Biology subjects

Schirmer, T.

Publications and source records attributed to Schirmer, T..

9 recordsLinked to original sources

bioRxiv2024

Full-length structure of the host targeted bacterial effector Bep1 reveals a novel structural domain conserved in FIC effector proteins from Bartonella

Bacterial effector proteins translocated via a type-IV secretion system (T4SS) typically harbor a C-terminal segment required for recognition by the type-IV secretion coupling protein 1. In the -proteobacterial pathogen Bartonella, the signal is bipartite being composed of a BID (Bep intracellular delivery) domain and a positively charged C-terminal tail 2. Here, we show the crystal structure of full length Bartonella effector protein 1 (Bep1), which shows a novel FIC - OB - BAS(BID) domain arrangement conserved in the majority of Beps with the BID domain inserted into the newly discovered BAS parent domain. We propose that the BAS domain is necessary for the overall "boomerang"-like shape of Bep1 and that it plays a role during translocation through the T4SS.

microbiology↗

bioRxiv2022

High-resolution crystal structure of a metabolic switch protein in a complex with monomeric c-di-GMP reveals a potential mechanism for c-di-GMP dimerization

Bacterial second messengers c-di-GMP and (p)ppGpp have broad functional repertoires ranging from growth and cell cycle control to the regulation of biofilm formation and virulence. The recent identification of SmbA, an effector protein from Caulobacter crescentus that is jointly targeted by both signaling molecules, has opened up studies on how these global bacterial networks interact. C-di-GMP and (p)ppGpp compete for the same SmbA binding site, with a dimer of the former ligand inducing a conformational change of loop 7 leading to downstream signaling. Here, we report a crystal structure of a partial loop 7 deletion mutant, SmbA{Delta}loop in complex with c-di-GMP determined at 1.4 [A] resolution. SmbA{Delta}loop binds monomeric c-di-GMP strengthening the view that loop 7 is required for c-di-GMP dimerization. In the crystal, SmbA{Delta}loop forms a 2-fold symmetric dimer via isologous interactions with the two symmetric halves of c-di-GMP. Structural comparisons of SmbA{Delta}loop with wild-type SmbA in complex with dimeric c-di-GMP or ppGpp support the idea that loop 7 is critical for SmbA function by interacting with downstream partners. These results underscore the flexibility of c-di-GMP in binding to the symmetric interface between protein subunits. It is envisaged that such isologous interactions of c-di-GMP will be observed in hitherto unrecognized targets.

biochemistry↗

bioRxiv2022

Intermediaries in phosphotransfer: structural features discriminating hybrid histidine kinase Rec domains from response regulator homologs

In two-component systems, the information gathered by the histidine kinase (HK) is relayed to the cognate response regulator (RR). Thereby, the phosphoryl group of the autophosphorylated HK is transferred to the receiver domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec domain (Recinter) that is part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about Recinter domains and their potentially discriminating features. As a bona-fide Recinter domain, here we have studied the C-terminal Rec domain of the hybrid HK CckA (CckARec) from Caulobacter crescentus by X-ray crystallography and NMR spectroscopy. CckARec exhibits the canonical Rec-fold, though with a degenerated 4 helix, in which all active site residues are pre-arranged for phosphoryl-binding. BeF3- binding does not alter secondary structure nor the oligomeric state, indicating the absence of allosteric changes, the hall mark of RRs. Based on structural modeling and sequence co-variation analysis, we present a detailed picture for the intramolecular association of the CckA DHp/Rec domains and discuss the role of a FATGUY motif, a distinguishing feature of CckARec orthologs.

biochemistry↗

bioRxiv2021

Digital control of c-di-GMP in E. coli balances population-wide developmental transitions and phage sensitivity

Nucleotide-based signaling molecules (NSMs) are widespread in bacteria and eukaryotes, where they control important physiological and behavioral processes. In bacteria, NSM-based regulatory networks are highly complex, entailing large numbers of enzymes involved in the synthesis and degradation of active signaling molecules. How the converging input from multiple enzymes is transformed into robust and unambiguous cellular responses has remained unclear. Here we show that Escherichia coli converts dynamic changes of c-di-GMP into discrete binary signaling states, thereby generating heterogeneous populations with either high or low c-di-GMP. This is mediated by an ultrasensitive switch protein, PdeL, which senses the prevailing cellular concentration of the signaling molecule and couples this information to c-di-GMP degradation and transcription feedback boosting its own expression. We demonstrate that PdeL acts as a digital filter that facilitates precise developmental transitions, confers cellular memory, and generates functional heterogeneity in bacterial populations to evade phage predation. Based on our findings, we propose that bacteria apply ultrasensitive regulatory switches to convert dynamic changes of NSMs into binary signaling modes to allow robust decision-making and bet-hedging for improved overall population fitness.

microbiology↗

bioRxiv2020

A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation.

The reactive oxygen species produced during inflammation through the neutrophilic respiratory burst play profound roles in combating bacterial pathogens and regulating the microbiota. Among these, the neutrophilic oxidant bleach, hypochlorous acid (HOCl), is the most prevalent and strongest oxidizer and kills bacteria through non-specific oxidation of proteins, lipids, and DNA. Thus, HOCl can be viewed as a host-specific cue that conveys important information about what bacterial physiology and lifestyle programs may be required for successful colonization. Nevertheless, bacteria that colonize animals face a molecular challenge in how to achieve highly selective detection of HOCl due to its reactive and transient nature and chemical similarity to more benign and non-host-specific oxidants like hydrogen peroxide (H2O2). Here, we report that in response to increasing HOCl levels E. coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We show the molecular mechanism of this activation to be specific oxidation of a conserved cysteine that coordinates the zinc of its regulatory chemoreceptor zinc-binding (CZB) domain, forming a zinc-cysteine redox switch 685-fold more sensitive to oxidation by HOCl over H2O2. Dissection of the signal transduction mechanism through quantum mechanics, molecular dynamics, and biochemical analyses reveal how the cysteine redox state alters the delicate equilibrium of competition for Zn++ between the CZB domain and other zinc binders to relay the presence of HOCl through activating the associated GGDEF domain to catalyze c-di-GMP. We find biofilm formation and HOCl-sensing in vivo to be regulated by the conserved cysteine, and point mutants that mimic oxidized CZB states increase production of the biofilm matrix polymer poly-N-acetylglucosamine and total biofilm. We observe CZB-regulated diguanylate cyclases and chemoreceptors in phyla in which host-associated bacteria are prevalent and are possessed by pathogens that manipulate host inflammation as part of their colonization strategy. A phylogenetic survey of all known CZB sequences shows these domains to be conserved and widespread across diverse phyla, suggesting CZB origin predates the bacterial last universal common ancestor. The ability of bacteria to use CZB protein domains to perceive and thwart the host neutrophilic respiratory burst has implications for understanding the mechanisms of diseases of chronic inflammation and gut dysbiosis.

microbiology↗

bioRxiv2020

Activation mechanism of a small prototypic Rec-GGDEF diguanylate cyclase

Diguanylate cyclases (DGCs) synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of DGCs. Full-length crystal structures revealed that BeF3- pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many DGCs with N-terminal coiled-coil linkers.

biochemistry↗

bioRxiv2020

Structural basis for selective targeting of Rac subfamily GTPases by a bacterial effector protein

SummaryRas-homology (Rho) family GTPases are conserved molecular switches controlling fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, molecular mechanisms of target selectivity have remained elusive. Here, we report a bacterial effector protein that targets all four Rac subfamily members of Rho family GTPases, but none of the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 facilitate modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural paradigm for target selectivity towards Rac subfamily GTPases and provides a highly selective tool for their functional analysis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

Precise transcription timing by a second-messenger drives a bacterial G1/S cell cycle transition

Bacteria adapt their growth rate to their metabolic status and environmental conditions by modulating the length of their quiescent G1 period. But the molecular mechanisms controlling G1 length and exit from G1 are poorly understood. Here we identify a key role for the second messenger c-di-GMP, and demonstrate that a gradual increase in c-di-GMP concentration determines precise gene expression during G1/S in Caulobacter crescentus. We show that c-di-GMP strongly stimulates the kinase ShkA, activates the TacA transcription factor, and initiates a G1/S-specific transcription program leading to cell morphogenesis and S-phase entry. C-di-GMP activates ShkA by binding to its central pseudo-receiver domain uncovering this wide-spread domain as a novel signal input module of bacterial kinases. Activation of the ShkA-dependent genetic program also causes c-di-GMP to reach peak levels, which triggers S-phase entry and, in parallel, promotes proteolysis of ShkA and TacA. Thus, a gradual increase of c-di-GMP results in a precisely tuned ShkA-TacA activity window enabling G1/S specific gene expression before cells commit to replication initiation. By defining a regulatory mechanism for G1/S control, this study contributes to understanding bacterial growth control at the molecular level.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC=\"FIGDIR/small/675330v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (46K):\norg.highwire.dtl.DTLVardef@1cb0c69org.highwire.dtl.DTLVardef@19bc477org.highwire.dtl.DTLVardef@17f8c89org.highwire.dtl.DTLVardef@e3aa6e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Hybrid histidine kinase activation by cyclic di-GMP- mediated domain liberation

Cytosolic hybrid histidine kinases (HHKs) constitute major signalling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In Caulobacter crescentus, the HHK ShkA is essential for accurate timing of the G1-S cell cycle transition and is regulated by the corresponding increase in the level of the second messenger c-di-GMP. Here, we use a combination of X-ray crystallography, NMR spectroscopy, functional analyses and kinetic modelling to reveal the regulatory mechanism of ShkA. In the absence of c-di-GMP, ShkA predominantly adopts a compact domain arrangement that is catalytically inactive. C-di-GMP binds to the dedicated pseudo-receiver domain Rec1 thereby liberating the canonical Rec2 domain from its central position where it obstructs the large-scale motions required for catalysis. Thus, c-di-GMP cannot only stabilize domain interactions, but also engage in domain dissociation to allosterically control activity. Enzyme kinetics data are consistent with conformational selection of the ensemble of active domain constellations by the ligand.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC=\"FIGDIR/small/675454v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (11K):\norg.highwire.dtl.DTLVardef@8304borg.highwire.dtl.DTLVardef@19a9d61org.highwire.dtl.DTLVardef@1d27000org.highwire.dtl.DTLVardef@b74d75_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗