Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography.

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography.

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio.

Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog.

P-glycoprotein is an ATP-binding cassette multidrug transporter that actively transports chemically diverse substrates across the lipid bilayer. The precise molecular mechanism underlying transport is not fully understood. Here, we present crystal structures of a eukaryotic P-glycoprotein homolog, CmABCB1 from Cyanidioschyzon merolae, in two forms: unbound at 2.6-Å resolution and bound to a unique allosteric inhibitor at 2.4-Å resolution. The inhibitor clamps the transmembrane helices from the outside, fixing the CmABCB1 structure in an inward-open conformation similar to the unbound structure, confirming that an outward-opening motion is required for ATP hydrolysis cycle. These structures, along with site-directed mutagenesis and transporter activity measurements, reveal the detailed architecture of the transporter, including a gate that opens to extracellular side and two gates that open to intramembranous region and the cytosolic side. We propose that the motion of the nucleotide-binding domain drives those gating apparatuses via two short intracellular helices, IH1 and IH2, and two transmembrane helices, TM2 and TM5.

Crystal structures of ß-primeverosidase in complex with disaccharide amidine inhibitors.

ß-Primeverosidase (PD) is a disaccharide-specific ß-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes ß-primeveroside (6-O-ß-d-xylopyranosyl-ß-d-glucopyranoside) at the ß-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones. PD only accepts primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. We determined the crystal structure of PD complexes using highly specific disaccharide amidine inhibitors, N-ß-primeverosylamidines, and revealed the architecture of the active site responsible for substrate specificity. We identified three subsites in the active site: subsite -2 specific for 6-O-ß-d-xylopyranosyl, subsite -1 well conserved among ß-glucosidases and specific for ß-d-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu-470, Ser-473, and Gln-477 act as the specific hydrogen bond donors for 6-O-ß-d-xylopyranosyl in subsite -2. On the other hand, subsite +1 was a large hydrophobic cavity that accommodates various aromatic aglycones. Compared with aglycone-specific ß-glucosidases of the glycoside hydrolase family 1, PD lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-ß-d-xylopyranosyl together. PD recognizes the ß-primeverosides in subsites -1 and -2 by hydrogen bonds, whereas the large subsite +1 loosely accommodates various aglycones. The glycone-specific activity of PD for broad aglycone substrates results in selective and multiple release of temporally stored alcoholic volatile aglycones of ß-primeveroside.

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Šis successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

Synthesis and inhibitory activity of substrate-analog fructosyl peptide oxidase inhibitors.

Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a-c were successfully designed and synthesized. These inhibitors mimic N(α)-fructosyl-L-valine (Fru-Val), [N(α)-fructosyl-L-valyl]-L-histidine (Fru-ValHis), and N(ε)-fructosyl-L-lysine (εFru-Lys), respectively. The secondary nitrogen atom in the natural substrates, linking fructose and amino acid or dipeptide moieties, was substituted in 1a-c with a sulfur atom to avoid enzymatic cleavage. Kinetic studies revealed that 1a-c act as competitive inhibitors against an FPOX obtained from Coniochaeta sp., and Ki values of 11.1, 66.8, and 782 µM were obtained for 1a-c, respectively.

Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p.

Peroxisomes require peroxin (Pex) proteins for their biogenesis. The interaction between Pex3p, which resides on the peroxisomal membrane, and Pex19p, which resides in the cytosol, is crucial for peroxisome formation and the post-translational targeting of peroxisomal membrane proteins (PMPs). It is not known how Pex3p promotes the specific interaction with Pex19p for the purpose of PMP translocation. Here, we present the three-dimensional structure of the complex between a cytosolic domain of Pex3p and the binding-region peptide of Pex19p. The overall shape of Pex3p is a prolate spheroid with a novel fold, the 'twisted six-helix bundle.' The Pex19p-binding site is at an apex of the Pex3p spheroid. A 16-residue region of the Pex19p peptide forms an α-helix and makes a contact with Pex3p; this helix is disordered in the unbound state. The Pex19p peptide contains a characteristic motif, consisting of the leucine triad (Leu18, Leu21, Leu22), and Phe29, which are critical for the Pex3p binding and peroxisome biogenesis.

Structural basis for gibberellin recognition by its receptor GID1.

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. A nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), has a primary structure similar to that of the hormone-sensitive lipases (HSLs). Here we analyse the crystal structure of Oryza sativa GID1 (OsGID1) bound with GA(4) and GA(3) at 1.9 A resolution. The overall structure of both complexes shows an alpha/beta-hydrolase fold similar to that of HSLs except for an amino-terminal lid. The GA-binding pocket corresponds to the substrate-binding site of HSLs. On the basis of the OsGID1 structure, we mutagenized important residues for GA binding and examined their binding activities. Almost all of them showed very little or no activity, confirming that the residues revealed by structural analysis are important for GA binding. The replacement of Ile 133 with Leu or Val-residues corresponding to those of the lycophyte Selaginella moellendorffii GID1s-caused an increase in the binding affinity for GA(34), a 2beta-hydroxylated GA(4). These observations indicate that GID1 originated from HSL and was further modified to have higher affinity and more strict selectivity for bioactive GAs by adapting the amino acids involved in GA binding in the course of plant evolution.

Crystallization and preliminary crystallographic analysis of two eukaryotic fructosyl peptide oxidases.

Fructosyl peptide oxidase (FPOX) catalyses the oxidation of α-glycated dipeptides such as N(α)-(1-deoxy-D-fructos-1-yl)-L-valyl-L-histidine (Fru-ValHis) and is used in the diagnosis of diabetes mellitus. Here, two thermostable mutants of FPOX, CFP-T7 and EFP-T5M, were crystallized by the sitting-drop vapour-diffusion method. The crystal of CFP-T7 belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 110.09, c = 220.48 Å, and that of EFP-T5M belonged to the monoclinic space group P2(1), with unit-cell parameters a = 43.00, b = 230.05, c = 47.27 Å, ß = 116.99°. The crystals of CFP-T7 and EFP-T5M diffracted to 1.8 and 1.6 Å resolution, respectively.

Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B from Geobacillus collagenovorans MO-1.

Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophile Geobacillus collagenovorans MO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Šat 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as `Kibo') at the International Space Station (ISS). The crystal belonged to the trigonal space group P3(1)21, with unit-cell parameters a = b = 87.64, c = 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.

Structural basis for the spectral difference in luciferase bioluminescence.

Fireflies communicate with each other by emitting yellow-green to yellow-orange brilliant light. The bioluminescence reaction, which uses luciferin, Mg-ATP and molecular oxygen to yield an electronically excited oxyluciferin species, is carried out by the enzyme luciferase. Visible light is emitted during relaxation of excited oxyluciferin to its ground state. The high quantum yield of the luciferin/luciferase reaction and the change in bioluminescence colour caused by subtle structural differences in luciferase have attracted much research interest. In fact, a single amino acid substitution in luciferase changes the emission colour from yellow-green to red. Although the crystal structure of luciferase from the North American firefly (Photinus pyralis) has been described, the detailed mechanism for the bioluminescence colour change is still unclear. Here we report the crystal structures of wild-type and red mutant (S286N) luciferases from the Japanese Genji-botaru (Luciola cruciata) in complex with a high-energy intermediate analogue, 5'-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA). Comparing these structures to those of the wild-type luciferase complexed with AMP plus oxyluciferin (products) reveals a significant conformational change in the wild-type enzyme but not in the red mutant. This conformational change involves movement of the hydrophobic side chain of Ile 288 towards the benzothiazole ring of DLSA. Our results indicate that the degree of molecular rigidity of the excited state of oxyluciferin, which is controlled by a transient movement of Ile 288, determines the colour of bioluminescence during the emission reaction.

[What Kind of Measurements Can Be Made with an X-ray Free Electron Laser at SACLA?]

Three-dimensional structural information is indispensable to understand the function of proteins in living organisms and X-ray crystallography plays a major role in determining the three-dimensional structure. X-ray free-electron laser (XFEL), which is intense and femtosecond X-ray pulses, enables us to obtain X-ray diffraction intensity data before the destruction of protein molecules, and is expected to be a technology to obtain dynamic structural information. This year marks the 10th anniversary of SPring-8 Angstrom Compact Free Electron Laser (SACLA), Japan's X-ray free electron laser facility. In this review, I describe the damage-free crystal structure analysis, de novo crystal structure determination using single wavelength anomalous dispersion by serial femtosecond crystallography (SFX), and time-resolved X-ray crystallography that have been performed at SACLA.

Crystal structure of CmABCB1 multi-drug exporter in lipidic mesophase revealed by LCP-SFX.

CmABCB1 is a Cyanidioschyzon merolae homolog of human ABCB1, a well known ATP-binding cassette (ABC) transporter responsible for multi-drug resistance in various cancers. Three-dimensional structures of ABCB1 homologs have revealed the snapshots of inward- and outward-facing states of the transporters in action. However, sufficient information to establish the sequential movements of the open-close cycles of the alternating-access model is still lacking. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has proven its worth in determining novel structures and recording sequential conformational changes of proteins at room temperature, especially for medically important membrane proteins, but it has never been applied to ABC transporters. In this study, 7.7 mono-acyl-glycerol with cholesterol as the host lipid was used and obtained well diffracting microcrystals of the 130 kDa CmABCB1 dimer. Successful SFX experiments were performed by adjusting the viscosity of the crystal suspension of the sponge phase with hy-droxy-propyl methyl-cellulose and using the high-viscosity sample injector for data collection at the SACLA beamline. An outward-facing structure of CmABCB1 at a maximum resolution of 2.22 Šis reported, determined by SFX experiments with crystals formed in the lipidic cubic phase (LCP-SFX), which has never been applied to ABC transporters. In the type I crystal, CmABCB1 dimers interact with adjacent molecules via not only the nucleotide-binding domains but also the transmembrane domains (TMDs); such an interaction was not observed in the previous type II crystal. Although most parts of the structure are similar to those in the previous type II structure, the substrate-exit region of the TMD adopts a different configuration in the type I structure. This difference between the two types of structures reflects the flexibility of the substrate-exit region of CmABCB1, which might be essential for the smooth release of various substrates from the transporter.

The exquisite structure and reaction mechanism of bacterial Pz-peptidase A toward collagenous peptides: X-ray crystallographic structure analysis of PZ-peptidase a reveals differences from mammalian thimet oligopeptidase.

Pz-peptidase A, from the thermophilic bacterium Geobacillus collagenovorans MO-1, hydrolyzes a synthetic peptide substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), which contains a collagen-specific tripeptide sequence, -Gly-Pro-X-, but does not act on collagen proteins themselves. The mammalian enzyme, thimet oligopeptidase (TOP), which has comparable functions with bacterial Pz-peptidases but limited identity at the primary sequence level, has recently been subjected to x-ray crystallographic analysis; however, no crystal structure has yet been reported for complexes of TOP with substrate analogues. Here, we report crystallization of recombinant Pz-peptidase A in complex with two phosphinic peptide inhibitors (PPIs) that also function as inhibitors of TOP and determination of the crystal structure of these complexes at 1.80-2.00 Å resolution. The most striking difference between Pz-peptidase A and TOP is that there is no channel running the length of bacterial protein. Whereas the structure of TOP resembles an open bivalve, that of Pz-peptidase A is closed and globular. This suggests that collagenous peptide substrates enter the tunnel at the top gateway of the closed Pz-peptidase A molecule, and reactant peptides are released from the bottom gateway after cleavage at the active site located in the center of the tunnel. One of the two PPIs, PPI-2, which contains the collagen-specific sequence, helped to clarify the exquisite structure and reaction mechanism of Pz-peptidase A toward collagenous peptides. This study describes the mode of substrate binding and its implication for the mammalian enzymes.

The crystal structure of the CmABCB1 G132V mutant, which favors the outward-facing state, reveals the mechanism of the pivotal joint between TM1 and TM3.

CmABCB1 is a homologue of human P-glycoprotein, which extrudes various substrates by iterative cycles of conformational changes between the inward- and outward-facing states. Comparison of the inward- and outward-facing structures of CmABCB1 suggested that pivotal joints in the transmembrane domain regulate the tilt of transmembrane helices. Transmembrane helix 1 (TM1) forms a tight helix-helix contact with TM3 at the TM1-3 joint. Mutation of Gly132 to valine at the TM1-3 joint, G132V, caused a 10-fold increase in ATPase activity, but the mechanism underlying this change remains unclear. Here, we report a crystal structure of the outward-facing state of the CmABCB1 G132V mutant at a 2.15 Å resolution. We observed structural displacements between the outward-facing states of G132V and the previous one at the region around the TM1-3 joint, and a significant expansion at the extracellular gate. We hypothesize that steric hindrance caused by the Val substitution shifted the conformational equilibrium toward the outward-facing state, favoring the dimeric state of the nucleotide-binding domains and thereby increasing the ATPase activity of the G132V mutant.

ABCB1/MDR1/P-gp employs an ATP-dependent twist-and-squeeze mechanism to export hydrophobic drugs.

ABCB1, also called MDR1 or P-glycoprotein, exports various hydrophobic compounds and plays an essential role as a protective physiological barrier in several organs, including the brain, testis, and placenta. However, little is known about the structural mechanisms that allow ABCB1 to recognize hydrophobic compounds of diverse structures or the coupling of ATP hydrolysis to uphill substrate export. High-resolution X-ray crystal structures of the pre- and post-transport states and FRET analyses in living cells have revealed that an aromatic hydrophobic network at the top of the inner cavity is key for the conformational change in ABCB1 that is triggered by a hydrophobic substrate. ATP binding, but not hydrolysis, induces a progressive network that results in a twisting motion of the whole protein, squeezing out the substrate directly to the extracellular space. This twist-and-squeeze mechanism by which ABCB1 exports hydrophobic substrates is distinct from those of other transporters.

Deleting two C-terminal alpha-helices is effective to crystallize the bacterial ABC transporter Escherichia coli MsbA complexed with AMP-PNP.

An MsbA deletion mutant DeltaC21 that lacks the two C-terminal alpha-helices was expressed in Escherichia coli strain C41 and purified by metal-affinity and gel-filtration chromatography. Purified DeltaC21 retained 26% of the activity of the wild-type ATPase and had a similar binding affinity to fluorescent nucleotide derivatives. Although crystals of wild-type MsbA complexed with adenosine 5'-(beta,gamma-imido)triphosphate could not be obtained, crystals of DeltaC21 that diffracted to 4.5 A resolution were obtained. The preliminary DeltaC21 structure had the outward-facing conformation, in contrast to the previously reported E. coli MsbA structure. This result suggests that deletion of the C-terminal alpha-helices may play a role in facilitating the outward-facing nucleotide-bound crystal structure of EcMsbA.

Crystal structure of the C-terminal clock-oscillator domain of the cyanobacterial KaiA protein.

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria, and KaiA activates kaiBC expression whereas KaiC represses it. Here we show that KaiA is composed of three functional domains, the N-terminal amplitude-amplifier domain, the central period-adjuster domain and the C-terminal clock-oscillator domain. The C-terminal domain is responsible for dimer formation, binding to KaiC, enhancing KaiC phosphorylation and generating the circadian oscillations. The X-ray crystal structure at a resolution of 1.8 A of the C-terminal clock-oscillator domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 shows that residue His270, located at the center of a KaiA dimer concavity, is essential to KaiA function. KaiA binding to KaiC probably occurs via the concave surface. On the basis of the structure, we predict the structural roles of the residues that affect circadian oscillations.

Inward- and outward-facing X-ray crystal structures of homodimeric P-glycoprotein CmABCB1.

P-glycoprotein extrudes a large variety of xenobiotics from the cell, thereby protecting tissues from their toxic effects. The machinery underlying unidirectional multidrug pumping remains unknown, largely due to the lack of high-resolution structural information regarding the alternate conformational states of the molecule. Here we report a pair of structures of homodimeric P-glycoprotein: an outward-facing conformational state with bound nucleotide and an inward-facing apo state, at resolutions of 1.9 Å and 3.0 Å, respectively. Features that can be clearly visualized at this high resolution include ATP binding with octahedral coordination of Mg2+; an inner chamber that significantly changes in volume with the aid of tight connections among transmembrane helices (TM) 1, 3, and 6; a glutamate-arginine interaction that stabilizes the outward-facing conformation; and extensive interactions between TM1 and TM3, a property that distinguishes multidrug transporters from floppases. These structural elements are proposed to participate in the mechanism of the transporter.

Improvement of Production and Isolation of Human Neuraminidase-1 in Cellulo Crystals.

In cellulo crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by in vitro crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of in cellulo crystallization because the process occurs spontaneously. Here we report a protocol to produce and extract in cellulo crystals of human lysosomal neuraminidase-1 (NEU1) in human cultured cells. Overexpression of NEU1 protein by the retransfection of cells pretransfected with neu1-overexpressing plasmid improved the efficiency of NEU1 crystallization. Microscopic analysis revealed that NEU1 proteins were not crystallized in the lysosome but in the endoplasmic reticulum (ER). Screening of the buffer conditions used to extract crystals from cells further improved the crystal yield. The optimal pH was 7.0, which corresponds to the pH in the ER. Use of a high-yield flask with a large surface area also yielded more crystals. These optimizations enabled us to execute a serial femtosecond crystallography experiment with a sufficient number of crystals to generate a complete data set. Optimization of the in cellulo crystallization method was thus shown to be possible.