Cu homeostasis in bacteria: The ins and outs

Copper (Cu) is an essential trace element for all living organisms and used as cofactor in key enzymes of important biological processes, such as aerobic respiration or superoxide dismutation. However, due to its toxicity, cells have developed elaborate mechanisms for Cu homeostasis, which balance Cu supply for cuproprotein biogenesis with the need to remove excess Cu. This review summarizes our current knowledge on bacterial Cu homeostasis with a focus on Gram-negative bacteria and describes the multiple strategies that bacteria use for uptake, storage and export of Cu. We furthermore describe general mechanistic principles that aid the bacterial response to toxic Cu concentrations and illustrate dedicated Cu relay systems that facilitate Cu delivery for cuproenzyme biogenesis. Progress in understanding how bacteria avoid Cu poisoning while maintaining a certain Cu quota for cell proliferation is of particular importance for microbial pathogens because Cu is utilized by the host immune system for attenuating pathogen survival in host cells

ER-associated Degradation and Cadmium Dependent Rescue of PCA1

Protein synthesis and proper folding is an essential process for all organisms. In eukaryotes proteins of the secretory pathway are synthesized and inserted into the lumen or membrane of the endoplasmic reticulum. Eukaryotic cells maintain a mechanism for removal of proteins unable to fold properly. This process is known as ER-associated degradation (ERAD). A poorly functioning ERAD can lead to a build-up of misfolded proteins which has been implicated in several degenerative diseases such as Alzheimer’s, Amyotrophic lateral sclerosis, and Parkinson’s. Thus, the study of how proteins are recognized, extracted from the ER, and degraded is essential for determining methods for maintaining protein solubility and stability, and prevention of toxic accumulation of protein aggregates. Our lab has previously identified Pca1, a cadmium exporting P1B-type ATPase in Saccharomyces cerevisiae. A genetic knockout screen led to the discovery that Pca1 expression is controlled post-translationally through the ERAD pathway. Specifically, the ERAD-Cytoplasm (ERAD-C, indicating the location of the misfolding) pathway utilizes the E3 ubiquitin ligase Doa10 to ubiquitinylate substrates. We further tested the mechanism by which Pca1 an eight transmembrane domain containing protein was extracted from the ER membrane for degradation in the cytoplasm. Surprisingly, we determined that the proteasome itself is essential for this process. Finally, we sought to determine the requirements of cadmium sensing and rescue from ERAD as well as the molecular factors involved in recognition of the degron of Pca1. Biophysical characterization revealed cadmium specific binding. A random-mutagenesis screen identified residues required for degradation of Pca1. Bioinformatical study of the Pca1 degron structure identified a hydrophobic patch that when broken with amino acid substitution stabilized the protein. It was also determined that interaction with a known recognition factor of ERAD, Ssa1, was much weaker in the presence of a hydrophilic substitution or cadmium supplementation. Collectively, our results revealed a mechanism in which Pca1 is regulated post-translationally through the ubiquitin proteasome system. We were also able to apply our findings of Pca1 to another ERAD-C substrate. Pca1 is an excellent model for the study of the ERAD-C pathway as it is short-lived and rapidly stabilized by the supplementation of cadmium. Adviser: Jaekwon Le

Spectroscopic Investigation of Conformational Transitions in the Copper-transporting P1B-ATPase CopA from Legionella pneumophila

All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover

Heterogeneous nuclear ribonucleoprotein A2/B1 regulates the abundance of the copper-transporter ATP7A, and its localization depends on cellular copper levels

Copper (Cu) is an essential micronutrient that plays roles in mammalian growth and development. Imbalance of Cu is implicated in several human diseases; thus, cellular Cu levels are tightly regulated by Cu-binding and -transporting proteins that act in concert. While the main Cu homeostasis proteins and their post-translational regulation have been well-characterized, their regulation at the mRNA-level is incompletely understood. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, an RNA-binding protein, was found to regulate Cu levels: its knockdown via small interfering RNA (siRNA) induced a significant decrease in cellular Cu. In this study, we investigated this phenomenon and found that the knockdown of hnRNP A2/B1 decreased Cu levels through increasing the abundance of the Cu-transporter ATP7A, likely through a mechanism involving the 3’ untranslated region (UTR) of the ATP7A transcript. Moreover, downregulation of just the exon 2-containing B-isoforms of hnRNP A2/B1 was sufficient to produce this effect, indicating that they may play a specific role in Cu regulation. This link between hnRNP A2/B1 and ATP7A was found to be true in reverse: increasing the expression of individual hnRNP A2/B1 isoforms lead to a decrease in ATP7A abundance, further suggesting that ATP7A expression is negatively regulated by hnRNP A2/B1. An increase in ATP7A abundance and decrease in hnRNP A2/B1 abundance and Cu levels was also observed in SH-SY5Y neuronal cells during retinoic acid-induced differentiation, highlighting an inverse relationship between hnRNP A2/B1 and ATP7A abundance that may play a role during neuronal differentiation. We also explored the effects of Cu accumulation on hnRNP A2/B1 and found that Cu elevation results in a rapid, dose-dependent increase in the amount of cytoplasmic hnRNP A2/B1. Interestingly, cytoplasmic hnRNP A2/B1 clustered into granules that did not stain for markers of common cytoplasmic granules, suggesting that they form as a unique response to Cu elevation. It is yet unclear whether the Cu-induced cytoplasmic accumulation of hnRNP A2/B1 is related to the hnRNP A2/B1-mediated regulation of ATP7A. However, our data show that there is a link between Cu homeostasis and RNA processing via hnRNP A2/B1 that could potentially be involved in a rapid and specific response to changing Cu levels

Managing the copper paradox: Protein stability, copper-binding, and inter-protein interactions of copper chaperones

To minimize copper (Cu) toxicity, organisms have evolved Cu transport pathways involving soluble metallochaperones that bind, transport, and deliver Cu+ to specific partner proteins, such as Cu-ATPases. The human Cu chaperone, Atox1, delivers Cu to the metal-binding domains of Menkes (MNK) and Wilson (WND) disease proteins that are Cu-ATPases in the Golgi network that transfer Cu to cuproenzymes (e.g., ceruloplasmin) that traverse the Golgi lumen. The metal binding motif, MetX1CysXXCys, and the ferredoxin-like fold appear conserved in both cytoplasmic Cu chaperones and the cytoplasmic metal-binding domains of the target Cu-ATPases from different organisms. The work reported here provides a basic understanding of in vitro holo- and apo-protein stability, Cu-dissociation mechanisms, and donor-acceptor interactions of key copper transport chaperones. Studies were conducted on purified protein variants using circular dichroism, fluorescence, and absorbance methods in equilibrium and time-resolved modes. We developed a kinetic assay to determine the Cu-dissociation mechanism of these proteins and a near-UV CD method for monitoring interactions between Atox1 and WND domains to complement NMR measurements and computer simulations. Despite the conservation of the overall structural fold, the chaperones Atox1 and its bacterial homolog, CopZ, and the metal-binding domains of WND, W2 and W4, have variable chemical and thermal stability in vitro. The role of residues proximal to the metal-binding site was determined using Atox1 as a prototypical Cu chaperone. Met10 is essential for structural stability of Atox1. Thr11 (position X1) seems to be conserved, not for integrity of protein structure, but for facilitating metal exchange between Atox1 and a receptor domain. The structural proximity of the charged side-chain of Lys60 neutralizes the Cu-thiolate center in Atox1. Replacement of Lys60 with an Ala or Tyr results in a higher rate and extent of loss of the metal to small molecule chelator, BCA, than those for wtAtox1. Lys60 also provides electrostatic interactions crucial for Atox1 interaction with W4. Thus, each proximal residue contributes to fine-tuning copper binding and its release mechanism to both the non-physiological Cu chelator, BCA, and the physiological acceptor of the WND protein, W4. Our new kinetic and spectral assays provide a comprehensive in vitro experimental platform for more advanced future mechanistic and kinetic studies

Metal binding to the Polaris protein associated with ethylene sensing by plants

Copper ions are essential to life, but toxic if not tightly regulated. In the model organism Arabidopsis thaliana, the ER-localised ethylene receptor, ETR1, requires Cu(I) at an intramembrane site, dependent on the Cu(I)-transporting P-type ATPase RAN1. However, the detailed biochemical mechanisms of Cu(I)-delivery, and ethylene binding, are unknown. The protein Polaris (PLS), a negative regulator of ethylene signalling, shares some characteristics of known Cu(I)-metallochaperones, and was proposed to be involved in correct Cu(I)-metalation of ETR1. Here, metal binding to PLS has been investigated in-vitro, allowing prediction of its likely metalation state in-vivo. PLS bound Cu(I) and Zn(II) in 2:1 protein:metal stoichiometries, with β2 affinities of 3.79 x1019 and 3.76 x1012 M-2 respectively. Recently developed metalation calculators, based on metal-availability read-out from calibrated bacterial cells, were adapted to use these constants. The metal affinities of the Arabidopsis cytosolic Cu(I) chaperone Atx1, showed Cu(I) bound in a 1:1 and Zn(II) a 2:1 stoichiometry, and its metalation was modelled. This work showed, in E. coli BL21(DE3), by reading out CueR-dependent copA transcripts, Atx1 overexpression decreased Cu(I)-availability, when calibrated using E. coli JM109, with implications for heterologous expression of metalloproteins in bacteria. Availabilities, measured here, were used to correctly predict the metal preference of Atx1 in E. coli, when tested post-extraction. Using the Atx1 Cu(I)-affinity of 5.47 x10-18 M as an estimate for the intracellular buffered Cu(I)-availability in the cytosol of Arabidopsis, the metalation of PLS as a function of Atx1 Cu(I)-metalation showed it was unlikely PLS extracts Cu(I) directly from the buffer, at least not as a 2:1 complex. This thesis speculates upon the putative roles of PLS in the biochemical activities of ETR1, and considers some of the implications and challenges associated with the potential formation of metal-dependent 2:1 ligand:metal complexes (with analogy to PLS2:Cu(I)) in biological systems, more broadly

De Novo Designed Metallopeptides to Investigate Metal Ion Homeostasis, Electron Transfer, and Redox Catalysis.

Protein design is a powerful way to interrogate the basic requirements for function of metal sites by systematically incorporating elements important for function. Single-stranded three-helix bundles with either thiolate-rich sites for spectroscopic characterization and electron transfer, or histidine-rich sites for redox catalysis are described. Using a previous design, two constructs were designed to incorporate a fourth cysteine residue to investigate thiolate-rich sites involved in metal ion homeostasis and electron transfer. Rational re-design replaced a putative coordinating histidine with a cysteine. A second construct embedded a CXXC binding motif into the helical scaffold. These two constructs show different UV-visisble, 113Cd NMR, and 111mCd PAC, which indicate that they form different proportions of CdS3O and CdS4. The spectroscopy of these sites sheds light on how Cd(II) bindis to CadC and suggests a dynamic site in fast exchange with the solvent. Previous attempts at the design of a rubredoxin site have focused on reproducing the peptide fold around or using flexible loop regions to define the site in addition to canonical CXXC motifs. However, the use of CXXC motifs embedded in an α-helical scaffold produces a rubredoxin site that reproduces the Mössbauer, MCD, and EPR of rubredoxin without the use of loop regions. This successful design is the largest deviation from consensus rubredoxin and zinc finger folds reported. Electron transfer rates through a de novo designed scaffold were studied by the design and synthesis of a ruthenium trisbipyridine derivative appended to an exterior cysteine residues. A redox-active tyrosine in the 70th position is implicated as a relay amino acid from the iron center and absence of the tyrosine decreases the rate of electron transfer from the metal site. This is the first photo-generated tyrosine radical in a designed protein. A construct, which was previously reported for CO2 hydration, is substituted with copper and its spectroscopic and nitrite reductase activity are studied. This is the first demonstration of nitrite reductase activity in a single-stranded designed peptide. This thesis provides insight into designed proteins and their applications and lays the groundwork for further studies to progress towards a unified multifunctional redox protein.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113513/1/agtebo_1.pd