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Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD.

ABSTRACT: SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals [Mn(II), Fe(II), Co(II), Cu(I), and Zn(II)] were examined by using a combination of optical spectroscopy and mass spectrometry. Binding of SlyD to Mn(II) or Fe(II) ions was not detected, but the protein coordinates multiple ions of Co(II), Zn(II), and Cu(I) with appreciable affinity (K(D) values in or below the nanomolar range), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ? Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed.

SUBMITTER: Kaluarachchi H

PROVIDER: S-EPMC3253375 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD.

Kaluarachchi Harini H Siebel Judith F JF Kaluarachchi-Duffy Supipi S Krecisz Sandra S Sutherland Duncan E K DE Stillman Martin J MJ Zamble Deborah B DB

Biochemistry 20111114 49

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of ...[more]

PMID: 22047179

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Project description:Control of protein folding is a conserved function in all domains of life. The ubiquitous peptidyl-prolyl cis-trans isomerases (PPIases) catalyze the cis/trans-isomerization of peptidyl-prolyl bonds, often a rate-limiting step for protein folding. PPIases are involved in central processes in eucaryotes but their functions in bacteria are poorly understood. We applied a large-scale two-hybrid screen to identify novel targets of SlyD, a metallochaperone PPIase, from the model organism Escherichia coli, and the important gastric pathogen Helicobacter pylori. SlyD from both organisms interacts with metal-containing proteins including TCA cycle enzymes and with enzymes driving the biogenesis of iron-sulfur (Fe-S) clusters. In H. pylori, a ∆slyD mutant contains strongly diminished amounts of MisSU, its sole Fe-S biogenesis proteins as well as diminished activity of the Fe-S aconitase. E. coli and H. pylori ∆slyD mutants are less susceptible to ampicillin and gentamicin antibiotics and under microaerobic conditions to the ROS-producing molecule hypochlorite. In addition, the intracellular ATP content and proton motive force are reduced in the H. pylori ∆slyD mutant, and its gentamicin and hypochlorite susceptibility phenotypes are rescued upon restoration of MisSU expression. Measurement of malate and fumarate in the H. pylori ∆slyD mutant suggests that the two enzymes before and after fumarase in the TCA cycle are affected. We conclude that SlyD regulates the activity of TCA cycle enzymes by controlling the efficiency of their Fe-S cluster formation. This is the first report of the role of a bacterial PPIase in central metabolism and susceptibility to stress and antibiotics. IMPORTANCE Correct folding of proteins represents a crucial step for their functions. Among the chaperones that control protein folding, the ubiquitous PPIases catalyze the cis/trans-isomerization of peptidyl-prolyl bonds. Only few protein targets of PPIases have been reported in bacteria. To fill this knowledge gap, we performed a large-scale two-hybrid screen to search for targets of the Escherichia coli and Helicobacter pylori SlyD PPIase-metallochaperone. SlyD from both organisms interacts with enzymes (i) containing metal cofactors, (ii) from the central metabolism tricarboxylic acid (TCA) cycle, and (iii) involved in the formation of the essential and ancestral Fe-S cluster cofactor. E. coli and H. pylori ∆slyD mutants present similar phenotypes of diminished susceptibility to antibiotics and to oxidative stress. In H. pylori, measurements of the intracellular ATP content, proton motive force, and activity of TCA cycle proteins suggest that SlyD regulates TCA cycle enzymes by controlling the formation of their indispensable Fe-S clusters.

Dataset Information

Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD.

Publications

Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD.

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