Structural and functional characterizations of the C-terminal domains of CzcD proteins

https://doi.org/10.1016/j.jinorgbio.2020.111087Get rights and content

Highlights

  • The metal-bound structures of the C-terminal domains of bacterial cation diffusion facilitator proteins are described.

  • All structures show conservation of the ‘C site’ described for the Yiip proteins from E. coli and Shewanella oneidensis.

  • In the Cuprividus metallidurans, Pseudomonas aeruginosa proteins, a new metal binding site is described that may promote dimerization and therefore play a role in the transport mechanisms of these proteins.

  • Metal binding at site ‘D’ may promote dimerization and function in the transport mechanisms of these proteins.

Abstract

Zinc is a potent antimicrobial component of the innate immune response at the host-pathogen interface. Bacteria subvert or resist host zinc insults by metal efflux pathways that include cation diffusion facilitator (CDF) proteins. The structural and functional examination of this protein class has been limited, with only the structures of the zinc transporter YiiP proteins from E. coli and Shewanella oneidensis described to date. Here, we determine the metal binding properties, solution quaternary structures and three dimensional architectures of the C-terminal domains of the metal transporter CzcD proteins from Cupriavidus metallidurans, Pseudomonas aeruginosa and Thermotoga maritima. We reveal significant diversity in the metal-binding properties and structures of these proteins and discover a potential novel mechanism for metal-promoted dimerization for the Cupriavidus metallidurans and Pseudomonas aeruginosa proteins.

Graphical abstract

The dimeric structures of the C-terminal domains (CTDs) of the metal transport CzcD proteins from Cupriavidus metallidurans (CmCzcDc), Pseudomonas aeruginosa (PaCzcDc) and Thermotoga maritima (TmCzcDc), including their metal binding sites (blue and gray spheres).

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Introduction

Zinc (Zn) is one of the most abundant metals in biology. It is essential for numerous cellular pathways that function in immune response, respiration, transcriptional regulation, cell proliferation and cell death, by serving catalytic and structural roles when bound to proteins and enzymes [1,2]. Zn is also a potent antimicrobial component of the innate immune response at the host-pathogen interface [3]. Phagocytic cells, such as macrophages, have been shown to use Zn within the phagolysosome to intoxicate invading bacterial pathogens. This was first established with Mycobacterium tuberculosis [4] but has since been shown to occur in response to a range of Gram-positive and Gram-negative bacterial pathogens including Streptococcus pyogenes, Escherichia coli, and Salmonella enterica serovar Typhimurium [5,6]. The antimicrobial activity of Zn has been associated with the inappropriate interaction of the metal ion with bacterial proteins in a process termed mismetallation. This can impair or inactivate proteins and has been attributed to disruption of manganese import, inappropriate alteration in gene expression and disruption of metabolic processes [7,8]. Subversion or resistance to host Zn insult in bacteria is achieved, at least in part, by metal efflux pathways [9] that include cation diffusion facilitator (CDF) proteins [[10], [11], [12]], P-type ATPases [13,14], resistance, nodulation and division (RNDs) efflux transporters [15,16] and ZRT (Zn regulated transport)/IRT (iron regulated transport)-like transporters (ZIPs) [[17], [18], [19], [20], [21]] depending on the organisms.

CDF family proteins are ubiquitous metal ion transporters present in all domains of life [10,12,17]. CDF proteins occur as homodimers and couple proton antiport to the export of divalent metal ions across the cytoplasmic membrane. Accordingly, they have been established to serve a crucial role in intracellular metal homeostasis and contribute to bacterial virulence [10,[22], [23], [24], [25], [26], [27], [28]]. The most extensively characterized CDF is the Zn transporter YiiP [29,30], which has been structurally characterized by X-ray crystallography for the E. coli protein (EcYiiP; 2.9 Å resolution, PDB 3H90 [31]) and by cryo-electron microscopy for the Shewanella oneidensis ortholog (SoYiiP; 4.1 Å resolution, PDB 5VRF [32]). Structural analyses of EcYiiP revealed a Y-shaped homodimeric architecture, comprised of a transmembrane domain (TMD) and an extended cytoplasmic C-terminal domain (CTD) [10,23,33,34] (Fig. S1). The TMDs of each CDF monomer are splayed apart at the membrane surface and the TMD/CTD interface. A salt bridge between residue Lys77 of one monomer and Asp207 of the other monomer forms the ‘charge interlock’, which contributes to homodimer stabilization [31,34]. SoYiiP shares the domain architecture of EcYiiP, but with the TMDs and CTDs being closely apposed [32].

Crystallographic studies of EcYiiP identified four metal-binding sites (A, B, C1 and C2) in each CDF monomer (Fig. S1) [34]. Site A is located in the TMD, with amino acid ligands provided by transmembrane helices (TM) 4 (Asp45, Asp49) and 5 (His153, Asp157; all residues are EcYiiP numbering) [22,32,34]. Site B is located in a short intracellular loop that connects TM2 and TM3 [28]. The C site is dinuclear, with two Zn atoms at positions C1 and C2, and is located within the CTD [22,34]. The Zn(II) atoms in the C site are bridged by a conserved Asp285 residue and coordinated by a series of histidine residues (His232, His248 and His283 from the same monomer, and His261 from the neighboring protomer).

Structural analyses also revealed that the CTD monomers are composed of a mixed α1-β1-β2-α2-β3, metallochaperone-like fold within the CTD stem-like structures of EcYiiP (EcYiiPc; Fig. S1) and SoYiiP (SoYiiPc) [31,34]. This fold has also been observed in the structures of CTDs from a number of bacterial CDF proteins, which have been studied as independent, soluble constructs. These include the CTDs of the Thermus thermophilus cadmium‑zinc resistance protein CzrB (TtCzrBc, 3BYP, 3BYR) [23], the Magnetospira sp. Magnetosome protein MamB QH-2 (MgMamBc, 5HO1, 5HO5) [35], the Thermotoga maritima cobalt‑zinc‑cadmium resistance protein CzcD (TmCzcDc, 2ZZT) [36] and the Magnetospirillum gryphiswaldense Magnetosome protein MamM (MgMamMc, 3W5X) [37] (Fig. S2). These CTD structures show similar dimeric arrangements, but with distinctly different relative monomer orientations. For example, the structures of metal-free TtCzrBc and Zn-bound TtCzrBc both show V-shaped homodimers, with metal binding accompanying a closer monomer association than in the metal-free state (Fig. S2) [12,23], whereas MgMamBc shows comparatively tight packing between monomers in both the metal-free and Zn-bound states (Fig. S2) [23,37,38]. Only the metal-free structures of the TmCzcDc and MgMamMc have been described to date, which both show open, splayed, dimeric structures (Fig. S2). The metal-binding properties of the CTDs also show considerable diversity. Most CTDs bind Zn at site C, and EcYiiP, SoYiiP and TtCzrBc contain dinuclear sites. In contrast, MgMamBc contains a mononuclear site that is entirely unique. Collectively, these data highlight the potential broad diversity of CTD structures, which also limits the generality of mechanistic insights for this protein family (Fig. S2).

Despite the contribution of CDFs to cellular metal homeostasis, questions regarding their structure and mechanisms remain. In particular, the link between sequence conservation and function remains unclear and there is also a paucity of knowledge on the relationship between metal-binding and oligomerization. Here, we have investigated the structure and properties of the CTDs of the bacterial CzcD efflux proteins from Cupriavidus metallidurans (CmCzcDc), Pseudomonas aeruginosa (PaCzcDc) and Thermotoga maritima (TmCzcDc). We reveal for the first time, the structures of these protein domains in their metal-bound states and provide insights into the thermodynamics governing metal binding by these proteins. Collectively, this work highlights the conservation of CTD metal binding sites among the CDF protein class and the mechanisms by which metal binding influences quaternary structures.

Section snippets

Expression and purification of the truncated CTDs

The CTD construct of TmCzcDc (UniProt code Q9WZX9, residues 206–306) used in this work was identical to that described for the published structure [36]. The CmCzcDc and PaCzcDc constructs were designed by a combination of sequence alignments with TmCzcDc and sequence analyses with the XtalPred web server [39]. The recombinant proteins CmCzcDc, PaCzcDc and TmCzcDc were overexpressed as glutathione-S-transferase (GST) and/or His6 fusion proteins and purified by affinity (reduce glutathione (GSH)

Conclusion

The characterizations of the Ni(II)-CmCzcDc, Zn(II)-PaCzcDc and Zn(II)-TmCzcDc proteins described here allow for a wide-ranging comparison of CDF CTD structures and properties, with a particular focus on the effect of metal binding.

The majority of structures described to date of the metal-bound CDF CTDs, both from the current study and those previously described, include a binding site equivalent to the C site, which was described for the EcYiiP structure. In the EcYiiP, SoYiiP and TtCzrBc

Materials and methods

Residue numbering for all proteins constructs described here including structural descriptions and the submitted Protein Data Bank (PDB) coordinates follow the PDB convention, numbered according to the full length proteins. All the proteins discussed in this work are the predicted C-terminal domains of full length proteins, unless otherwise noted.

Accession numbers

The coordinates and structure factors for the Ni(II)-CmCzcDc, Zn(II)-PaCzcDc and Zn(II)-TmCzcDc structures have been deposited in the Protein Data Bank (PDB) with accession codes 6VD9, 6VD8 and 6VDA, respectively.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

This research was funded by Australian Research Council (ARC) Discovery Project Grant DP170102102 to CAM and Future Fellowships to MJM (FT180100397) and CAM (FT170100006). This work was also supported by the National Health and Medical Research Council (NHMRC) Project Grants GNT1080784 and GNT1140554 to MJM and CAM. Part of this study was carried out using the MX2 beamline at the Australian Synchrotron, with is part of ANSTO, and made use of the ACRF detector. We thank the beamline staff for

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