YoeB–YefM, the widespread type II toxin–antitoxin (TA) module, binds to its own promoter to autoregulate its transcription: repress or induce transcription under normal or stress conditions, respectively. It remains unclear how YoeB–YefM regulates its transcription depending on the YoeB to YefM TA ratio. We find that YoeB–YefM complex from S.aureus exists as two distinct oligomeric assemblies: heterotetramer (YoeB–YefM2–YoeB) and heterohexamer (YoeB–YefM2–YefM2–YoeB) with low and high DNA-binding affinities, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain. At TA ratio of 1:2, unsaturated binding of YoeB to the C-terminal regions of YefM dimer forms an optimal heterohexamer for DNA binding, and two YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5′-TTGTACAN6AGTACAA-3′ palindromic sequence, resulting in transcriptional repression. In contrast, at TA ratio of 1:1, binding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which are unable to bind the promoter DNA optimally due to steric clashes, hence derepresses TA operon transcription.

Toxin–antitoxin (TA) systems are ubiquitous regulatory modules for bacterial growth and cell survival following stress. YefM–YoeB, the most prevalent type II TA system, is present in a variety of bacterial species. In Staphylococcus aureus, the YefM–YoeB system exists as two independent paralogous copies. Our previous research resolved crystal structures of the two oligomeric states (heterotetramer and heterohexamer–DNA ternary complex) of the first paralog as well as the molecular mechanism of transcriptional autoregulation of this module. However, structural details reflecting molecular diversity in both paralogs have been relatively unexplored. To understand the molecular mechanism of how Sa₂YoeB and Sa₂YefM regulate their own transcription and how each paralog functions independently, we solved a series of crystal structures of the Sa₂YoeB–Sa₂YefM. Our structural and biochemical data demonstrated that both paralogous copies adopt similar mechanisms of transcriptional autoregulation. In addition, structural analysis suggested that molecular diversity between the two paralogs might be reflected in the interaction profile of YefM and YoeB and the recognition pattern of promoter DNA by YefM. Interaction analysis revealed unique conformational and activating force effected by the interface between Sa₂YoeB and Sa₂YefM. In addition, the recognition pattern analysis demonstrated that residues Thr⁷ and Tyr¹⁴ of Sa₂YefM specifically recognizes the flanking sequences (G and C) of the promoter DNA. Together, these results provide the structural insights into the molecular diversity and independent function of the paralogous copies of the YoeB–YefM TA system.

Thiolases are vital enzymes which participate in both degradative and biosynthetic pathways. Biosynthetic thiolases catalyze carbon–carbon bond formation by a Claisen condensation reaction. The cytoplasmic acetoacetyl-CoA thiolase fromSaccharomyces cerevisiae, ERG10, catalyses carbon–carbon bond formation in the mevalonate pathway. The structure of aS. cerevisiaebiosynthetic thiolase has not previously been reported. Here, crystal structures of apo ERG10 and its Cys91Ala variant were solved at resolutions of 2.2 and 1.95 Å, respectively. The structure determined shows that ERG10 shares the characteristic thiolase superfamily fold, with a similar active-site architecture to those of type II thiolases and a similar binding pocket, apart from Ala159 at the entrance to the pantetheine-binding cavity, which appears to be a determinant of the poor binding ability of the substrate. Moreover, comparative binding-pocket analysis of moleculeBin the asymmetric unit of the apo structure with that of the CoA-bound complex of human mitochondrial acetoacetyl-CoA thiolase indicates the canonical binding mode of CoA. Furthermore, the steric hindrance revealed in a structural comparison of moleculeAwith the CoA-bound form raise the possibility of conformational changes that are associated with substrate binding.

Gene expression is mediated by a series of regulatory proteins, i.e., transcription factors. Under different growth conditions, the transcriptional regulation of structural genes is associated with the recognition of specific regulatory elements (REs) in promoter DNA. The manner by which transcription factors recognize distinctive REs is a key question in structural biology. Previous research has demonstrated that Ino2p/Ino4p heterodimer is associated with the transcriptional regulation of phospholipid biosynthetic genes. Mechanistically, Ino2p/Ino4p could specifically recognize the inositol/choline-responsive element (ICRE), followed by the transcription activation of the phospholipid biosynthetic gene. While the promoter DNA sequence for Ino2p has already been characterized, the structural basis for the mutual interaction between Ino2p/Ino4p and their binding interface with promoter DNA remain relatively unexplored. Here, we have determined the crystalline structure of the Ino2p^DBD/Ino4p^DBD/DNA ternary complex, which highlights some residues (Ino2p^{His12/Glu16/Arg20/Arg44} and Ino4p^{His12/Glu16/Arg19/Arg20}) associated with the sequence-specific recognition of promoter DNA. Our biochemical analysis showed that mutating these residues could completely abolish protein–DNA interaction. Despite the requirement of Ino2p and Ino4p for interprotein–DNA interaction, both proteins can still interact—even in the absence of DNA. Combined with the structural analysis, our in vitro binding analysis demonstrated that residues (Arg35, Asn65, and Gln69 of Ino2p^DBD and Leu59 of Ino4p^DBD) are critical for interprotein interactions. Together, these results have led to the conclusion that these residues are critical to establishing interprotein–DNA and protein–DNA mutual interactions.