Use of NMR to probe changes in the flexibility of acid‐stress chaperone HdeB from its inactive to active state

Abstract

HdeB is an intrinsically disordered periplasmic chaperone essential to the acid resistance of numerous pathogenic bacteria. It, along with its homolog, HdeA, binds to vulnerable periplasmic proteins at low pH and prevents them from irreversibly aggregating in the acidic stomach. The activity of these two proteins is responsible for the survivability of pathogenic bacteria and resulting proliferation of dysentery. At physiological pH, HdeB is an inactive folded homodimer and remains a structurally unchanged, fully folded dimer in its active state (at pH 4–5), suggesting that the mechanism of chaperone activation is a result of changes in its dynamics (internal motions). The long‐term goal of this study is to gain insight into HdeB's unique chaperoning mechanism by characterizing its pH dependent changes in structure and dynamics. In order to achieve atomic level specificity and detect subtle conformational changes, we are utilizing Nuclear Magnetic Resonance (NMR) spectroscopy to compare HdeB's flexibility and structure in its inactive and active states at pH 6.0 and 4.5, respectively. Isotopically labeled HdeB was recombinantly expressed and purified. Subsequently, protein backbone and side chain dynamics were examined at pH 6 and pH 4.5 utilizing R2 and R1 relaxation experiments, which identify fast (ps–ns) time scale motions within the structure; this indicates local motions, such as methyl rotations. Additionally, intermediate (μs‐ms) time scale motions, which reflect global conformational changes and flexibility, were identified using CPMG relaxation dispersion experiments. Notable changes in dynamics were observed with activation. At pH 6.0, most residues with μs‐ms motions are clustered at the dimer binding interface (BC loop and helix B). With the transition to pH 4.5, μs‐ms motions expand within the dimer binding interface (W27, T33, G37, T40, K53) and extend beyond it (L16δ, I55δ). Furthermore, at pH 4.5, Helix B decreases in solvent protection, suggesting decreased burial and a loosening of the dimer binding interface. Since this helix is extremely hydrophobic, loosening exposes residues that are well suited to interacting with the exposed hydrophobic regions of an unfolding client protein, likely enabling interactions crucial to substrate binding. Interestingly, the largest changes with activation were observed near multiple titratable residues, making us suspect a protonation change. Continuing research will focus on experimentally determining acid dissociation constants to confirm this.Support or Funding InformationWe gratefully acknowledge the support of the NIH for research funds (SC3‐GM116745) and BUILD PODER for research support to L.A., as well as the NSF for funding the purchase of our NMR spectrometer (CHE‐1040134).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.