Toshiaki Hosaka

A three-dimensional movie of structural changes in bacteriorhodopsin

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science, this issue p. 1552 Time-resolved serial crystallography using an x-ray free electron laser reveals structural changes in bacteriorhodopsin. 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.

Crystal structure of cucumisin, a subtilisin-like endoprotease from Cucumis melo L

Cucumisin is a plant serine protease, isolated as an extracellular glycoprotein from the melon fruit Cucumis melo L. var. Prince. Cucumisin is composed of multiple domain modules, including catalytic, protease-associated, and fibronectin-III-like domains. The crystal structure of cucumisin was determined by the multiwavelength anomalous dispersion method and refined at 2.75Å resolution. A structural homology search indicated that the catalytic domain of cucumisin shares structural similarity with subtilisin and subtilisin-like fold enzymes. According to the Z-score, the highest structural similarity is with tomato subtilase 3 (SBT3), with an rmsd of 3.5Å for the entire region. The dimer formation mediated by the protease-associated domain in SBT3 is a distinctive structural characteristic of cucumisin. On the other hand, analytical ultracentrifugation indicated that cucumisin is mainly monomeric in solution. Although the locations of the amino acid residues composing the catalytic triad are well conserved between cucumisin and SBT3, a disulfide bond is uniquely located near the active site of cucumisin. The steric circumstances of the active site with this disulfide bond are distinct from those of SBT3, and it contributes to the substrate preference of cucumisin, especially at the P2 position. Among the plant serine proteases, the thermostability of cucumisin is higher than that of its structural homologue SBT3, as determined by their melting points. A structural comparison between cucumisin and SBT3 revealed that cucumisin possesses less surface area and shortened loop regions. Consequently, the higher thermostability of cucumisin is achieved by its more compact structure.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

Significance This study shows successful experimental phasing methods (single-wavelength anomalous diffraction, single isomorphous replacement, and single isomorphous replacement with anomalous scattering) for crystal structure determination of a membrane protein by serial femtosecond crystallography with X-ray free electron lasers. Our iodine-containing detergent provided strong anomalous and isomorphous difference signals, which enabled experimental phasing using lower-resolution reflections (worse than 3 Å) from fewer indexed images than phasing attempts reported previously. The findings of this study will be applicable to a wide range of target proteins in structural biology, especially membrane proteins that often diffract to low resolution. The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3

The light-driven inward chloride ion-pumping rhodopsin Nonlabens marinus rhodopsin-3 (NM-R3), from a marine flavobacterium, belongs to a phylogenetic lineage distinct from the halorhodopsins known as archaeal inward chloride ion-pumping rhodopsins. NM-R3 and halorhodopsin have distinct motif sequences that are important for chloride ion binding and transport. In this study, we present the crystal structure of a new type of light-driven chloride ion pump, NM-R3, at 1.58 Å resolution. The structure revealed the chloride ion translocation pathway and showed that a single chloride ion resides near the Schiff base. The overall structure, chloride ion-binding site, and translocation pathway of NM-R3 are different from those of halorhodopsin. Unexpectedly, this NM-R3 structure is similar to the crystal structure of the light-driven outward sodium ion pump, Krokinobacter eikastus rhodopsin 2. Structural and mutational analyses of NM-R3 revealed that most of the important amino acid residues for chloride ion pumping exist in the ion influx region, located on the extracellular side of NM-R3. In contrast, on the opposite side, the cytoplasmic regions of K. eikastus rhodopsin 2 were reportedly important for sodium ion pumping. These results provide new insight into ion selection mechanisms in ion pumping rhodopsins, in which the ion influx regions of both the inward and outward pumps are important for their ion selectivities.

Identification of nucleotide binding sites in V-type Na+-ATPase from Enterococcus hirae.

A and B subunits of the V-type Na+-ATPase from Enterococcus hirae were suggested to possess nucleotide binding sites (Murata, T. et al., J. Biochem., 132, 789–794 (2002)), although the B subunit did not have the consensus sequence for nucleotide binding. To further characterize the binding sites in the V-ATPase, we did the photoaffinity labeling study using 8-azido-[α-32P]ATP. A and B subunits were labeled with 8-azido-[α-32P]ATP when analysed with SDS polyacrylamide gel electrophoresis. The peptide fragment of A subunit obtained by lysyl endopeptidase digestion after labeling showed a molecular size of 9 kDa and its amino acid sequencing revealed that it corresponded to residues Arg423-Lys494. The peptide fragment from B subunit after photoaffinity labeling and lysyl endopeptidase digestion showed the size of 5 kDa and corresponded to residues Phe404-Lys443. In our structure model, these peptides were close to the adenine ring of ATP. We suggest that non-catalytic B subunit of E. hirae V-ATPase has a nucleotide binding site, similarly to eukaryotic V-ATPases and F-ATPases.

Crystal structural characterization reveals novel oligomeric interactions of human voltage‐dependent anion channel 1

Voltage‐dependent anion channel 1 (VDAC1), which is located in the outer mitochondrial membrane, plays important roles in various cellular processes. For example, oligomerization of VDAC1 is involved in the release of cytochrome c to the cytoplasm, leading to apoptosis. However, it is unknown how VDAC1 oligomerization occurs in the membrane. In the present study, we determined high‐resolution crystal structures of oligomeric human VDAC1 (hVDAC1) prepared by using an Escherichia coli cell‐free protein synthesis system, which avoided the need for denaturation and refolding of the protein. Broad‐range screening using a bicelle crystallization method produced crystals in space groups C222 and P22121, which diffracted to a resolution of 3.10 and 3.15 Å, respectively. Each crystal contained two hVDAC1 protomers in the asymmetric unit. Dimer within the asymmetrical unit of the crystal in space group C222 were oriented parallel, whereas those of the crystal in space group P22121 were oriented anti‐parallel. From a model of the crystal in space group C222, which we constructed by using crystal symmetry operators, a heptameric structure with eight patterns of interaction between protomers, including hydrophobic interactions with β‐strands, hydrophilic interactions with loop regions, and protein–lipid interactions, was observed. It is possible that by having multiple patterns of interaction, VDAC1 can form homo‐ or hetero‐oligomers not only with other VDAC1 protomers but also with other proteins such as VDAC2, VDAC3 and apoptosis‐regulating proteins in the Bcl‐2 family.

Structural Basis of Cucumisin Protease Activity Regulation by Its Propeptide

Cucumisin [EC 3.4.21.25], a subtilisin-like serine endopeptidase, was isolated from melon fruit, Cucumis melo L. Mature cucumisin (67 kDa, 621 residues) is produced by removal of the propeptide (10 kDa, 88 residues) from the cucumisin precursor by subsequence processing. It is reported that cucumisin is inhibited by its own propeptide. The crystal structure of mature cucumisin is reported to be composed of three domains: the subtilisin-like catalytic domain, the protease-associated domain and the C-terminal fibronectin-III-like domain. In this study, the crystal structure of the mature cucumisin•propeptide complex was determined by the molecular replacement method and refined at 1.95 Å resolution. In this complex, the propeptide had a domain of the &agr;–&bgr; sandwich motif with four-stranded antiparallel &bgr;-sheets, two helices and a strand of the C-terminal region. The &bgr;-sheets of the propeptide bind to two parallel surface helices of cucumisin through hydrophobic interaction and 27 hydrogen bonds. The C-terminus of the propeptide binds to the cleft of the active site as peptide substrates. The inhibitory assay suggested that the C-terminal seven residues of the propeptide do not inhibit the cucumisin activity. The crystal structure of the cucumisin•propeptide complex revealed the regulation mechanism of cucumisin activity.

Mutagenesis of the residues forming an ion binding pocket of the NtpK subunit of Enterococcus hirae V-ATPase

The crystal structures of the Na(+)- and Li(+)-bound NtpK rings of Enterococcus hirae V-ATPase have been obtained. The coupling ion (Na(+) or Li(+)) was surrounded by five oxygen atoms contributed by residues T64, Q65, Q110, E139, and L61, and the hydrogen bonds of the side chains of Q110, Y68, and T64 stabilized the position of the E139 γ carboxylate essential for ion occlusion (PDB accession numbers 2BL2 and 2CYD). We previously indicated that an NtpK mutant strain (E139D) lost tolerance to sodium but not to lithium at alkaline pHs and suggested that the E139 residue is indispensable for the enzymatic activity of E. hirae V-ATPase linked with the sodium tolerance of this bacterium. In this study, we examined the activities of V-ATPase in which these four residues, except for E139, were substituted. The V-ATPase activities of the Q65A and Y68A mutants were slightly retained, but those of the T64A and Q110A mutants were negligible. Among the residues, T64 and Q110 are indispensable for the ion coupling of E. hirae V-ATPase, in addition to the essential residue E139.