Structural and functional diversity calls for a new classification of ABC transporters

Members of the ATP‐binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP‐binding cassette in the nucleotide‐binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs

Crystallization and preliminary diffraction analysis of Wzi, a member of the capsule export and assembly pathway in Escherichia coli

Wzi is a membrane protein from E. coli thought to be involved in the attachment of capsular polysaccharides to the bacterial surface. This reports describes recombinant Wzi’s purification, crystallization and the results of initial diffraction studies

Structure determination of an integral membrane protein at room temperature from crystals in situ

The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs

For the successfulgenerativeengineeringof functionalartificialcells,a convenientandcontrollablemeansof deliveringmembraneproteinsinto membranelipidbilayersis necessary.Herewereporta deliverysystemthatachievesthis by employingmembraneprotein-carryingnanodiscsandthecalcium-dependentfusionofphosphatidylserinelipidmembranes.We showthat lipidnanodiscscanfuse a transportedlipidbilayerwith the lipidbilayersof smallunilamellarvesicles(SUVs)or giantunilamellarvesicles(GUVs)whileavoidingrecipientvesiclesaggregation.Thisis triggeredby a simple,transientincreasein calciumconcentration,whichresultsin efficientand rapidfusionin a one-potreaction.Furthermore,nanodiscscan be loadedwithmembraneproteinsthatcan be deliveredintotargetSUVor GUVmembranesin a detergent-independentfashionwhileretainingtheirfunctionality.Nanodiscshavea provenabilityto carrya widerangeof membraneproteins,controltheiroligomericstate,and arehighlyadaptable.Giventhis, our approachmay be the basisfor the developmentof usefultoolsthat will allowbespokedeliveryofmembraneproteinsto protocells,equippingthemwith the cell-likeabilityto exchangematerialacrossouter/subcellularmembranes

Ray‐tracing analytical absorption correction for X‐ray crystallography based on tomographic reconstructions

Processing of single-crystal X-ray diffraction data from area detectors can be separated into two steps. First, raw intensities are obtained by integration of the diffraction images, and then data correction and reduction are performed to determine structure-factor amplitudes and their uncertainties. The second step considers the diffraction geometry, sample illumination, decay, absorption and other effects. While absorption is only a minor effect in standard macromolecular crystallography (MX), it can become the largest source of uncertainty for experiments performed at long wavelengths. Current software packages for MX typically employ empirical models to correct for the effects of absorption, with the corrections determined through the procedure of minimizing the differences in intensities between symmetry-equivalent reflections; these models are well suited to capturing smoothly varying experimental effects. However, for very long wavelengths, empirical methods become an unreliable approach to model strong absorption effects with high fidelity. This problem is particularly acute when data multiplicity is low. This paper presents an analytical absorption correction strategy (implemented in new software AnACor) based on a volumetric model of the sample derived from X-ray tomography. Individual path lengths through the different sample materials for all reflections are determined by a ray-tracing method. Several approaches for absorption corrections (spherical harmonics correction, analytical absorption correction and a combination of the two) are compared for two samples, the membrane protein OmpK36 GD, measured at a wavelength of λ = 3.54 Å, and chlorite dismutase, measured at λ = 4.13 Å. Data set statistics, the peak heights in the anomalous difference Fourier maps and the success of experimental phasing are used to compare the results from the different absorption correction approaches. The strategies using the new analytical absorption correction are shown to be superior to the standard spherical harmonics corrections. While the improvements are modest in the 3.54 Å data, the analytical absorption correction outperforms spherical harmonics in the longer-wavelength data (λ = 4.13 Å), which is also reflected in the reduced amount of data being required for successful experimental phasing

Altered Antibiotic Transport in OmpC Mutants Isolated from a Series of Clinical Strains of Multi-Drug Resistant E. coli

Antibiotic-resistant bacteria, particularly Gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane

Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35)

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95±0.89 μM peptide and a Vmax of 53.91±3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein

TAEKWONDO TECHNIQUES AND COMPETITION CHARACTERISTICS INVOLVED IN TIME-LOSS INJURIES

The purpose of this study was to assess time-loss injuries in young and adult taekwondo athletes. Participants were 2739 children (11-13 years), Junior (14-17 years) and adult males and females (18 years and older) competing in the national Greek championships. Injury data were collected by project staff with all diagnoses made by the tournament physician. Odds ratios were computed as well as 95% confidence intervals around the injury rates. The female Juniors had a higher time-loss injury rate (Fisher's Exact Test p = 0.033) than their adult counterparts. However, they were not at a higher risk of incurring a time-loss injury: OR = 0.143, 95% CI: 0.018-1.124. Collapsed over age, the females as a group recorded more time-loss injuries [11.36/1,000 A-E (95% CI: 6.25-16.47) versus 7.40/1,000 A-E (95% CI: 4.44-10.36)], but this was not significant (OR = 0.703, 95% CI: 0.383-1.293). In the Juniors, the boys only incurred time-loss injuries to the head and neck. There was no difference in the Junior girls in the distribution of time-loss injuries across body region, although they were at higher risk of sustaining an injury to the head and neck (OR = 1.510, 95% CI: 0.422-5.402) but this was not statistically significant. Although there were no statistical differences among age groups within gender, the Junior boys and girls (11-13 years) sustained more cerebral concussions. The Junior boys were at a higher risk of incurring a cerebral concussion than the boys (OR = 7.871, 95% CI: 0.917-67.583, Fisher's Exact Test p = 0.036). In the males, there was no difference between the men and Junior boys in injury rate for swing kicks compared to other techniques (OR = 2.000, 95% CI = 0.397-28.416). There also was no difference between the men and boys (OR = 4.800, 95% CI: 0.141-58.013). To help reduce the incidence of time-loss injuries in taekwondo, especially cerebral concussions, it is suggested for coaches to emphasize blocking skills. Educating referees, coaches and athletes plays an important role in helping to reduce taekwondo time-loss injurie