The proteobacterial antimicrobial compound efflux (PACE) family of transport proteins was only recently described. PACE family transport proteins can confer resistance to a range of biocides used as disinfectants and antiseptics, and are encoded by many important Gram-negative human pathogens. However, we are only just beginning to appreciate the range of functions and the mechanism(s) of transport operating in these proteins. Genes encoding PACE family proteins are typically conserved in the core genomes of bacterial species rather than on recently acquired mobile genetic elements, suggesting that they confer important core functions in addition to biocide resistance. Three-dimensional structural information is not yet available for PACE family proteins. However, PACE proteins have several very highly conserved amino acid sequence motifs that are likely to be important for substrate transport. PACE proteins also display strong amino acid sequence conservation between their N— and C-terminal halves, suggesting that they evolved by duplication of an ancestral protein comprised of two transmembrane helices. In light of their drug resistance functions in Gram-negative pathogens, PACE proteins should be the subject of detailed future investigation.
The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane.
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