Crystal structure of the DegS stress sensor: How a PDZ domain recognises misfolded protein and activates a protease

Gram-negative bacteria respond to misfolded proteins in the cell envelope with the σE-driven expression of periplasmic proteases/chaperones. Activation of σE is controlled by a proteolytic cascade that is initiated by the DegS protease. DegS senses misfolded protein in the periplasm, undergoes autoactivation, and cleaves the antisigma factor RseA. Here, we present the crystal structures of three distinct states of DegS from E. coli. DegS alone exists in a catalytically inactive form. Binding of stress-signaling peptides to its PDZ domain induces a series of conformational changes that activates protease function. Backsoaking of crystals containing the DegS-activator complex revealed the presence of an active/inactive hybrid structure and demonstrated the reversibility of activation. Taken together, the structural data illustrate in molecular detail how DegS acts as a periplasmic stress sensor. Our results suggest a novel regulatory role for PDZ domains and unveil a novel mechanism of reversible protease activation

The 1.3 A crystal structure of the flavoprotein YqjM reveals a novel class of Old Yellow Enzymes

Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (alpha/beta)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the beta-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs

Regulation of the σE stress response by DegS: how the PDZ domain keeps the protease inactive in the resting state and allows integration of different OMP-derived stress signals upon folding stress

The unfolded protein response of Escherichia coli is triggered by the accumulation of unassembled outer membrane proteins (OMPs) in the cellular envelope. The PDZ-protease DegS recognizes these mislocalized OMPs and initiates a proteolytic cascade that ultimately leads to the σE-driven expression of a variety of factors dealing with folding stress in the periplasm and OMP assembly. The general features of how OMPs activate the protease function of DegS have not yet been systematically addressed. Furthermore, it is unknown how the PDZ domain keeps the protease inactive in the resting state, which is of crucial importance for the functioning of the entire σE stress response. Here we show in atomic detail how DegS is able to integrate the information of distinct stress signals that originate from different OMPs containing a ϕ-x-Phe C-terminal motif. A dedicated loop of the protease domain, loop L3, serves as a versatile sensor for allosteric ligands. L3 is capable of interacting differently with ligands but reorients in a conserved manner to activate DegS. Our data also indicate that the PDZ domain directly inhibits protease function in the absence of stress signals by wedging loop L3 in a conformation that ultimately disrupts the proteolytic site. Thus, the PDZ domain and loop L3 of DegS define a novel molecular switch allowing strict regulation of the σE stress response system

Safety and efficacy of eculizumab in anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis (REGAIN): a phase 3, randomised, double-blind, placebo-controlled, multicentre study

Background Complement is likely to have a role in refractory generalised myasthenia gravis, but no approved therapies specifically target this system. Results from a phase 2 study suggested that eculizumab, a terminal complement inhibitor, produced clinically meaningful improvements in patients with anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis. We further assessed the efficacy and safety of eculizumab in this patient population in a phase 3 trial. Methods We did a phase 3, randomised, double-blind, placebo-controlled, multicentre study (REGAIN) in 76 hospitals and specialised clinics in 17 countries across North America, Latin America, Europe, and Asia. Eligible patients were aged at least 18 years, with a Myasthenia Gravis-Activities of Daily Living (MG-ADL) score of 6 or more, Myasthenia Gravis Foundation of America (MGFA) class II\ue2\u80\u93IV disease, vaccination against Neisseria meningitides, and previous treatment with at least two immunosuppressive therapies or one immunosuppressive therapy and chronic intravenous immunoglobulin or plasma exchange for 12 months without symptom control. Patients with a history of thymoma or thymic neoplasms, thymectomy within 12 months before screening, or use of intravenous immunoglobulin or plasma exchange within 4 weeks before randomisation, or rituximab within 6 months before screening, were excluded. We randomly assigned participants (1:1) to either intravenous eculizumab or intravenous matched placebo for 26 weeks. Dosing for eculizumab was 900 mg on day 1 and at weeks 1, 2, and 3; 1200 mg at week 4; and 1200 mg given every second week thereafter as maintenance dosing. Randomisation was done centrally with an interactive voice or web-response system with patients stratified to one of four groups based on MGFA disease classification. Where possible, patients were maintained on existing myasthenia gravis therapies and rescue medication was allowed at the study physician's discretion. Patients, investigators, staff, and outcome assessors were masked to treatment assignment. The primary efficacy endpoint was the change from baseline to week 26 in MG-ADL total score measured by worst-rank ANCOVA. The efficacy population set was defined as all patients randomly assigned to treatment groups who received at least one dose of study drug, had a valid baseline MG-ADL assessment, and at least one post-baseline MG-ADL assessment. The safety analyses included all randomly assigned patients who received eculizumab or placebo. This trial is registered with ClinicalTrials.gov, number NCT01997229. Findings Between April 30, 2014, and Feb 19, 2016, we randomly assigned and treated 125 patients, 62 with eculizumab and 63 with placebo. The primary analysis showed no significant difference between eculizumab and placebo (least-squares mean rank 56\uc2\ub76 [SEM 4\uc2\ub75] vs 68\uc2\ub73 [4\uc2\ub75]; rank-based treatment difference \ue2\u88\u9211\uc2\ub77, 95% CI \ue2\u88\u9224\uc2\ub73 to 0\uc2\ub796; p=0\uc2\ub70698). No deaths or cases of meningococcal infection occurred during the study. The most common adverse events in both groups were headache and upper respiratory tract infection (ten [16%] for both events in the eculizumab group and 12 [19%] for both in the placebo group). Myasthenia gravis exacerbations were reported by six (10%) patients in the eculizumab group and 15 (24%) in the placebo group. Six (10%) patients in the eculizumab group and 12 (19%) in the placebo group required rescue therapy. Interpretation The change in the MG-ADL score was not statistically significant between eculizumab and placebo, as measured by the worst-rank analysis. Eculizumab was well tolerated. The use of a worst-rank analytical approach proved to be an important limitation of this study since the secondary and sensitivity analyses results were inconsistent with the primary endpoint result; further research into the role of complement is needed. Funding Alexion Pharmaceuticals