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

がんの多剤排出の原因となっているABCトランスポーターの立体構造をSACLAのX線自由電子レーザーを用いて決定. 京都大学プレスリリース. 2021-12-23.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 mono­acyl­glycerol 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 hy­droxy­propyl methyl­cellulose 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 Å is 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

An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography.

SACLAのX線自由電子レーザーを用いた新規タンパク質立体構造決定に世界で初めて成功. 京都大学プレスリリース. 2015-09-14.Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing. Only about 20,000 patterns in total were needed for SIRAS phasing while single wavelength anomalous dispersion (SAD) phasing was unsuccessful with more than 80,000 patterns of derivative crystals. We employed high energy X-rays from SACLA (12.6 keV) to take advantage of the large anomalous enhancement near the LIII absorption edge of Hg, which is one of the most widely used heavy atoms for phasing in conventional protein crystallography. Hard XFEL is of benefit for de novo phasing in the use of routinely used heavy atoms and high resolution data collection

大腸菌K-12株由来アスパラギン合成酵素の構造学的研究

京都大学0048新制・課程博士博士(農学)甲第6909号農博第927号新制||農||740(附属図書館)学位論文||H9||N3033(農学部図書室)UT51-97-H293京都大学大学院農学研究科農芸化学専攻(主査)教授 小田 順一, 教授 林 力丸, 教授 江崎 信芳学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA

Crystal Structure of Asparagine Synthetase Reveals a Close Evolutionary Relationship to Class II Aminoacyl-tRNA Synthetase (MOLECULAR BIOFUNCTION-Functional Molecular Conversion)

The crystal structure of E. coli asparagine synthetase has been determined by X-ray diffraction analysis at 2.5 A resolution. The overall structure of the enzyme is remarkably similar to that of the catalytic domain of yeast aspartyl-tRNA synthetase despite low sequence similarity. These enzymes have a common reaction mechanism that implies the formation of aminoacyl-adenylate intermediate. The active site architecture and most of the catalytic residues are also conserved in both enzymes. These enzymes have probably evolved from a common ancestor even though their sequence similarities are small

Potent Transition-State Analogue Inhibitor of Esherichia coli Asparagine Synthetase A (MOLECULAR BIOFUNCTION-Functional Molecular Conversion)

A potent and slow-binding inhibitor of E. coli asparagine synthetase A (AS-A) was synthesized, and its inhibition behavior was characterized. The enzyme complexed with the inhibitor was analyzed by X-ray diffraction analysis to identify several key amino acid residues responsible for catalysis as well as for substrate recognition. AS-A catalyzes the formation of L-Asn from L-Asp and ammonia coupled with the hydrolysis of ATP to AMP and pyrophosphate. The reaction catalyzed by this enzyme is prototypic of mammalian asparagine synthetase (AS-B) which utilizes glutamine as a nitrogen source. In addition, asparagine synthetase is a potential target for chemotherapy to treat certain leukemias. We therefore designed and synthesized a transition-state analogue, N-adenylated S-methyl-L-cysteine sulfoximine 1, based on the proposed reaction mechanisms of AS-A. The compound 1 strongly inhibited the E. coli AS-A in a time-dependent manner with an overall inhibition constant (Ki*) of 67 nM and with an onset rate of inactivation of 3.27 s-1 mM-1. The inhibition was almost irreversible and no regain of enzyme activity was observed in 10 days after gel filtration. The inhibitor 1 was also used as a ligand for X-ray diffraction analysis of AS-A. The X-ray crystal structure of AS-A complexed with 1 revealed several key amino acid residues such as Arg 100, Gln 116 and Asp 46 responsible for catalysis as well as those for substrate recognition. An attempt to inhibit AS-A by each diastereomer of S-methyl-L-cysteine sulfoximine and ATP is also described. Since AS-A is prototypic of asparagine synthetases in terms of the chemistry in substrate activation, compound 1 should formulate a basis for future inhibitor design of asparagine synthetase B

Crystal structures of two tropinone reductases: Different reaction stereospecificities in the same protein fold. (MOLECULAR BIOFUNCTION-Functional Molecular Conversion)

A pair of tropinone reductases (TRs) share 64% identical amino acid residues, and belong to the shortchain dehydrogenase/reductase family. In the synthesis of tropane alkaloids in several medicinal plants, the TRs reduce a carbonyl group of an alkaloid intermediate, tropinone, to hydroxy groups having different diastereomeric configurations. To clarify the structural basis for their different reaction stereospecificities, we determined the crystal structures of the two enzymes at 2.4- and 2.3-A resolutions. The overall folding of the two enzymes was almost identical. The substrate binding site was composed mostly of hydrophobic amino acids in both TRs, but the presence of different charged residues conferred different electrostatic environments on the two enzymes