EXPLORING PROTEIN ALLOSTERY AND DYNAMICS IN THE METALLOREGULATOR CZRA

DAIANA ANDREA CAPDEVILA; JOSEPH BRAYMER; KATHERINE EDMONDS; HONGWEI WU; DAVID GIEDROC

Vermont

Conferencia; Gordon Research Conference: Cell Biology of Metals; 2015

Gordon Research Conference

In bacteria, metalloregulatory proteins are central to transition metal ion balance. These proteins employ allostery to connect metal binding to DNA binding, which regulates transcription of genes that control metal homeostasis. The homodimeric Zn(II) sensor CzrA from Staphylococcus aureus is a prototypical example used in understanding metal-mediated bacterial transcriptional regulation. Previous work provides evidence for a ?pathway? that physically connects the zinc-binding site to the DNA recognition helix. Biophysical studies support this hypothesis where perturbations of key residues along this pathway, e.g., V66, L68, result in reduced free energies of allosteric coupling. However, these mutants exhibit significant entropy-enthalpy compensation in Zn(II) binding; therefore, it has been proposed that conformational entropy is a major driving force for the allosteric mechanism. To test this hypothesis, we measured methyl chemical shift perturbation (CSP) and residue-specific dynamics for apo, DNA- and Zn(II)-bound states for the mutants and WT. The CSP maps for Zn(II) binding are very similar between WT and the mutants, revealing that all ?switch? conformations. While there is no evidence of µs-ms motion in either the apo- or zinc-bound states, the ps-ns dynamics as measured by the methyl group axial order parameter and a reporter for site-specific conformational entropy, reveal distinct methyl dynamics signatures for Zn(II) binding when comparing wild-type CzrA with allosterically compromised V66A/L68V (AV) and V66A/L68A (AA) mutants. These data suggest that uncoupling of Zn(II) and DNA binding in the AV and AA mutants occurs as a result of a non-native redistribution of conformational entropy that is incompatible with single-pathway allosterism.