Moving metals: how microbes deliver metal cofactors to metalloproteins

Graphical Abstract

Transition metals are required nutrients for all forms of life, as 30–40% of proteins are predicted to require the binding of a specific metal ion to support proper protein function. Several recent advancements have shaped our evolving understanding of how bacterial proteins bind the correct cognate metal ion while avoiding mismetallation. Herein we summarize our understanding of how bacteria distribute metal ions to metalloproteins and speculate on future research directions in bacterial metallobiology.

Introduction

First row d-block metal ions, manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), and zinc (Zn), are mandatory for all Kingdoms of life as they serve as required cofactors for numerous essential enzymes. Approximately 30–40% of proteins are predicted to require metal binding for function (1, 2). While nutrient transition metals are required for life, they are also extremely toxic in excess (3). Metals cannot be synthesized by living organisms and must be obtained from the environment, therefore, single-cellular organisms like bacteria are particularly susceptible to fluctuations in metal availability as they inhabit environmental niches where they face both extreme metal limitation and excess (4). Thus, bacteria have evolved numerous systems to sense, acquire, and maintain the intracellular concentrations of metals within specific bounds that meet cellular requirements while avoiding toxicity. These systems include sensory mechanisms that monitor the intra- and extracellular environments for metal concentrations; transcriptional regulatory elements to respond to fluctuations in these concentrations; and acquisition, efflux, chaperone, and storage systems to modulate the intracellular pool of bioavailable metals (5).

In particular, pathogenic bacteria face conditions of both extreme nutrient metal limitation and intoxication during host infection. Vertebrate hosts exploit the nutrient metal requirements of bacterial pathogens by using a suite of mechanisms that actively limit access of bioavailable nutrient metals to invading pathogens in a process known as nutritional immunity (5, 6). Conversely, the toxicity of excess transition metals is highlighted by the fact that vertebrates leverage the lethal effects of metals as a defense strategy against infection. Innate immune cells mobilize high concentrations of metals to intoxicate phagocytosed pathogens (7, 8). These aspects of pathogenesis have resulted in decades of research to understand the mechanisms that bacteria employ to maintain metal homeostasis (9–11).

While the presence of Fe and Cu ions contribute to cellular toxicity through oxidative stress due to their ability to efficiently decompose H₂O₂ to HO• through Fenton-like pathways (12, 13), excess metal also contributes to cellular toxicity through the mis-metalation of metalloproteins (14). Paradoxically the intrinsic metal-binding affinities of enzymes often do not match their metal requirements (15–17), and proteins frequently bind non-cognate metals in vitro, primarily dictated by the Irving–Williams series. This mis-metalation can result in enzymes that are catalytically inactive (18). Bacteria employ at least three strategies to ensure that metalloproteins bind the correct cognate metal: i) controlling the cellular accumulation of metals at an inverse concentration to their protein complex stabilities (17, 19), ii) segregating protein maturation to cellular compartments that contain differential metal availabilities (18), and iii) encoding for specialized proteins to govern the distribution of metals to cognate target proteins, known as metallochaperones. Metallochaperones function as specialized delivery machinery that serve two functions: the sequestration of reactive transition metal ions from binding to non-target proteins, and post-translational metal cofactor insertion to target client proteins. The activities of metallochaperones allow bacterial cells to maintain viability when cellular nutrient metal concentrations fluctuate by prioritizing the distribution of metal cofactors to essential enzymes while avoiding metal-mediated toxicity.

Intracellular free metal pools are maintained at extremely low concentrations (20) and labile metal concentrations change in response to environmental shifts (21–23). This suggests a requirement for specific metallochaperones to ensure the loading of the correct metal ions to their cognate binding proteins to promote enzymatic activity and mitigate toxicity while relative intracellular metal availability fluctuates (19, 24, 25). Since the first description of a bacterial metallochaperone, Enterococcus hirae CopZ (26, 27), numerous proteins have been identified that function in the maturation of metallocenters in metalloproteins. Several of these confirmed and putative metallochaperones are induced during conditions of both metal limitation and intoxication (28–30) implicating them in the maintenance of cellular transition metal homeostasis under conditions of metal stress.

In recent years, increased interest in bacterial metallochaperones has resulted in several exciting findings in the field, including the implication of new classes of proteins in metal transfer. This perspective will summarize our understanding of the mechanisms utilized by bacteria to distribute required metal cofactors to specific cognate metalloproteins while highlighting these recent advancements. We will also discuss open questions in the field and speculate on future research directions.

Body

Copper storage and chaperone systems

Chaperones that deliver Cu to target proteins across numerous domains of life were the first metallochaperones described in the literature (26, 27, 31, 32). Due to the ability of Cu ions to mis-metalate metalloproteins (16) it is modeled that free Cu ions do not exist within bacterial cells. Cu metallochaperones sequester Cu ions and shield them from inadvertent reactions with non-target proteins (33). Unlike other metallochaperones discussed later which utilize nucleoside triphosphate hydrolysis to drive their metal delivery function, Cu metallochaperones typically rely on protein affinity gradients for metal cofactor delivery (34). Delivery of Cu to Cu-requiring bacterial proteins is the most well-studied mechanism in nutrient transition metal distribution, thus, several classes of bacterial proteins have been implicated in the delivery of Cu to client proteins. Below we discuss multiple classes of bacterial Cu chaperones, including the recently discovered copper sequestering proteins (Csps).

CopZ chaperones

CopZ from Enterococcus hirae was the first described metallochaperone (26, 27). CopZ-like proteins are small, soluble metallochaperones that adopt a βαββαβ ferredoxin-like fold and contain a MxCxxC Cu binding site (35). These proteins are universally conserved across diverse species from humans to yeast and bacteria (27). The first structurally described CopZ-like chaperone was MerP, a periplasmic protein which binds Hg and transfers it to the membrane transport protein MerT (36). Since the initial finding that E. hirae CopZ functions in resistance to excess Cu (37) the role of CopZ-like metallochaperones in bacterial metal homeostasis has been studied in several organisms (38–41). It appears that CopZ chaperones serve two primary conserved functions in Cu resistance: the delivery of Cu to P-type ATPases for movement of the metal ions across biological membranes (35, 42, 43), and the intracellular detoxification of excess Cu ions, likely through sequestration (44–46). However, CopZ proteins have also been shown to transfer Cu to bacterial Cu-responsive transcriptional regulators that serve in mounting the adaptive response to elevated Cu concentrations (26, 47). This work, coupled with the finding that Bacillus licheniformis CopZ supports Cu incorporation into B. licheniformis multicopper oxidase CotA when both proteins are co-expressed in E. coli (48), indicates that CopZ-like Cu chaperones may serve unappreciated functions in the delivery of Cu to client proteins other than P-type ATPases.

Csps

A recently described class of bacterial proteins, Csps, were discovered due to their unique capacity to bind large quantities of Cu ions with high affinity (49). Structural analysis of Csp1 from the methanotroph Methylosinus trichosporium and the model organism Bacillus subtilis revealed that Csps are bundle-forming, largely alpha-helical proteins that contain several Cys residues, which are involved in binding up to 80 Cu ions (49, 50). Phenotypic analysis of a csp mutant of M. trichosporium indicated that Csps store and possibly deliver Cu to particulate methane monooxygenase (pMMO) enzymes, the major Cu-requiring proteins within the cell (49). However, direct interaction between the two proteins has not been established. Recent work indicates that the B. subtilis cytoplasmic Csp, so called Csp3, supplies Cu to the endospore multicopper oxidase CotA (51), collectively implicating Csps in delivery of Cu to Cu-requiring proteins. Mining of bacterial genomes revealed that Csp3s are present within the genomes of over 4,000 species, including several important human pathogens (52), suggesting that Csp-mediated Cu delivery to metalloproteins may be conserved in these species. The finding that a Csp produced by Acinetobacter baumannii is enriched upon exposure to the metal-chelating immune effector protein complex calprotectin suggests that Csps may play a role at the host-pathogen interface (53). However, there are no reports of the function of a Csp in a pathogen. The mechanism of Cu transfer between Csps and Cu-requiring target proteins remains unclear, indicating that future studies will need to determine if other proteins are required for Cu transfer from Csps. The finding that CotA metalation appears to be limited to interaction with Csp, and not other Cu-binding proteins (51), suggests that Cu transfer between Csps and client proteins may be specific. Given that the suite of Cu-requiring proteins within bacterial proteomes is limited (54), future work will need to identify if other Cu-requiring enzymes are metalated by Csps. Interestingly, in Streptomyces lividans, CopZ delivers Cu to a cytoplasmic Csp, suggesting that these Cu homeostasis and delivery mechanisms may work in concert with one another, an exciting possible future direction for the field of bacterial Cu delivery (55).

Nickle chaperones

Ni-Fe hydrogenases and ureases are rare Ni-requiring classes of enzymes that necessitate specific suites of accessory proteins for their metalation. Investigation of Ni-Fe hydrogenases in E. coli has developed one of the clearer understandings of chaperone-mediated metallocenter maturation in bacteria. Ni-Fe hydrogenase activity is dependent on the P-loop G3E GTPase HypB (56–58). HypB is believed to gather Ni from the cellular Nik transporter system and bind the accessory protein HypA, which is thought to serve as a docking protein to deliver HypB to a target hydrogenase precursor (59). HypB transfers Ni to HypA, ultimately inserting the metal to the target hydrogenase to facilitate enzymatic activity (60). The finding that the presence of increased concentrations of Ni can overcome the loss of hyp genes suggests that their role in hydrogenase maturation does not involve any chemical transformation, but instead is to facilitate nickel delivery (59). Nickel acquired from the Nik transport system is largely inserted into hydrogenases, and mostly unavailable to the Ni-responsive transcriptional regulator NikR (61). Direct delivery of imported Ni to hydrogenases may serve to shield imported Ni from deleterious interactions with other metalloproteins while maintaining hydrogenase activity. The HypB system also delivers Ni to NiFe hydrogenases in Helicobacter pylori (62–64) and Aeromonas spp. (65). Similar mechanisms have been suggested for Ni transfer by UreG during urease assembly (66, 67).

Cobalt chaperones

The COG0523 protein family is a subset of the ubiquitous G3E family of GTPases that are encoded within all Kingdoms of life and have recently emerged as an important group of metallochaperones (68, 69). COG0523 family members contain a GTPase domain consisting of canonical Walker A and Walker B motifs, and a conserved putative metal-binding CXCC motif (70). The first identified member of the COG0523 family was CobW from Pseudomonas denitrificans, so named due to its role in the biosynthesis of the cobalt-containing metabolite, cobalamin (71). The observation that the expression of several genes encoding for COG0523 family proteins are induced in response to cellular nutrient metal limitation suggests a role in the maintenance of metal homeostasis under conditions of metal starvation (29, 68, 72). Since this initial discovery the COG0523 family of proteins have been the subject of investigation. This work has resulted in a model where the COG0523 enzyme utilizes the energy from GTP hydrolysis to drive the delivery of transition metal ions to cognate recipient metalloprotein clients. In agreement with this model, recent work illustrated that a eukaryotic COG0523 family protein, ZNG1, binds to and delivers Zn to the client protein METAP1 in a GTP hydrolysis-dependent manner, activating the enzymatic activity of METAP1 (73, 74). This work was the first to demonstrate direct metal transfer from a COG0523 family protein to a client metalloprotein.

Since the initial observation that the COG0523 family protein CobW is required for synthesis of cobalamin (71), it has been speculated that CobW functions in Co delivery. However, CobW-dependent Co transfer has not been established (75). Recent work on the E. coli CobW has indicated that CobW binds Co within the intracellular environment, specifically when CobW is also bound to GTP. The calculated Co occupancy of CobW correlates with cellular cobalamin synthesis, collectively implicating CobW in the insertion of Co during cobalamin synthesis (76). Although cellular requirements for Co are low, Co within bacteria is primarily incorporated into cobalamin, so it remains unknown if CobW serves any metal transfer functions to other clients (77, 78).

Zinc Chaperones

Several bacterial COG0523 family members are under the transcriptional regulation of the Zn master metalloregulator Zur and are therefore induced under conditions of Zn limitation. This observation suggests that these enzymes function in bacterial Zn homeostasis when bacteria are starved for Zn (29, 68, 72). Consistent with this hypothesis, Zn-regulated COG0523 family proteins in multiple organisms, including several important human pathogens, bind Zn and participate in survival when Zn is limiting. These proteins include A. baumannii ZigA (28), Staphylococcus aureus ZigA (79), B. subtilis ZagA (80), and E. coli YeiR (81). These findings suggest that Zn-regulated COG0523 enzymes may reallocate Zn to metalloenzymes that are essential to survival during Zn starvation. While the identities of COG0523 metal-recipient client proteins have remained mostly elusive, recent interrogation of Zn-responsive COG0523 proteins have suggested interacting partners. A. baumannii ZigA impacts both cellular Zn and histidine levels, possibly through interaction with HutH, a Zn-binding histidine ammonia-lyase (28). Further, studies of B. subtilis ZagA indicate that it interacts with FolE, a Zn-dependent GTP cyclohydrolase IB enzyme that catalyzes the first step in folate synthesis. The finding that the Acinetobacter baylyi ZagA homolog interacts with the B. subtilis FolE, coupled with the observation that Zur-regulated COG0523 proteins are often encoded near the folEB genes in bacterial genomes, suggests that Zn transfer between ZagA and FolE may be an evolutionally conserved interaction (80).

Some bacterial species encode multiple enzymes that putatively function as metallochaperones (68). Notably, A. baumannii encodes two COG0523 proteins, one induced under conditions of Zn limitation, and one that is not (28), suggesting that these proteins likely function within different environments. The conditions under which COG0523 proteins that are not regulated in response to metal concentrations are required remains unclear (82), and how bacterial cells coordinate the function of several metallochaperones also remains unknown.

Iron chaperones

Several bacterial proteins require Fe in the form of the pre-assembled Fe-cofactors heme and Fe-S clusters. The mechanisms of biogenesis and insertion of these cofactors will not be discussed here as they are beyond the scope of this perspective and have been the subject of recent reviews (83–85). Bacterial enzymes, such as nitrile hydratases (NHases), require Fe ions in the absence of these Fe-cofactors (86). The finding that expression of the Rhodococcus equi COG0523-family GTPase Nha3 is required for hydratase activity implicated Nha3 in the delivery of Fe ions to NHases (87, 88). Consistent with this hypothesis, the co-expression of the Pseudomonas chlororaphis Nha3 homologue, NhpC, with NHase genes in E. coli induces NHase activity in cell-free extracts, and increases the Fe content of purified NHase enzymes by over four-fold (89). However, GTP-dependent Fe delivery has not been established. Further, the function of either Nha3 or NhpC within their native hosts has not been reported.

Conclusions and perspective

Under conditions of differential metal availabilities metallochaperones may represent an underappreciated means of post-translational enzymatic regulation by inserting cognate metal ions to specific client proteins to enable enzymatic activity, while reducing the toxic effects of mis-mismetallation. If individual metallochaperones have multiple client proteins and how metal delivery is prioritized to specific clients are major questions in the field. Further, whether chaperones can gather metals directly from other proteins to reallocate to specific clients during metal restriction is also unknown. These questions are particularly interesting in the context of infection, where invading bacteria face conditions of both extreme metal intoxication and limitation. Extracellularly, bacterial pathogens experience simultaneous multi-metal limitation through nutritional immunity (6), and within phagosomes bacterial cells are intoxicated with high concentrations of Cu and Zn (90–92). Under these conditions metallochaperones may serve as key mediators of bacterial adaptability by governing the metalation of required proteins. This hypothesis is supported by findings that several putative metallochaperones metalate proteins required for host colonization and are implicated in virulence phenotypes (28, 65, 93–96).

Recent years have seen exciting new advancements in our understanding of the mechanisms that bacteria employ to distribute required metal cofactors to target enzymes. However, the metallobiology field has struggled with identifying metallochaperone client proteins and demonstrating direct metal transfer between chaperone and clients. Recent analyses in eukaryotic cells resulted in the first studies to identify a bona fide client protein of a COG0523 metallochaperone and demonstrate direct metal transfer (73, 74). These studies may serve as a blueprint for more mechanistic analysis of bacterial metallochaperones moving forward. It remains unclear if these chaperones interact with one specific target client protein, or several. Yeast-two-hybrid studies of eukaryotic COG0523 proteins revealed several protein binding partners, suggesting that COG0523 proteins may have multiple clients (73). If individual bacterial chaperones are, in fact, capable of metalating several client proteins, gaining global understandings of these interactions may reveal the impacts these proteins have on bacterial physiology.

The recent findings that a newly identified class of proteins, Csps, participate in metal delivery to target proteins indicate a possibility that there are yet unidentified proteins outside of those discussed here that serve functions in metal transfer. Studies aimed at defining bacterial metalloproteomes have identified novel metal-binding proteins (18, 50, 97), similar strategies may be fruitful in the identification of previously unknown proteins that participate in metalloprotein metalation. Collectively, these questions represent exciting new areas of research in the burgeoning field of bacterial nutrient metal allocation.