Identification and characterisation of a novel family of copper storage proteins from Methylosinus trichosporium OB3b

PhD ThesisMethane oxidizing bacteria (MOB) use methane as their main source of carbon and energy. The main methane oxidizing enzyme in MOB is the copper-containing particulate methane monooxygenase (pMMO), a rare example of cytoplasmic copper enzyme. Some ‘switchover’ strains are capable of differentially expressing pMMO as well as a soluble iron-containing form (sMMO), and the switchover is regulated by copper. MOB secrete methanobactin (mb) which mediates copper uptake and is internalized in the cytoplasm. Despite this pathway for copper import, as well as copper regulating components such as CopA, CopZ and CueR being present in MOB, little is known on how these bacteria handle the large amounts of copper required for methane oxidation by pMMO. Through metalloproteomic analysis of soluble extracts from the switchover MOB M. trichosporium OB3b a large number of soluble copper pools were visualized and a novel copper protein, Csp1, was identified. Two more homologues, Csp2 and Csp3, were identified in M. trichosporium OB3b through bioinformatics. In vitro characterization of Csp1 and the homologue Csp3 showed these proteins are tetramers of 4-helix bundles that bind 13 and 18 Cu(I) ions per monomer, respectively, all of which are stored inside the core of the 4-helix bundle and are coordinated mostly by Cys residues. Csp1 binds tightly at least 10 Cu(I) ions whereas Csp3 has an average Cu(I) affinity at the order of 1017 M-1. Csp1 and Csp3 do not remove Cu(I) from Cu(I)-mb, however it is likely that apo-mb, which removes Cu(I) from these proteins, notably at very different rates, transports Cu(I) to pMMO. Csp1 is thought to be exported from the cytosol potentially to the intra-cytoplasmic membranes, where pMMO is localised, to store copper for the enzyme. Csp3 is thought to be cytosolic and either sequesters copper to prevent copper-induced toxicity or, more likely, supplies copper to unknown cytosolic copper enzymes, consistent with the large number of soluble copper pools visualized in the organism. Csp1 protein homologues are present in other bacteria, including MOB, while homologues of the cytosolic Csp3 are widely distributed in members of all major bacterial phyla. The presence of Csp3 in the bacterial cytosol implies either a function as a defense mechanism against copper-induced toxicity or more likely copper storage for supply to cytosolic copper enzymes, yet to be identified. The latter possibility challenges the present model according to which bacteria do not have a cytosolic requirement for copper

A four-helix bundle stores copper for methane oxidation

Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO). Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location

Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

Abstract Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis . These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1–2) × 10 17 M −1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria

The Ig-like domain of Punctin/MADD-4 is the primary determinant for interaction with the ectodomain of neuroligin NLG-1: Molecular bases of Punctin/MADD-4 interaction with NLG-1

International audiencePunctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin NLG-1, itself a postsynaptic organizer of inhibitory synapses. To identify the molecular bases of their partnership, we generated recombinant forms of the two proteins and carried out a comprehensive biochemical and biophysical study of their interaction, complemented by an in vivo localisation study. We show that spontaneous proteolysis of MADD-4B first generates a shorter N-MADD-4B form, which comprises four thrombospondin (TSP) and one Ig-like domains and binds NLG-1. A second processing event eliminates the C-terminal Ig-like domain along with the ability of N-MADD-4B to bind NLG-1. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro. We further demonstrate in vivo that this Ig-like domain is essential, albeit not sufficient per se, for efficient recruitment of GABAA receptors at GABAergic synapses in C. elegans. The interaction of N-MADD-4B with NLG-1 is also disrupted by heparin, used as a surrogate for the extracellular matrix component, heparan sulphate, and whose high-affinity binding to the Ig-like domain may proceed from surface charge complementarity, as suggested by homology 3D modelling. These data point to N-MADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms that can regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses