Metallochaperones and metalloregulation in bacteria

Abstract

Bacterial transition metal homoeostasis or simply 'metallostasis' describes the process by which cells control the intracellular availability of functionally required metal cofactors, from manganese (Mn) to zinc (Zn), avoiding both metal deprivation and toxicity. Metallostasis is an emerging aspect of the vertebrate host-pathogen interface that is defined by a 'tug-of-war' for biologically essential metals and provides the motivation for much recent work in this area. The host employs a number of strategies to starve the microbial pathogen of essential metals, while for others attempts to limit bacterial infections by leveraging highly competitive metals. Bacteria must be capable of adapting to these efforts to remodel the transition metal landscape and employ highly specialized metal sensing transcriptional regulators, termed metalloregulatory proteins,and metallochaperones, that allocate metals to specific destinations, to mediate this adaptive response. In this essay, we discuss recent progress in our understanding of the structural mechanisms and metal specificity of this adaptive response, focusing on energy-requiring metallochaperones that play roles in the metallocofactor active site assembly in metalloenzymes and metallosensors, which govern the systems-level response to metal limitation and intoxication.

Keywords: allosteric regulation; metallochaperone; metalloregulation.

Figure 1. Overview of the central concepts of transition metal homoeostasis

(A) Biologically important late d-block first-row transition metals extracted from the periodic table from Mn to Zn. The competitiveness in a cellular environment is inversely related to ‘bioavailability’ of each metal in the cell. Bioavailability is roughly based on reported metal sensor affinities for their cognate metal and is not a direct measure of rapidly exchangeable metal in cells [23]. (B) Schematic representation of bacterial response to metal-induced stress, highlighting the roles of sensor proteins and metallochaperones discussed here. Note that a metallochaperone may not necessarily function via a direct interaction with the target or client protein; in fact, much of the work on the G3E GTPases discussed here argues against a direct insertion mechanism (see text for details).

Figure 2. Ribbon representation of the structure of homodimeric Helicobacter pylori HypB (4LPS) with one subunit shaded grey, the other shaded white as representative of a G3E P-loop GTPase

Signature motifs G1–G5 are indicated on the structure shaded cyan, orange, magenta, blue and yellow for G1–G5 respectively. G1, P-loop/Walker A motif, GxxxxGKS/T; G2, switch I, with only a threonine nearly universally conserved; G3, switch II/Walker B motif, with the minimal sequence DxxG, but ExxG in G3E family GTPases; G4, base recognition motif/base specificity loop, (S/N/T)KxD; G5, guanine base specificity determinant, with the sequence motif SAK, but only weakly conserved in G3E GTPases. Mg^II ions, green; GTP, red stick; tetrathiolate (S₄) subunit bridging transition metal-binding site bound to Ni^II (shaded black); Zn^II in a related bacterial HypB is also known to bind here with a different coordination structure and stoichiometry [208].

Figure 3. G3E family P-loop GTPases involved in the maturation of metalloenzymes

(A) H. pylori urease accessory protein UreG (protomers shaded grey and white) bound to GDP (pdb code 4hi0). (B) H. pylori urease metallochaperone complex of UreD (light blue), UreF (green) and UreG (grey) (4hi0), including dimeric UreE (orange; 3yn0) in an orientation described in a previously reported docking model [56]. (C) The trimer-of-trimers urease apoprotein (UreA, UreB, UreC; cyan, 4z42) from Yersinia enterocolitica either sequentially binds UreD, UreF and UreG or binds a preformed UreD₂F₂G₂ complex. Formation of the active enzyme requires GTP binding and hydrolysis by UreG and Ni^II delivery by UreE [27]. (D) Thermococcus kodakarensis homodimeric HypB (grey and white) bound to ADP (5aun) [48]. (E) T. kodakarensis chaperone complex HypA₂B₂ (HypB dimer shaded pink) (5aun) [48]. (F) E. coli mature [NiFe] hydrogenase (5a4f) highlighting the active site. (G) Human MeaB GDP bound homodimer (2www). (H) Cupriavidus metallidurans chaperone complex formed by a MeaB monomer in a complex with the holo-Cbl-binding domain (yellow) (4xc6). (I) C. metallidurans chaperone complex with the methylmalonyl-CoA mutase (MCM, cyan; 4xc6). (J) E. coli Zn^II-bound dimer of YjiA, a COG0523 subfamily member of unknown function (4ixm) [74]. Nucleotides are shown in red stick and metals are shown as black spheres. Zn^II ligand C66 is derived from the C64-x-C66-C67 (CxCC) motif conserved in COG0523 proteins, which is close to the G2 loop (switch I) containing the Zn^II ligand E37; ?, chaperone-accessory protein and chaperone-target protein complexes for YjiA and other COG0523 GTPases are as yet unknown. The coordination structures of the bound metals are expanded in each panel.

Figure 4. Structural families of metal efflux regulators

For each family, boxes for metals that are known to be sensed are shaded red on the abbreviated periodic table, while boxes on the right denote family members that are known to react with small molecule reactive species, also following the order of the periodic table as C, reactive electrophile species (RES); N, reactive nitrogen species (RNS); O, reactive oxygen species (ROS); and S, reactive sulfur species (RSS). Boxes identifying putative metal and non-metal sensors that are likely not physiologically relevant are shaded pink or yellow respectively. The four-letter designations for individual proteins that perform the function listed in the nearby box are shown (see text for details). Ribbon representations of representative sensors in the DNA-bound state are shown on the right with individual protomers shaded white and blue in each case, with the DNA-binding motif shaded red on both protomers. Metal ions are shaded in black. Structures are from top to bottom are (A) Staphylococcus aureus QsrR in the apo form (4hqe) [209] (B) E. coli Cu^I-sensor CueR with the apo (right, 4wls) and Cu^I-bound forms shown, with Cu^I ions in black (left, 4wlw) [105] (C) Geobacillus thermodenitrificans Cu^I-sensor copper-sensitive operon repressor (CsoR) with regulatory Cu^I ions shown The DNA cartoon is shown to represent the proposed DNA-binding mode (4m1p) [128] (D) S. aureus MecI as a model for Enterococcus copper sensor (CopY) (2d45) [210].

Figure 5. Structural families of metal uptake regulators

Boxes, colours and labels follow the same convention as in Figure 4. Structures are from top to bottom: (A) Magnetospirillum gryphiswaldense MSR-1 ferric uptake regulator (Fur)-Mn²⁺ (4rb1) (B) M. tuberculosis IdeR (2isz) (C) E. coli NikR (2hzv).

Figure 6. Other structural families of transcriptional regulators where metallosensors have been identified

Boxes, colours and labels follow the same convention as in Figure 4. Structures are from top to bottom: (A) Lactococcus lactis HrtR (3vok) (B) Bacillus subtilis OhrR (1z9c) (C) Vibrio cholerae FadR as a model of the DNA bound form of metal-binding proteins from the FadR family. (4p9u) (D) E. coli ModE in molybdate-bound form without DNA (1b9m).

Figure 7. Ribbon representations of five representative metalloregulatory protein families

The approximate location of metal sites schematized on each structure, and the chelate or disulfide of representative members of each family shown in atomic detail on the insets. Subunits are shaded blue and grey, with the regulatory family indicated on top, and the specific protein designation indicated directly below. Ligand sets are as defined for the specific protein indicated. For MarR and TetR proteins (panels B and C) the pockets for organic ligands are displayed with a yellow line. Structures are from top to bottom: (A) S. aureus CzrA in the Zn form (2m30), Xylella fastidiosa BigR in the oxidized form (inset, 3pqk), S. aureus QsrR-menadione complex (inset, 4hqm) (B) S. pneumoniae adhesin competence regulator (AdcR) in the Zn form (inset, 3tgn) (C) Streptococcus agalactiae SczA in the metal-bound form (3kkc) [3], L. lactis HrtR in the haem-bound form (inset, 3vp5) (D) Pseudomonas aeruginosa MerR in the Hg bound form (5crl), E. coli CueR in the Cu bound form (inset, 1q05), Ralstonia metallidurans PbrR in the Pb bound form (inset, 5gpe) (E) Geobacillus thermodenitrificans CsoR in the Cu bound form (4m1p), formaldehyde-treated E. coli FmrR containing a methylene bridge (CH₂) that links C35′ and the N-terminal P2 residue of the adjacent protomer (inset, 5lbm).