Impact of a surface methyl substitution on correlated motions in metalloregulatory protein

DAIANA A. CAPDEVILA; DAVID GIEDROC

Carbondale

Congreso; Gibbs Conference on Biothermodynamics; 2017

Gibbs Society of Biological Thermodynamics

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 model system used in understanding metal-mediated bacterial transcriptional regulation. Previous work on this protein [1] suggests that Zn-induced allosteric inhibition of DNA operator binding is driven by perturbations in a network of coupled equilibrium ps-ns motions that give rise to slow-timescale (µs-ms) conformational interconversion and affect conformational entropy.To test this idea, we explore the origin of allosteric inhibition in a cavity mutation (L34A) in a solvent expose hydrophobic core, located in the periphery of the protein, distal to both Zn and DNA binding sites. This mutant has been previously shown to be allosterically impaired for releasing DNA upon Zn binding [1], despite having Zn(II) binding and DNA binding affinities only minimally affected from wild type.In order to determine the structural origin of this compromised allosteric linkage we solved the crystal structure of this mutant to 2.0 Å resolution and study the changes in the NMR spectra upon Zn addition. We show that the global structure is conserved (rmsd = 0.5 Å) and the protein structurally ?switches? upon Zn coordination, as measured by nearly identical chemical shift perturbation maps. Previous studies have shown that Zn binding forms a quaternary structural hydrogen bond between the Zn and DNA binding sites and locks the protein in a single conformation. The His97′ NHε2???O=C His67 H-bond is only present in the crystal in the L34A mutant and not in solution although the mutation site is in the opposite site of the molecule.NMR was used to assess dynamical changes in the apo-, and Zn-bound states, by measuring order parameters (S2axis, sensitive to ps-ns motion, and a reporter for site-specific conformational entropy) and relaxation dispersion (sensitive to slower µs-ms motions) for each state. Our NMR data confirms that L34A in the Zn state retains the ability of the Apo protein to visit an excited state capable of binding DNA and shows the same degree of correlated motion as the Apo protein responsible for the entropic driving force of DNA binding. Collectively, these data suggest that Zn fails to lock L34A CzrA in a single conformation impaired for binding DNA, as it was previously shown for the wild-type protein, and this mutation impact mainly internal protein dynamics.1 Capdevila, D. A., Braymer, J. J., Edmonds, K. A., Wu, H. and Giedroc, D. P. (2017) Proc. Natl. Acad. Sci. 114, 4424?9.