Structural and mechanistic basis of zinc regulation across the E. coli Zur regulon.

Benjamin A Gilston,Mason D Marcus,Mónica A Canalizo-Hernández,Suning Wang,Yi Xue,Alfonso Mondragón,Thomas V O'Halloran,Elden P Swindell,Ann M Stock

doi:10.1371/journal.pbio.1001987

Abstract

Author SummaryZinc is an essential nutrient for most organisms, with the Zn2+ ion performing numerous structural, regulatory, and catalytic roles in a range of proteins. However, this nutrient can neither be synthesized nor degraded and individual cells need to be able to maintain steady levels of zinc in the face of near-zero or excessively high environmental concentrations. Here we look at how the bacterium E. coli does this, by examining the structure and function of Zur, a transcriptional repressor that is exquisitely sensitive to Zn2+ concentration. Although the structures of related Zur proteins on their own are known, here we show how E. coli protein binds to DNA and explain its extreme sensitivity and specificity (it responds to Zn2+ concentrations in the femtomolar range). Our results reveal how the Zur protein switches on and off a bank of bacterial genes that control zinc physiology. Extensive analysis of protein-DNA interactions revealed both a surprising degree of cooperativity and an extremely large range of Zur-DNA binding affinities across the set of genes known as the Zur regulon. The results provide strong support for a controversial idea that the thermodynamics of an ensemble of protein-DNA interactions play a dominant role in the physiological control of gene regulation networks. In addition, we have used our structural and thermodynamic analysis to identify a novel gene target of Zur regulation.

Highlights

Zinc fluxes are involved in regulating a wide variety of cellular functions, including host immune activation [1], malaria parasite invasion of erythrocytes [2], oocyte maturation and fertilization [3,4], glucose-induced insulin secretion [5], as well as the expression of a wide range of microbial genes responsible for metal homeostasis and pathogenicity [6,7]
Overall Fold and Characterization of Zinc Binding Sites The structure of E. coli zinc uptake regulator (Zur) (EcZur) protein in complex with a 31 bp duplex derived from the znuABC operator (PznuABC) was determined by X-ray crystallography using multiwavelength anomalous dispersion (MAD) data collected at both high energy and the zinc absorption edge and refined to 2.50 A (Table 1)
We addressed whether the degree of repression at each promoter PznuABC, PzinT, PL31p, and PpliG correlates with the affinity of Zur for the operators

Summary

Introduction

Zinc fluxes are involved in regulating a wide variety of cellular functions, including host immune activation [1], malaria parasite invasion of erythrocytes [2], oocyte maturation and fertilization [3,4], glucose-induced insulin secretion [5], as well as the expression of a wide range of microbial genes responsible for metal homeostasis and pathogenicity [6,7]. I.e., the number of atoms per cell, for essential transition metals such as zinc are tightly controlled in the face of changing metal concentrations in the surrounding growth environment [9,10]. Microbes use a diverse set of metal-specific sensors known as metalloregulatory proteins to respond to changes in metal concentration in the immediate environment [7,11,12]. These transcription factors control expression of many diverse factors including membrane bound metal ion transporters that optimize cellular physiology in the face of dynamic shifts in metal availability

Methods

Results

Conclusion