Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

Abstract

The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding ␤-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to ␤-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when ␤-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0-and 2.7-Å resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix ␣3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.␤-Lactam antibiotics are becoming less effective therapy for treating staphylococcal infections as resistance to them increases. Resistance is mediated by a ␤-lactamase (encoded by blaZ) that hydrolyzes penicillins (4) and an alternate penicillin binding protein target (PBP2a, encoded by mecA) to which ␤-lactam antibiotics bind poorly (17). The transcription of blaZ and mecA is corepressed by related regulators (encoded by blaI and mecI). Both BlaI and MecI bind to palindromic promoteroperator sequences, presumably as dimers, and can interchangeably repress the transcription of either target gene (8,26,36). Signal transduction through either of two transmembrane inducers, BlaR1 or MecR1, leads to proteolytic autocleavage of the cytoplasmic domains of these molecules. Autocleavage of the signal transducer is followed by cleavage of the cognate repressor (BlaI or MecI, respectively) and induction of the transcription of blaZ or mecA (42). The exact mechanism of cleavage is unclear at present. The current hypothesis (1, 42) is that ␤-lactam binding to the extramembrane sensor of BlaR1 or MecR1 triggers autocleavage of its cytoplasmic domain at a single site, leaving the putative cytoplasmic protease te...

Coordinated Regulation by AgrA, SarA, and SarR To Control agr Expression in Staphylococcus aureus

“…Nestled between the two sets of direct repeats are the palindrome and the overlapping inverted repeats to which winged helix structures such as SarA and SarR bind, respectively ( Fig. 1) (20,21,31). To dissect the contribution of these binding sites to agr P2 and P3 transcription, we have mutated the AgrA boxes and truncated and mutated the SarA/SarR binding site of an agr promoter fragment on a plasmid carrying the GFP uvr fusion in strain SH1000 (Fig.…”

β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

“…The two systems are remarkably similar in structure and function [18][19][20][21], yet still retain distinct identities [22,23]. Of the two systems, the S. aureus bla regulatory system is the better characterized, as our understanding is complemented by that of a homologous system for b-lactamase expression found in the non-pathogenic Gram-positive bacterium Bacillus lichenformis [24][25][26][27][28]. The mec system is believed to parallel the better-characterized bla gene regulatory system, and therefore the bla system forms the working model for regulation of both components of resistant Staphylococci.…”

“…The metalloprotease cleaves, at a specifi c sequence, the two 14-kDa repressors into the DNA binding domain and dimerization domains. X-ray structure confi rms a bound homodimeric repressor (fi g. 2) [28,69]. The N-terminal domain of each binds to the DNA with winged helixturn-helix topology, independently binding to the DNA from its counterpart on the other monomer.…”

Solution structure of a multifunctional DNA- and protein-binding motif of human Werner syndrome protein

“…Second, the helix 5 likely mediates protein-protein interactions. Homodimerization of DNA-binding domains through one or two ␣-helices has been observed in a number of WH domains, including MarR, RTP, and BlaI (36)(37)(38). These ␣-helices are usually located on the other side from the DNA-binding surface of the protein and are highly solvent-exposed even in the protein-protein complexes.…”