Coupled ATPase-adenylate kinase activity in ABC transporters

ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters

Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP

ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetric inward-facing state, and we show that the C-terminal helices, arranged in a zipper-like fashion, play a crucial role in guiding the conformational changes associated with substrate transport. In conclusion, TmrAB can be regarded as a model system for asymmetric ABC exporters in general, and for TAP in particular.publishe

EIGER detector : application in macromolecular crystallography

The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine φ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed

Liquid-Crystalline Properties of Thioesters

An extension of a new method for forming thioesters with mesomorphic properties has been described, whereby the treatment of aryl tert-butylthioethers with long-chain acid chlorides in the presence of bismuth triflate afforded simple derivatives in good yields. This method in the case of 1-cyanoazulenes was, however, complicated by a competitive Friedel-Crafts-type acylation side reaction at the 3-position. Long-chain derivatives of cyanobiphenyl attached through a thioester linkage exhibited mesophases comparable with their ester analogues. The use of shorter chains to decorate the cyanobiphenyl moiety did not produce mesophasic behaviour, unlike their ester analogues. The cyanobiphenyl derivatives showing mesophasic behaviour were studied by small-angle X-ray diffraction, showing alignment for molecules possessing a smectic A phase. It was found that the layer spacing in the smectic A phase was ∼1.5 times the length of the molecule, owing to antiparallel pairing arising from π-stacking between neighbouring units. Calculations suggest these derivatives should exhibit large dielectric anisotropy; however, instability made an exact value difficult to determine. For potential use in a liquid crystal display (LCD), the thioester holds some promise, although this may be limited by the relative stability of the thioester functionality