SITE SPECIFIC DYNAMICS MODULATE ALLOSTERY IN THE ZINC METALLOREGULATOR CZRA

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

Carbondale

Congreso; 2015 Gibbs Conference on Biothermodynamics; 2015

Gibbs Society of Biological Thermodynamics

In bacteria, metalloregulatory proteins are central to transition metal ion balance. Theseproteins employ allostery to connect metal binding to DNA binding, which regulatestranscription of genes that control metal homeostasis. The homodimeric Zn(II) sensorCzrA from Staphylococcus aureus is a prototypical example used in understandingmetal-mediated bacterial transcriptional regulation. Previous work on this proteinsuggests that in order to bind DNA CzrA needs to access a bent conformation, and thatZn binding might compromise the ability to explore this bent state without significantlyaffecting the protein structure.To test this idea, NMR was used to assess structural and dynamical changes amongthe apo-, DNA-, and Zn-bound states, by measuring methyl chemical shift perturbations(CSP), order parameters (S2axis, sensitive to ps-ns motion, and a reporter for sitespecificconformational entropy) and relaxation dispersion (sensitive to slower μs-msmotions) for each state.Interestingly, our NMR data show no evidence of μs-ms motion in either the apo- or Znboundstates, suggesting that exploring different conformations is not necessary.However Zn and DNA binding significantly affect the sidechains? internal ps-ns motions,as reflected in the site-specific order parameters of the methyl groups. DNA bindingincreases sidechain mobility, whereas Zn binding induces stiffening in a considerablenumber of residues. In order to test the role of these mobility changes in allostery, wehave studied allosterically compromised CzrA mutants that show reduced free energiesof allosteric coupling, yet have identical Zn-binding and DNA-binding affinities. The CSPmaps for Zn binding are very similar between WT and the mutants, revealing that thesmall structural changes are present in all cases and, therefore, not determiningallostery. The ps-ns dynamics reveal distinct methyl dynamics signatures for Zn bindingwhen comparing wild-type CzrA with the mutants. This result suggests that uncouplingof Zn and DNA binding in the mutants occurs as a result of a non-native redistribution ofconformational entropy.One possible model compatible with these results is that the CzrA structure has evolvedto distribute the heat released upon DNA binding among the internal motions of theprotein to prevent the complex from disassembling, while the binding of Zn imparts apartial restriction of the sidechain mobility and a release of heat to the environment thatcould dissociate the protein from DNA. This hypothesis derives from directmeasurement of sidechain mobility for CzrA and it will be discussed in the context ofother transcriptional regulators: TrxR, a novel dithiol ArsR-family regulator that is likelyto sense oxidative stress, and AdcR, a homodimeric MarR-family repressor in which theligand (Zn) functions as an allosteric activator, in contrast to CzrA, where Zn functionsas an allosteric inhibitor.