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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.

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semi-rational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodel a network of aromatic-aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor co-evolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes.

It has been shown that phages have evolved anti-CRISPR (Acr) proteins to inhibit host CRISPR-Cas systems. Most acr genes are located upstream of anti-CRISPR-associated (aca) genes, which is instrumental for identifying these acr genes. Thus far, eight Aca families (Aca1–Aca8) have been identified, all proteins of which share low sequence homology and bind to different target DNA sequences. Recently, Aca1 and Aca2 proteins were discovered to function as repressors by binding to acr-aca promoters, thus implying a potential anti-anti-CRISPR mechanism. However, the structural basis for the repression roles of Aca proteins is still unknown. Here, we elucidated apo-structures of Aca1 and Aca2 proteins and their complex structures with their cognate operator DNA in two model systems, the Pseudomonas phage JBD30 and the Pectobacterium carotovorum template phage ZF40. In combination with biochemical and cellular assays, our study unveils dimerization and DNA-recognition mechanisms of Aca1 and Aca2 family proteins, thus revealing the molecular basis for Aca1- and Aca2-mediated anti-CRISPR repression. Our results also shed light on understanding the repression roles of other Aca family proteins and autoregulation roles of acr-aca operons.

During signal transduction, the G protein, Gαq, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gαq, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gαq. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gαq with low micromolar affinity and inhibits Gαq by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gαq, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gαq, and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gαq/i with high affinity in vitro (KD = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gαq in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions.

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1-P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix-turn-helix domain (amino acids I13-R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1-D12) did not bind. The N-terminal M1-P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1-M234) and Tnp26 M1-P56 fragment. These findings indicate that the helix-turn-helix and disordered domains of Tnp26 play a role in Tnp26-TIR complex formation. Both domains are conserved in all members of the IS26 family.

Proteins have highly conserved domains that determine their functionality. Out of the thousands of domains discovered so far across all living forms, some of the predominant clinically-relevant domains include IENR1, HNHc, HELICc, Pro-kuma_activ, Tryp_SPc, Lactamase_B, PbH1, ChtBD3, CBM49, acidPPc, G3P_acyltransf, RPOL8c, KbaA, HAMP, HisKA, Hr1, Dak2, APC2, Citrate_ly_lig, DALR, VKc, YARHG, WR1, PWI, ZnF_BED, TUDOR, MHC_II_beta, Integrin_B_tail, Excalibur, DISIN, Cadherin, ACTIN, PROF, Robl_LC7, MIT, Kelch, GAS2, B41, Cyclin_C, Connexin_CCC, OmpH, Bac_rhodopsin, AAA, Knot1, NH, Galanin, IB, Elicitin, ACTH, Cache_2, CHASE, AgrB, PRP, IGR, and Antimicrobial21. These domains are distributed in nucleases/helicases, proteases, esterases, lipases, glycosylase, GTPases, phosphatases, methyltransferases, acyltransferase, acetyltransferase, polymerase, kinase, ligase, synthetase, oxidoreductase, protease inhibitors, nucleic acid binding proteins, adhesion and immunity-related proteins, cytoskeletal component-manipulating proteins, lipid biosynthesis and metabolism proteins, membrane-associated proteins, hormone-like and signaling proteins, etc. These domains are ubiquitous stretches or folds of the proteins in pathogens and allergens. Pathogenesis alleviation efforts can benefit enormously if the characteristics of these domains are known. Hence, this review catalogs and discusses the role of such pivotal domains, suggesting hypotheses for better understanding of pathogenesis at molecular level., Highlights • Proteins have highly conserved regions or domains across pathogens and allergens. • Knowledge on these critical domains can facilitate our understanding of pathogenesis mechanisms. • Such immune manipulation-related domains include IENR1, HNHc, HELICc, ACTIN, PROF, Robl_LC7, OmpH etc. • These domains are presnt in enzyme, transcription regulators, adhesion proteins, and hormones. • This review discusses and hypothesizes on these domains.

Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation., Graphical Abstract Unlabelled Image, Highlights • Mechanism of transcription inhibition by σ54 • Coupled load and unwind model for transcription initiation • Role of bacterial enhancer-binding proteins in isomerization to the open complex • Comparison of structural and functional differences between σ70 and σ54

Phospholipase C β (PLCβ), which is activated by the Gq family of heterotrimeric G proteins, hydrolyzes the inner membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). Because Gq and PLCβ regulate many crucial cellular processes and have been identified as major disease drivers, activation and termination of PLCβ signaling by the Gαq subunit have been extensively studied. Gq-coupled receptor activation induces intense and transient PIP2 hydrolysis, which subsequently recovers to a low-intensity steady-state equilibrium. However, the molecular underpinnings of this equilibrium remain unclear. Here, we explored the influence of signaling crosstalk between Gq and Gi/o pathways on PIP2 metabolism in living cells using single-cell and optogenetic approaches to spatially and temporally constrain signaling. Our data suggest that the Gβγ complex is a component of the highly efficient lipase GαqGTP–PLCβ–Gβγ. We found that over time, Gβγ dissociates from this lipase complex, leaving the less-efficient GαqGTP–PLCβ lipase complex and allowing the significant partial recovery of PIP2 levels. Our findings also indicate that the subtype of the Gγ subunit in Gβγ fine-tunes the lipase activity of Gq–PLCβ, in which cells expressing Gγ with higher plasma membrane interaction show lower PIP2 recovery. Given that Gγ shows cell- and tissue-specific subtype expression, our findings suggest the existence of tissue-specific distinct Gq–PLCβ signaling paradigms. Furthermore, these results also outline a molecular process that likely safeguards cells from excessive Gq signaling.

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that produce a wide range of bioactive peptides, such as siderophores, toxins, and antibacterial and insecticidal agents. NRPSs are dynamic proteins characterized by extensive interdomain communications as a consequence of their assembly-line mode of synthesis. Hence, crystal structures of multidomain fragments of NRPSs have aided in elucidating crucial interdomain interactions that occur during different steps of the NRPS catalytic cycle. One crucial yet unexplored interaction is that between the reductase (R) domain and the peptide carrier protein (PCP) domain. R domains are members of the short-chain dehydrogenase/reductase family and function as termination domains that catalyze the reductive release of the final peptide product from the terminal PCP domain of the NRPS. Here, we report the crystal structure of an archaeal NRPS PCP-R didomain construct. This is the first NRPS R domain structure to be determined together with the upstream PCP domain and is also the first structure of an archaeal NRPS to be reported. The structure reveals that a novel helix-turn-helix motif, found in NRPS R domains but not in other short-chain dehydrogenase/reductase family members, plays a major role in the interface between the PCP and R domains. The information derived from the described PCP-R interface will aid in gaining further mechanistic insights into the peptide termination reaction catalyzed by the R domain and may have implications in engineering NRPSs to synthesize novel peptide products.

Cellular organisms in different domains of life employ structurally unrelated, non-homologous DNA primases for synthesis of a primer for DNA replication. Archaea and eukaryotes encode enzymes of the archaeo-eukaryotic primase (AEP) superfamily, whereas bacteria uniformly use primases of the DnaG family. However, AEP genes are widespread in bacterial genomes raising questions regarding their provenance and function. Here, using an archaeal primase–polymerase PolpTN2 encoded by pTN2 plasmid as a seed for sequence similarity searches, we recovered over 800 AEP homologs from bacteria belonging to 12 highly diverse phyla. These sequences formed a supergroup, PrimPol-PV1, and could be classified into five novel AEP families which are characterized by a conserved motif containing an arginine residue likely to be involved in nucleotide binding. Functional assays confirm the essentiality of this motif for catalytic activity of the PolpTN2 primase–polymerase. Further analyses showed that bacterial AEPs display a range of domain organizations and uncovered several candidates for novel families of helicases. Furthermore, sequence and structure comparisons suggest that PriCT-1 and PriCT-2 domains frequently fused to the AEP domains are related to each other as well as to the non-catalytic, large subunit of archaeal and eukaryotic primases, and to the recently discovered PriX subunit of archaeal primases. Finally, genomic neighborhood analysis indicates that the identified AEPs encoded in bacterial genomes are nearly exclusively associated with highly diverse integrated mobile genetic elements, including integrative conjugative plasmids and prophages., Graphical Abstract Image 1, Highlights • Primases of the archaeo-eukaryotic primase (AEP) superfamily are widespread in bacteria. • We describe five new AEP families in bacteria belonging to 12 diverse phyla. • The new AEP families display a conserved signature motif likely involved in nucleotide binding. • The primase domains are fused to diverse functional domains, revealing new families of putative helicases. • The novel primases are encoded within highly diverse integrated mobile genetic elements.

Eukaryotic chromosomes are capped by telomeres, nucleoprotein complexes that prevent chromosome end-to-end fusions and control cell ageing. Two proteins in this complex, telomere repeat binding factors (TRF1 and TRF2), specifically recognise the double-stranded TTAGGG tandem repeat sequence. TRF1 is a homodimer with roles governing DNA architecture and negatively regulating telomere length. We explore the conformational space of this protein–DNA complex using molecular dynamics and, for the first time, generate a complete model of TRF1–DNA recognition that has not been possible on the basis of crystallographic and NMR data alone. The results reconcile previous conflicting experimental models for the sequence selectivity of the recognition process, by confirming many of the findings while identifying important new interactions and behaviour. This improved characterisation also reveals extensive indirect readout, which suggests that recognition will be affected by changes to DNA helical parameters such as bending., Graphical abstract, Highlights • X-ray and NMR data on TRF1–DNA fail to fully explain sequence-dependent recognition. • Molecular dynamics simulations reveal a much richer array of dynamic interactions. • The effects of changes in the DNA sequence on binding affinity are explained.

ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.

Bacterial cells continuously sense and respond to their environment using their inherent signalling and gene regulatory networks. Cells are equipped with parallel signalling pathways, which can specifically cope with individual input signals, while interconnectivities between pathways lead to an enhanced complexity of regulatory responses that enable sophisticated adaptation. In principle, any cell signalling pathway may be rewired to respond to non-cognate signals by exchanging and recombining their underlying cognate signalling components. In the present article, we review the engineering strategies and use of chimaeric regulatory proteins in cell signalling pathways, especially the TCS (two-component signalling) system in bacteria, to achieve novel customized signalling or regulatory functions. We envisage that engineered chimaeric regulatory proteins can play an important role to aid both forward and reverse engineering of biological systems for many desired applications.