Homology- and coevolution-consistent structural models of bacterial copper-tolerance protein CopM support a ‘metal sponge’ function and suggest regions for metal-dependent protein-protein interactions

Abstract

Abstract.Copper is essential for life but toxic, therefore all organisms control tightly its intracellular abundance. Bacteria have indeed whole operons devoted to copper resistance, with genes that code for efflux pumps, oxidases, etc. Recently, the CopM protein of the CopMRS operon was described as a novel important element for copper tolerance in Synechocystis. This protein consists of a domain of unknown function, and was proposed to act as a periplasmic/extracellular copper binder. This work describes a bioinformatic study of CopM including structural models based on homology modeling and on residue coevolution, to help expand on its recent biochemical characterization. The protein is predicted to be periplasmic but membrane-anchored, not secreted. Two disordered regions are predicted, both possibly involved in protein-protein interactions. The 3D models disclose a 4-helix bundle with several potential copper-binding sites, most of them largely buried inside the bundle lumen. Some of the predicted copper-binding sites involve residues from the disordered regions, suggesting they could gain structure upon copper binding and thus possibly modulate the interactions they mediate. All models are provided as PDB files in the Supporting Information and can be visualized online at http://lucianoabriata.altervista.org/modelshome.html Note (January 2017): Recent X-ray structures of apo, copper-and silver-bound CopM are < 3Å RMSD away from the models, and reveal metal-dependent structural flexibility (Zhao et al Acta Crystallogr D Struct Biol. 2016)

Assessment of hard target modeling in CASP12 reveals an emerging role of alignment‐based contact prediction methods

“…More globally, alignment‐based contact prediction methods are becoming important sources of information in structural biology, used not only to model proteins but also to derive restrains for modeling complexes, to seek out possible alternative conformations, and to understand amino acid variability in proteins . In fact there are already several clear applications to specific proteins in studies where modeling at least overall topologies and interactions through contact prediction is enough to answer certain questions and drive research forward …”

Applications of sequence coevolution in membrane protein biochemistry

“…While evolutionary couplings provide some evidence for a dimer, the B. bronchiseptica ZIP does not form a parallel dimer in the crystals, leaving this question unresolved. In another example, homology modeling using I-TASSER of the membrane-tethered periplasmic copper-binding protein CopM was refined using coevolving pair constraints from EVFold [123]. Finally, the conformation of specific secondary structure elements of the cone tetrameric cyclic nucleotide-gated (CNG) ion channels that was built from homology models was confirmed through inspection of coevolving residue pairs [124].…”

Cloning of a copper resistance gene cluster from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery

“…The cloned gene cluster was subsequently used to complement a copper-sensitive E. coli mutant, although only the pcopM gene was necessary for the increase in copper tolerance observed. Thus, the copper-binding protein CopM is active in a heterologous bacterial species through a mechanism that is likely to involve the sequestration of copper ions [14,28].…”