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

Ras-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

Recommendations for physical activity and exercise in persons living with Systemic Lupus Erythematosus (SLE): consensus by an international task force

Objective: This international task force aimed to provide healthcare professionals and persons living with systemic lupus erythematosus (SLE) with consensus-based recommendations for physical activity and exercise in SLE. Methods: Based on evidence from a systematic literature review and expert opinion, 3 overarching principles and 15 recommendations were agreed on by Delphi consensus. Results: The overarching principles highlight the importance of shared decision-making and the need to explain the benefits of physical activity to persons living with SLE and other healthcare providers. The 15 specific recommendations state that physical activity is generally recommended for all people with SLE, but in some instances, a medical evaluation may be needed to rule out contraindications. Pertaining to outdoor activity, photoprotection is necessary. Both aerobic and resistance training programmes are recommended, with a gradual increase in frequency and intensity, which should be adapted for each individual, and ideally supervised by qualified professionals. Conclusion: In summary, the consensus reached by the international task force provides a valuable framework for the integration of physical activity and exercise into the management of SLE, offering a tailored evidence-based and eminence-based approach to enhance the well-being of individuals living with this challenging autoimmune condition

Fic-mediated adenylylation : catalysis and regulation

Adenylylation, also referred to as adenylation or AMPylation, is the process by which adenosine-5‘-monophosphate (AMP) is covalently attached to a protein, a nucleic acid, or a small molecule. It is a widespread post-translational modification employed by a large variety of enzymes to regulate multiple cellular functions. This modification, originally discovered in the 1960s, recently re-emerged as a modification used by bacterial effector proteins to regulate key host signaling events upon infection. The Type Three Secretion System effectors VopS and IbpA from Vibrio parahaemolyticus and Histophilus somni, respectively, harbor a FIC domain, which catalyzes adenylylation of Rho family GTPases. The covalent attachment of the bulky AMP moiety abrogates binding of downstream effectors resulting in actin cytoskeleton collapse and concomitant host cell death. The FIC domain is not only present in a pathogenic setting but is also found in proteins that play a role in intrinsic signaling processes where its deleterious adenylylation activity needs to be tightly regulated. In this study, I performed structural and biochemical analyses to unravel the structural determinants and mechanisms governing catalysis and regulation of Fic proteins as widespread signaling proteins. First, by solving various structures of FIC domain-containing proteins I could show that FIC utilizes conserved active site features to favorably orientate the ATP substrate relative to the target protein, allowing AMP transfer to occur. We suggest that the catalytic mechanism can be generalized and extrapolated to all adenylylation-competent Fic proteins. Second, I deciphered the structural mechanism controlling Fic-catalyzed adenylylation. An alpha helix characterized by a conserved [S/T]xxxE[G/N] motif tightly associates with the Fic active site and perturbs adenylylation-competent ATP binding. This alpha helix can be part of the Fic protein itself as an N- or a C-terminal extension, or can be provided by a separate antitoxin. Based on structural homology modeling, this regulation mechanism was found to be conserved from bacteria to higher eukaryotes. The structural insights on the protein-ATP substrate binding specificity accumulated in this study will prove useful for coming efforts on rational drug design. Furtheremore, knowledge of the universal catalytic and inhibitory mechanism of Fic mediated AMP transfer will now pave the way for further studies towards the physiological roles of Fic proteins and particularly the identification of their protein targets

Adenylylation of Gyrase and Topo IV by FicT Toxins Disrupts Bacterial DNA Topology

Toxin-antitoxin (TA) modules are ubiquitous molecular switches controlling bacterial growth via the release of toxins that inhibit cell proliferation. Most of these toxins interfere with protein translation, but a growing variety of other mechanisms hints at a diversity that is not yet fully appreciated. Here, we characterize a group of FIC domain proteins as toxins of the conserved and abundant FicTA family of TA modules, and we reveal that they act by suspending control of cellular DNA topology. We show that FicTs are enzymes that adenylylate DNA gyrase and topoisomerase IV, the essential bacterial type IIA topoisomerases, at their ATP-binding site. This modification inactivates both targets by blocking their ATPase activity, and, consequently, causes reversible growth arrest due to the knotting, catenation, and relaxation of cellular DNA. Our results give insight into the regulation of DNA topology and highlight the remarkable plasticity of FIC domain proteins

Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1)

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control 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, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in 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 G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis

Type II Toxin-Antitoxin Loci: The fic Family

FIC domain containing proteins (Fic proteins) are present in all domains of life but particularly widespread among prokaryotes. FIC domains with a fully conserved HxFx[D/E]GNGRxxR active site motif catalyze adenylylation (also known as AMPylation), the transfer of an adenosine 50-monophosphate moiety onto target proteins. Adenylylation activity is tightly controlled by an inhibitory α-helix (αinh) that can either be part of the Fic protein (intramolecular inhibition) or encoded on a different polypeptide chain (intermolecular inhibition), the latter constituting a novel class of type II toxin-antitoxin (TA) modules represented by VbhT-VbhA of Bartonella schoenbuchensis and FicT-FicA of Escherichia coli. The helix αinh harbors a [S/T]xxxE[G/N] motif with the conserved glutamate partially obstructing the ATP-binding site and forcing ATP to bind in a catalytically incompetent conformation. Release of inhibition by removal of the antitoxin component or by mutation of the conserved glutamate in αinh converts Fic proteins into toxins that severely impair bacterial growth

The human IL-17A/F heterodimer: a two-faced cytokine with unique receptor recognition properties

IL-17A and IL-17F are prominent members of the IL-17 family of cytokines that regulates both innate and adaptive immunity. IL-17A has been implicated in chronic inflammatory and autoimmune diseases, and anti-IL-17A antibodies have shown remarkable clinical efficacy in psoriasis and psoriatic arthritis patients. IL-17A and IL-17F are homodimeric cytokines that can also form the IL-17A/F heterodimer whose precise role in health and disease remains elusive. All three cytokines signal through the assembly of a ternary complex with the IL-17RA and IL-17RC receptors. Here we report the X-ray analysis of the human IL-17A/F heterodimer that reveals a two-faced cytokine closely mimicking IL-17A as well as IL-17F. We also present the crystal structure of its complex with the IL-17RA receptor. Unexpectedly in view of the much higher affinity of this receptor toward IL-17A, we find that IL-17RA is bound to the "F-face" of the heterodimer in the crystal. Using site-directed mutagenesis, we then demonstrate that IL-17RA can also bind to the "A-face" of IL-17A/F with similar affinity. Further, we show that IL-17RC does not discriminate between the two faces of the cytokine heterodimer either, thus enabling the formation of two topologically-distinct heterotrimeric complexes with potentially different signaling properties

Structural Analysis Reveals that the Cytokine IL-17F Forms a Homodimeric Complex with Receptor IL-17RC to Drive IL-17RA-Independent Signaling

IL-17, a crucial cytokine for chronic inflammatory diseases, forms a heteromeric complex with IL-17RA and IL-17RC receptors for signaling. Goepfert et al. determine the structure of human IL-17F bound to IL-17RC and reveal a homodimeric assembly that contrasts with the prevailing signaling paradigm and suggests IL-17RA-independent roles for IL-17RC. Interleukin-17A (IL-17A), IL-17F, and IL-17A/F heterodimers are key cytokines of the innate and adaptive immune response. Dysregulation of the IL-17 pathway contributes to immune pathology, and it is therefore important to elucidate the molecular mechanisms that govern IL-17 recognition and signaling. The receptor IL-17RC is thought to act in concert with IL-17RA to transduce IL-17A-, IL-17F-, and IL-17A/F-mediated signals. We report the crystal structure of the extracellular domain of human IL-17RC in complex with IL-17F. In contrast to the expected model, we found that IL-17RC formed a symmetrical 2:1 complex with IL-17F, thus competing with IL-17RA for cytokine binding. Using biophysical techniques, we showed that IL-17A and IL-17A/F also form 2:1 complexes with IL-17RC, suggesting the possibility of IL-17RA-independent IL-17 signaling pathways. The crystal structure of the IL-17RC:IL-17F complex provides a structural basis for IL-17F signaling through IL-17RC, with potential therapeutic applications for respiratory allergy and inflammatory bowel diseases

Crystal structures of E->G mutated Fic proteins representing classes I to III in complex with substrate or substrate analog.

<p>A, VbhA_E24G/VbhT(FIC) in complex with ATP/Mg²⁺; B, SoFic_E73G, C, NmFic_E186G, both in complex with AMPPNP/Mg²⁺. Representation as in Fig. 2 with magnesium ions shown as magenta spheres. The 2Fo-Fc simulated annealing omit maps covering the nucleotide/Mg²⁺ ligands are contoured at 1.1 σ. D, Stereo view of the superposition of the ligand structures shown in panels B and C onto the VbhA_E24G/VbhT(FIC) complex (same as in panel A). Note that the nucleotides of the various complexes are distinguished by their carbon color (VbhA_E24G/VbhT(FIC) ATP in green, SoFic_E73G AMPPNP in orange and NmFic_E186G AMPPNP in pink). The residues of the HxFx(D/E)GNGRxxR signature motif are labeled as in Fig. 2C with the phenylalanine not shown. Also shown is the modifiable hydroxyl side-chain Y32 of Cdc42 (blue) after superposition of the IbpA(FIC2)/Cdc42 complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064901#pone.0064901-Xiao1" target="_blank">[4]</a> onto VbhA_E24G/VbhT(FIC). For the superposition, only the Fic active site loops were used. The α-phosphate moieties appear well-suited for in-line attack of the target hydroxyl group (broken line in magenta).</p

Data collection and refinement statistics.

<p>Values for the highest resolution shell are shown in brackets.</p