Structure of the mannose phosphotransferase system (man-PTS) complexed with microcin E492, a pore-forming bacteriocin

Abstract

Bacteriocins comprise various protein classes produced and secreted by bacteria that have a toxic effect on strains closely related to the producer strain 1 . Their functions are diverse, including pore-formation, DNases, RNases, and inhibition of protein synthesis or DNA replication. Bacteriocins produced by Gram-negative bacteria are divided based on their molecular masses into colicins (high molecular mass, 30–80 kDa) and microcins (Mccs; low molecular mass, < 10 kDa) 2 . Mccs are hydrophobic peptides that are highly resistant to heat, extreme pH, and proteases with potent antibacterial activity 3 . These properties are considered essential for the role that Mcc may play in intestinal microbiota, specifically in bacterial displacements. Mccs can be classified into two classes. Class I encompasses highly posttranslationally modified very-low-molecular-mass peptides (< 5 kDa) which must cross the inner membrane and target intracellular enzymes responsible for the DNA/RNA structure or synthesis. Class II, including high-molecular-mass peptides (ranging from 5 to 10 kDa), is subdivided into two subclasses: (1) class IIa Mccs need no further posttranslational modification (MccL, MccV, and Mcc24); and (2) class IIb Mccs are linear peptides that may carry a C-terminal posttranslational modification (MccE492, MccM, MccH47, and MccI47). MccE492 (UniProt: Q9Z4N4) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492 4 . MccE492 undergoes posttranslational modification with the covalent linkage of a catechol-type siderophore at the C-terminal end with the glycine–serine motif 5 (Supplementary Fig. S1 ). The N-terminal leader peptide of MccE492 is cleaved 6 ; the secreted, modified protein is called active MccE492, and its polypeptide backbone is referred to as MceA 7 . The C terminus of active MccE492 (siderophore) is recognized by the outer membrane catecholate siderophore receptor FepA, Fiu, and Cir of several species of Enterobacteriaceae 4 and translocated across the outer membrane by using the “Trojan horse” strategy. Once in the periplasm, MccE492 inserts itself into the cytoplasmic membrane and stably associates with ManYZ, the inner membrane component of the mannose permease (man-PTS) 8 . Subsequently, it kills target cells through depolarization and permeabilization of their inner membrane, thereby disrupting the proton motive force 9 . MccE492-producing bacteria synthesize a 95-amino acid immunity protein (MceB), which renders the cell resistant to MccE492 10 . The predicted structure of MceB shows three hydrophobic domains and suggests an integral membrane protein, implying that MceB sequesters or blocks the entry of active MccE492. To elucidate the specific interaction between MccE492 (MceA + siderophore) and ManYZ, we sought to resolve the structure of the MceA–ManYZ complex, which is necessary for designing improved toxic molecules for attacking specific targets.

Topics & Concepts