Imperfect coordination chemistry facilitates metal ion release in the Psa permease

Abstract

The relative stability of divalent first-row transition metal ion complexes, as defined by the Irving-Williams series, poses a fundamental chemical challenge for selectivity in bacterial metal ion acquisition. Here we show that although the substrate-binding protein of Streptococcus pneumoniae, PsaA, is finely attuned to bind its physiological substrate manganese, it can also bind a broad range of other divalent transition metal cations. By combining high-resolution structural data, metal-binding assays and mutational analyses, we show that the inability of open-state PsaA to satisfy the preferred coordination chemistry of manganese enables the protein to undergo the conformational changes required for cargo release to the Psa permease. This is specific for manganese ions, whereas zinc ions remain bound to PsaA. Collectively, these findings suggest a new ligand binding and release mechanism for PsaA and related substrate-binding proteins that facilitate specificity for divalent cations during competition from zinc ions, which are more abundant in biological systems.

Characterizing the conformational dynamics of metal-free PsaA using molecular dynamics simulations and electron paramagnetic resonance spectroscopy

“…The unwinding of the linking helix, characteristic of all metal-bound structures crystallised thus far, [24] was not observed in our simulations of metal-free PsaA, suggesting that this conformational change is only induced upon metal binding. This study characterizes the intrinsic flexibility of metal-free PsaA in solution, providing additional structural information that cannot be obtained from the available crystal structures.…”

“…Therefore, it remains to be ascertained how the crystallographic structure of metal-free PsaA compares to its conformational flexibility in solution. Previous MD studies of PsaA have examined the structural changes that are induced by removing the metal ion from the metal-bound protein [24]. The series of 50 ns simulations from that work showed that the observed structural changes were consistent with the proposed spring-hammer mechanism [24].…”

“…PsaA has a single metal-binding site located ~1.5 nm beneath the solvent accessible surface of the protein (Figure 1a), which is able to bind ~1 mol of Mn 2+ per mol of protein [24,37]. As shown in Figure 1, the cysteine substitutions used for MTSL spin-labelling are located outside of the metal binding site; nevertheless an equilibrium-binding assay was used to verify that incorporation of the point mutations did not affect Mn 2 -binding.…”

“…In contrast to the large movements observed in the LivJ structures, the metal-free and metal-bound structures of PsaA only showed a relatively minor (~13°) rotation of the C-terminal domain, in which the pivoting was centered on the last segment of the linking helix (residues 190 -194). As such, this distinct conformational transition, which suggests a relatively rigid protein, was likened to a 'spring-hammer' binding mechanism [24]. However, crystal structures represent static snapshots of proteins, usually captured in a single conformation within the crystal lattice.…”

“…SBPs [24]. Based on these crystal structures, it was proposed that the helical linking region restricts the extent of conformational movement within PsaA.…”