Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

The cellulose synthesizing terminal complex consisting of subunits A, B, C, and D in Acetobacter xylinum spans the outer and inner cell membranes to synthesize and extrude glucan chains, which are assembled into subelementary fibrils and further into a ribbon. We determined the structures of subunit D (AxCeSD/AxBcsD) with both N- and C-terminal His 6 tags, and in complex with cellopentaose. The structure of AxCeSD shows an exquisite cylinder shape (height: ∼65  Å , outer diameter: ∼90  Å , and inner diameter: ∼25  Å ) with a right-hand twisted dimer interface on the cylinder wall, formed by octamer as a functional unit. All N termini of the octamer are positioned inside the AxCeSD cylinder and create four passageways. The location of cellopentaoses in the complex structure suggests that four glucan chains are extruded individually through their own passageway along the dimer interface in a twisted manner. The complex structure also shows that the N-terminal loop, especially residue Lys6, seems to be important for cellulose production, as confirmed by in vivo assay using mutant cells with axcesD gene disruption and N-terminus truncation. Taking all results together, a model of the bacterial terminal complex is discussed.

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Background:The details of the catalytic mechanism of cellobiose 2-epimerase (CE) remains unclear. Results: The crystal structures of Rhodothermus marinus CE in the apo form and complexes with its substrates/products 4-O-␤-D-glucopyranosyl-D-mannnose, epilactose, or cellobiitol (reaction intermediate analog) were elucidated. Conclusion: Epimerization catalyzed by CE proceeds through ring opening, deprotonation/reprotonation, carbon-carbon bond rotation, and ring closure. Significance: This study yielded structural insights into epimerization catalyzed by CE.

Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OXR structures in complex with selective antagonists and previously determined OXR/OXR structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OXR. The importance of these residues for binding selectivity to OXR was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.

Edited by Judit OvádiKeywords: X-ray crystallography Cellobiose 2-epimerase Deprotonation Unmodified sugar Structure a b s t r a c t Enzymatic epimerization is an important modification for carbohydrates to acquire diverse functions attributable to their stereoisomers. Cellobiose 2-epimerase (CE) catalyzes interconversion between D-glucose and D-mannose residues at the reducing end of b-1,4-linked oligosaccharides. Here, we solved the structure of Ruminococcus albus CE (RaCE). The structure of RaCE showed strong similarity to those of N-acetyl-D-glucosamine 2-epimerase and aldose-ketose isomerase YihS with a high degree of conservation of residues around the catalytic center, although sequence identity between them is low. Based on structural comparison, we found that His184 is required for RaCE activity as the third histidine added to two essential histidines in other sugar epimerases/isomerases. This finding was confirmed by mutagenesis, suggesting a new catalytic mechanism for CE involving three histidines. Structured summary of protein interactions:RaCE and X-ray crystallography (View interaction)

In some Proteobacteria and Firmicutes such as Pseudomonas aeruginosa, Vibrio cholerae, Xanthomonas campestris, and Clostridium difficile, cyclic dimeric guanosine monophosphate (c-di-GMP) is known to regulate cellular processes, including motility, biofilm formation, and virulence, as a second messenger. Cellulose production in

A high-viscosity cartridge-type injector for serial femtosecond crystallography has been developed at SPring-8 Angstrom Compact Free-Electron Laser.