Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.

Antonio N Calabrese,Lynsey N Jones,Scott M Jackson,Sheena E Radford,Maria Kokkinidou,Alison E Ashcroft,Florian Heinkel,Arwen R Pearson,David Sharples,Joerg Gsponer,Peter J F Henderson,Marta Sans,Oliver Beckstein

doi:10.1021/acs.analchem.7b01310

Abstract

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion–substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein’s conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.

Highlights

S econdary active membrane transport proteins exploit the potential energy of ion gradients to drive the transport of solutes across membranes.[1]
The conformational state(s) of such proteins can be determined by means of X-ray crystallography, but elucidating the conformational state(s) and intermediates adopted in solution, and how the binding of ligands influences the conformational equilibrium of the protein, is of vital importance to enable full characterization of the transport cycle.[7−10]
Membrane transport proteins of these families are involved in processes such as neurotransmitter, sugar, amino acid, and drug transport.[42−44] The diversity of their biological functions has led to a burgeoning field of research pertaining to unravelling the structural basis by which this class of proteins transport their assorted substrates.[2−4,6] Importantly, structures of proteins in the 5-helix inverted repeat (5HIRT)/LeuT superfamily differ completely from those in the major facilitator superfamily, a small number of which have been studied by Mass Spectrometry (MS) previously.[35,45]

Summary

■ RESULTS AND DISCUSSION

The observation that significantly more protein remains unlabeled in the presence of higher concentrations of NaCl suggests that the conformational equilibrium of Mhp[1] is shifted even further to the outward-facing form by higher NaCl concentrations From these data we propose that the mass distribution after NEM labeling can be used as a conformational “fingerprint” to deduce the conformational state of the protein under varied conditions of Na+ and ligand concentrations. Detection of unfolding does not necessarily rely on the Cys residues being in strategically placed positions, as would be required to monitor inward-to-outward (or the reverse) interconversion

■ CONCLUSION

■ ACKNOWLEDGMENTS

■ REFERENCES