Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I.

G. Marius Clore,Vitali Tugarinov,Alexander Grishaev,Charles D. Schwieters,Vincenzo Venditti

doi:10.1038/ncomms6960

Abstract

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here, we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate (PEP) and the inhibitor α-ketoglutarate (αKG), on the structure and dynamics of EI using NMR, small-angle X-ray scattering (SAXS) and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

Highlights

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date
Experiments were acquired on the wild-type protein (EIWT) and on mutants of EI in which the active site H189 was replaced by Gln (EIQ) or Ala (EIA)
Addition of 10 mM aKG to EIWT, EIQ or EIA does not result in any significant chemical shift perturbation within the EIN domain (Fig. 2b and Supplementary Fig. 1); the only chemicalshift changes observed are located within the EIC domain, and closely resemble the chemical shift perturbations induced by aKG on isolated EIC (Supplementary Fig. 2)[8]

Summary

Introduction

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. The enzyme adopts a closed conformation (state B) in which the EIN domains of the two subunits are in direct contact, and the active site H189 is inserted in the PEP-binding pocket of the EIC domain (Fig. 1a). Modelling the position of PEP by replacing oxalate and the phosphoryl group indicates that the closed conformation is fully consistent with in-line phosphoryl transfer from PEP bound to the EIC domain to H189 on the a/b subdomain of EIN11 (Fig. 1c). It is not clear whether the closed conformation corresponds to the holo form of the enzyme or is only transiently sampled to permit the phosphoryl transfer reaction. Using solution NMR and small-angle X-ray scattering (SAXS) we show that binding of PEP to the EIC domain induces a

Methods

Results

Conclusion