MacB is an ABC transporter that collaborates with the MacA adaptor protein and TolC exit duct to drive efflux of antibiotics and enterotoxin STII out of the bacterial cell. Here we present the structure of ATP-bound MacB and reveal precise molecular details of its mechanism. The MacB transmembrane domain lacks a central cavity through which substrates could be passed, but instead conveys conformational changes from one side of the membrane to the other, a process we term mechanotransmission. Comparison of ATP-bound and nucleotide-free states reveals how reversible dimerization of the nucleotide binding domains drives opening and closing of the MacB periplasmic domains via concerted movements of the second transmembrane segment and major coupling helix. We propose that the assembled tripartite pump acts as a molecular bellows to propel substrates through the TolC exit duct, driven by MacB mechanotransmission. Homologs of MacB that do not form tripartite pumps, but share structural features underpinning mechanotransmission, include the LolCDE lipoprotein trafficking complex and FtsEX cell division signaling protein. The MacB architecture serves as the blueprint for understanding the structure and mechanism of an entire ABC transporter superfamily and the many diverse functions it supports.

Ferritin proteins function to detoxify, solubilize and store cellular iron by directing the synthesis of a ferric oxyhydroxide mineral solubilized within the protein's central cavity. Here, through the application of X-ray crystallographic and kinetic methods, we report significant new insight into the mechanism of mineralization in a bacterioferritin (BFR). The structures of nonheme iron-free and di-Fe(2+) forms of BFR showed that the intrasubunit catalytic center, known as the ferroxidase center, is preformed, ready to accept Fe(2+) ions with little or no reorganization. Oxidation of the di-Fe(2+) center resulted in a di-Fe(3+) center, with bridging electron density consistent with a mu-oxo or hydro bridged species. The mu-oxo bridged di-Fe(3+) center appears to be stable, and there is no evidence that Fe(3+)species are transferred into the core from the ferroxidase center. Most significantly, the data also revealed a novel Fe(2+) binding site on the inner surface of the protein, lying approximately 10 A directly below the ferroxidase center, coordinated by only two residues, His46 and Asp50. Kinetic studies of variants containing substitutions of these residues showed that the site is functionally important. In combination, the data support a model in which the ferroxidase center functions as a true catalytic cofactor, rather than as a pore for the transfer of iron into the central cavity, as found for eukaryotic ferritins. The inner surface iron site appears to be important for the transfer of electrons, derived from Fe(2+) oxidation in the cavity, to the ferroxidase center. Bacterioferritin may represent an evolutionary link between ferritins and class II di-iron proteins not involved in iron metabolism.

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/ thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.Cell wall-anchored proteins play critical roles in the virulence of most Gram-positive bacterial pathogens by acting as adhesins or invasins and/or interfering with various arms of the host innate or specific immune defenses. The vast majority of these virulence proteins are retained at the bacterial surface after secretion by a mechanism that involves the covalent linkage of target proteins to the peptidoglycan layer of the cell wall. This linkage is catalyzed by membrane-associated transpeptidases called sortases (1, 2). Proteins destined for cell-surface attachment contain a sorting signal recognized by these enzymes. As this mechanism is unique to Gram-positive pathogens, inhibiting the reaction is an attractive target for the development of novel antibacterials (3, 4). The sortase-mediated transpeptidation reaction is also being increasingly used in a variety of biotechnology applications (5-8).The sorting signal that targets proteins for cell surface attachment is located at the C terminus of substrates and comprises a pentapeptide motif, typically LPXTG (where X is any amino acid), followed by a hydrophobic region and a tail of positively charged residues that locates the substrate to the cell surfacefollowingsecretion(2,9).Inonecurrentmodelofsortasedependent transpeptidation, the LPXTG motif is specifically recognized by the enzyme (10), and the thiolate group of an essential active sit...

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking.