The Repressor, Open reading frame, Kinase (ROK) class comprises a large family of bacterial proteins that exhibit sugar kinase and/or transcriptional repressor activities. The best studied ROK repressor is probably the Escherichia coli protein MLC, which serves as a central coordinator of carbon metabolism in a number of gram-negative bacteria. In contrast to the strict kinases, ROK repressors contain an N-terminal DNA-binding domain with a helix-turn-helix motif.
Analogous to the well-known lactose operon repressor (LacR), genes that encode for ROK repressors are frequently found on an operon alongside genes that function in the uptake and metabolic breakdown of a specific carbohydrate substrate. Such repressors auto-regulate the operon they are contained on, activating transcription when the substrate for proteins encoded on the operon is present.
As part of a study conducted to determine the metabolism of starch derivatives in the foodborne pathogen Listeria monocytogenes, the NIAID-supported Center for Structural Genomics of Infectious Diseases (CSGID) has determined multiple crystal structures of a disaccharide regulated ROK repressor. These structures reveal provide insight into the molecular underpinnings of ROK repressor function.
A structure that contains a pair of ROK homodimers bound to the operator DNA reveals the basis of repression. These dimers bind overlapping and specific sequences and recognize the DNA in an interesting way. In addition to engaging the major groove in a typical helix-turn-helix fashion, the first helix in the DNA-binding domain inserts into the central minor groove and, on the flanks, large hairpin “wings” insert into the peripheral minor grooves.
A second crystal structure of the ROK repressor in complex with isomaltose (its disaccharide activator) reveals the mechanism of transcriptional activation. This structure illustrates that the sugar binds between the effector subdomains, occupying the site that the substrate assumes in homologous ROK kinases. The binding of sugar induces conformational changes, which necessitates the moving apart of the DNA-binding domains. These structural changes cause the DNA domains to become disordered, making them unresolvable in the experimental electron density maps, and takes them out of position to simultaneously engage the DNA. Interestingly, this structure and conformational changes are conserved in ROK kinases, which implies that the evolution of ROK repression elegantly arose by the simple fusion of DNA binding domains and the coopting of pre-existing induced fit conformational changes.
Listeria monocytogenes ROK repressor structures. (A) Structure of ROK repressor bound to operator DNA depicted from two vantage points. (B) Structure of ROK repressor bound to the disaccharide inducer isomaltose. The disordered helix-turn-helix domains are depicted as spheres. Reprinted by permission from Macmillan Publishers Ltd: Nat Microbiol, 2016 Nov 7;2:16202. doi: 10.1038/nmicrobiol.2016.202.
Coordinates are available in the Protein Data Bank, www.rsb.org, PDB IDs 5S7P, 5S7R, 5S7Q.
Light SH, Cahoon LA, Halavaty AS, Freitag NE, Anderson WF. Structure to function of an α-glucan metabolic pathway that promotes Listeria monocytogenes pathogenesis. Nat Microbiol. 2016 Nov 7;2:16202. doi: 10.1038/nmicrobiol.2016.202.