Synthetic and structural aspects of metal coordination to pyrimidines, purines and nucleotides

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Iron is a ligand of SecA-like metal-binding domains in vivo

Funding: JEL thanks the Royal Society for a University Research Fellowship and the Wellcome Trust for the Q-band EPR spectrometer (099149/Z/12/Z).The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modelling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.PostprintPeer reviewe

Mutational and topological analysis of the Escherichia coli BamA protein

The multi-protein β-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential

Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial

BackgroundAnterior cruciate ligament (ACL) rupture is a common debilitating injury that can cause instability of the knee. We aimed to investigate the best management strategy between reconstructive surgery and non-surgical treatment for patients with a non-acute ACL injury and persistent symptoms of instability.MethodsWe did a pragmatic, multicentre, superiority, randomised controlled trial in 29 secondary care National Health Service orthopaedic units in the UK. Patients with symptomatic knee problems (instability) consistent with an ACL injury were eligible. We excluded patients with meniscal pathology with characteristics that indicate immediate surgery. Patients were randomly assigned (1:1) by computer to either surgery (reconstruction) or rehabilitation (physiotherapy but with subsequent reconstruction permitted if instability persisted after treatment), stratified by site and baseline Knee Injury and Osteoarthritis Outcome Score—4 domain version (KOOS4). This management design represented normal practice. The primary outcome was KOOS4 at 18 months after randomisation. The principal analyses were intention-to-treat based, with KOOS4 results analysed using linear regression. This trial is registered with ISRCTN, ISRCTN10110685, and ClinicalTrials.gov, NCT02980367.FindingsBetween Feb 1, 2017, and April 12, 2020, we recruited 316 patients. 156 (49%) participants were randomly assigned to the surgical reconstruction group and 160 (51%) to the rehabilitation group. Mean KOOS4 at 18 months was 73·0 (SD 18·3) in the surgical group and 64·6 (21·6) in the rehabilitation group. The adjusted mean difference was 7·9 (95% CI 2·5–13·2; p=0·0053) in favour of surgical management. 65 (41%) of 160 patients allocated to rehabilitation underwent subsequent surgery according to protocol within 18 months. 43 (28%) of 156 patients allocated to surgery did not receive their allocated treatment. We found no differences between groups in the proportion of intervention-related complications.InterpretationSurgical reconstruction as a management strategy for patients with non-acute ACL injury with persistent symptoms of instability was clinically superior and more cost-effective in comparison with rehabilitation management

Secondary structure and 1H, 13C and 15N resonance assignments of the Escherichia coli YaeT POTRA domain

We report the first H, C and N chemical shift assignments and secondary structure of the Escherichia coli YaeT POTRA domain; a domain found in the Omp85 family of proteins which is critical for insertion and folding of outer membrane proteins in Gram-negative bacteria

Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport

Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a β-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β-barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion