Inferring the transport stoichiometry of the proton/zinc transporter YiiP from microscale thermophoresis and simulations.

Abstract

The transport stoichiometry of the Zn2+/H+ antiporter YiiP remains an open question. We used experimental microscale thermophoresis (MST) zinc binding data in combination with a microscopic thermodynamic model and constant pH simulation in order to infer the likely microstates of H+ and Zn2+ binding. The pH dependence of Zn2+ binding in different binding sites was measured with MST. For these sites, we constructed microscopic, thermodynamically consistent models based on the pKas of the binding residues and zinc binding affinities. Such a model relates the macroscopically measured MST binding curves to microscopic protonation states and zinc binding in a concentration and pH-dependent manner. We inferred microscopic model parameters from the experimental data with a Monte Carlo approach. We compared the MST-inferred microscopic parameters to results from atomistic constant pH MD simulations in the absence of Zn2+. For zinc binding site B on a solvent-exposed loop between transmembrane helices 2 and 3, MST and simulation data broadly agree with each other. For the transport site A in the transmembrane domain, the CpHMD simulations indicate that the binding site His residue is protonated before any Asp. Using the CpHMD results as a starting point for the MST inference approach, we find that the microstate distribution that best fits the experimental data indicates competitive binding between either one Zn2+ ion or two or three protons bound, with the one-proton state suppressed, possibly due to high cooperativity between the Asp and His residues. The microscopic model indicates a transport stoichiometry of 1:2, or possibly 1:3 at lower pH.