistent with prior EPR work done using both the R1 and Cu II ‐iminodiacetic acid label [12c] …”

“…The differences in rotational rates may be due to the helix existing in two distinct states with different reorientational rates or due to the label experiencing two distinct local interactions leading to changes in label dynamics. An examination of the structure of the helix [12c, 28] (cf. Figure S12,13) shows that the dHis motif is solvent exposed.…”

A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications

“…[50,53] The lack of a flexible linker means dH Cu II -labelling is appealing for structural studies in systems with subtle conformational changes [54] or nuanced conformational equilibria. [55] Furthermore, use of Cu II -NTA in conjunction with the commercially available methanethiosulfonate spin label MTSL, in the 5-pulse dead-time free Relaxation Induced Dipolar Modulation Enhancement (RIDME) experiment [56] yields superb concentration sensitivity, down to hundreds of nM. [28,57] Taken together, this makes dH Cu IIlabelling a powerful tool for future applications in PDEPR spectroscopy.…”

A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications

“…[50,53] The lack of a flexible linker means dH Cu II -labelling is appealing for structural studies in systems with subtle conformational changes [54] or nuanced conformational equilibria. [55] Furthermore, use of Cu II -NTA in conjunction with the commercially available methanethiosulfonate spin label MTSL, in the 5-pulse dead-time free Relaxation Induced Dipolar Modulation Enhancement (RIDME) experiment [56] yields superb concentration sensitivity, down to hundreds of nM. [28] Taken together, this makes dH Cu IIlabelling a powerful tool for future applications in PDEPR spectroscopy.…”