The Crystal Structure of Mlc, a Global Regulator of Sugar Metabolism in Escherichia coli

Abstract

Mlc from Escherichia coli is a transcriptional repressor controlling the expression of a number of genes encoding enzymes of the phosphotransferase system (PTS), including ptsG and manXYZ, the specific enzyme II for glucose and mannose PTS transporters. In addition, Mlc controls the transcription of malT, the gene of the global activator of the mal regulon. The inactivation of Mlc as a repressor is mediated by binding to an actively transporting PtsG (EIICB Glc ). Here we report the crystal structure of Mlc at 2.7 Å resolution representing the first described structure of an ROK (repressors, open reading frames, and kinases) family protein.Mlc forms stable dimers thus explaining its binding affinity to palindromic operator sites. The N-terminal helix-turn-helix domain of Mlc is stabilized by the amphipathic C-terminal helix implicated earlier in EIICBGlc binding. Furthermore, the structure revealed a metal-binding site within the cysteine-rich ROK consensus motif that coordinates a structurally important zinc ion. A strongly reduced repressor activity was observed when two of the zinc-coordinating cysteine residues were exchanged against serine or alanine, demonstrating the role of zinc in Mlc-mediated repressor function. The structures of a putative fructokinase from Bacillus subtilis, the glucokinase from Escherichia coli, and a glucomannokinase from Arthrobacter sp. showed high structural homology to the ROK family part of Mlc.Mlc (makes large colonies) has been discovered as a regulator protein curbing the utilization of glucose in Escherichia coli (1, 2). Mlc, acting as a transcriptional repressor, controls the expression of malT, encoding the central transcriptional activator of the maltose system (3). In addition, Mlc controls the expression of two operons encoding PTS 1 -dependent transporters for glucose ptsG (4, 5) and mannose manXYZ (6) as well as the genes encoding the general components of the PTS (7-9). In contrast to the classical mode of repressor inactivation by a cognate inducer, Mlc is inactivated by the sequestrating interaction with the actively transporting glucose transporter, the EIICB Glc protein of the PTS (10 -12). The interaction occurs at the EIIB Glc domain of the transporter encompassing a critical cysteine residue . This cysteine residue is phosphorylated in the resting transporter and becomes readily dephosphorylated during glucose transport by the transfer of the phosphoryl group onto the incoming glucose. Mlc binds only to the dephosphorylated form of EIIB Glc (13). The membranebound state of EIIB Glc is essential for Mlc inactivation. Soluble EIIB Glc , even though able to interact with Mlc (12, 13), does not prevent Mlc from binding to its operator regions and from its repressing activity. However, EIIB Glc attached to the membrane by any lipophilic anchor, even unrelated to EIICB Glc , binds Mlc in a fashion that prevents binding to the operator regions (13). This indicates that Mlc, when it is in close contact with the membrane, alters its conformation to suppress operator ...

Analyses of Mlc–IIB Glc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration

“…The dimerization appears to be related to the structural instability of the mutant protein. It has been shown that mutations of residues that coordinate a zinc ion in O-domain disturb the DNA-binding activity of Mlc (19). The D-domain of Mlc, which is responsible for DNA binding, is far away from O-domain (Fig.…”

“…A central sheet consisting of five strands is flanked by three ␣-helices and two short ␤-strands. Residues 195-380 form O-domain with a four-stranded sheet sandwiched asymmetrically by eight ␣-helices on one face and by one ␣-helix on the other face and contains a zinc ion known to play a structural role (19).…”

“…The physiological importance of the glucose-specific enzyme II and the discovery of this regulatory circuit led to structural studies about dephosphorylated EIIB (17,18) and an Mlc mutant (19). However, the underlying mechanisms of how the phosphorylation of EIICB Glc affects the interaction with Mlc and why Mlc loses its DNA-binding activity when localized to the membrane still remain unexplained.…”

Structure-Function Studies of the Staphylococcal Methicillin Resistance Antirepressor MecR2

“…It further has an adjacent regulatory zinc-binding site, which is required for repressor activity (60) and is provided by the segment topologically equivalent to the protruding ␤-ribbon ␤13␤14 in MecR2. In contrast to the latter, however, this segment is folded back toward the body of the molecule in Mlc, in a fashion similar to that in ROK glucokinase from E. coli (70) and glucomannokinase from Arthrobacter sp.…”

“…However, there is no structural data on XylR available. E. coli protein Mlc is the only functionally and structurally characterized ROK family protein with DNA repressor function (60,67). Mlc is a dimeric/tetrameric transcriptional repressor that controls the utilization of glucose in E. coli (68).…”