Multidimensional free energy landscapes reveal mechanism of metal ion discrimination in PsaA

MacDermott-Opeskin HI and O'Mara ML

Australian National University.

Manganese homeostasis is crucial for the viability of S. pneumoniae, protecting against oxidative stress and aiding cellular metabolism. Manganese uptake is mediated uniquely by the PsaBCA ATP Binding Cassette import system, whose substrate binding component, PsaA, controls selective uptake of manganese. PsaA lacks a metal chelating cofactor and faces significant competition from other d block metal species. Competitive and irreversible binding by other d block metals has been identified as a mechanism for bacterial susceptibility to zinc and cadmium. In this work we conduct a range of molecular dynamics experiments including free energy calculations to reveal mechanisms of cognate and competitive metal binding to PsaA. We demonstrate that manganese is scavenged more effectively than competing ligands from solution via a series of mobile carboxylates. We also demonstrate that ligand exchange of bound metals with water control reversibility of binding and that propensity to exchange ligands differentiates between metal species. We rationalise experimentally observed affinities and kinetics for multiple metal species on converged two dimensional free energy landscapes. We also compare and benchmark a range of molecular dynamics ion models as well as conducting quantum mechanical calculations to probe model ligand substitution reactions at the PsaA active site. Our work reveals that the coordination chemistry of PsaA is tightly balanced to provide selectivity and reversibility, utilising ligand exchange reactions and favourable kinetics to achieve transport of manganese despite significant competition. These atomic resolution insights into metal ion selectivity have implications for the de novo design of metalloproteins as well as for the development of novel inhibitors for metal uptake in S. pneumoniae.