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.

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.

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.

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.

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.

X-ray crystallographic study of an HIV-1 fusion inhibitor with the gp41 S138A substitution.

The S138A substitution of fusion inhibitory peptides derived from the C-terminal heptad repeat (C-HR) of the human immunodeficiency virus type 1 (HIV-1) gp41 leads to enhanced binding affinity to the N-terminal heptad repeat (N-HR). As such, these peptides exhibit highly potent anti-HIV-1 activity. X-ray crystallographic analysis was performed to understand the effect of the substitution on binding affinity. The comparison of the native and S138A crystal structures indicated that the increase in the hydrophobicity of the S138A substitution may aid the stabilization of the N-HR/C-HR complex through additional hydrophobic contacts. Free-energy calculations suggest that the difference between the desolvation free energies of the C-HR-derived peptides with and without the S138A mutation dominates the observed difference in anti-HIV-1 activity.

Functional role of the linker region in purified human P-glycoprotein.

Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a 'linker region' of approximately 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size-exclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleavage increased the k(cat) values both with substrate (k(sub)) and without substrate (k(basal)), but decreased the k(sub)/k(basal) values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity constants [k(sub)/K(m(D))] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp.

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.

Crystal structure of gamma-glutamylcysteine synthetase: insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis.

Gamma-glutamylcysteine synthetase (gammaGCS), a rate-limiting enzyme in glutathione biosynthesis, plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer. We have determined the crystal structures of Escherichia coli gammaGCS unliganded and complexed with a sulfoximine-based transition-state analog inhibitor at resolutions of 2.5 and 2.1 A, respectively. In the crystal structure of the complex, the bound inhibitor is phosphorylated at the sulfoximido nitrogen and is coordinated to three Mg2+ ions. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure, an open space exists around the representative cysteine-binding site and is probably responsible for the competitive binding of glutathione. Upon inhibitor binding, the side chains of Tyr-241 and Tyr-300 turn, forming a hydrogen-bonding triad with the carboxyl group of the inhibitor's cysteine moiety, allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket. This movement is caused by a conformational change of a switch loop (residues 240-249). Based on this crystal structure, the cysteine-binding sites of mammalian and parasitic gammaGCSs were predicted by multiple sequence alignment, although no significant sequence identity exists between the E. coli gammaGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel gammaGCS inhibitors.

Crystal structure of the PsbP protein of photosystem II from Nicotiana tabacum.

PsbP is a membrane-extrinsic subunit of the water-oxidizing complex photosystem II (PS II). The evolutionary origin of PsbP has long been a mystery because it specifically exists in higher plants and green algae but not in cyanobacteria. We report here the crystal structure of PsbP from Nicotiana tabacum at a resolution of 1.6 A. Its structure is mainly composed of beta-sheet, and is not similar to any structures in cyanobacterial PS II. However, the electrostatic surface potential of PsbP is similar to that of cyanobacterial PsbV (cyt c(550)), which has a function similar to PsbP. A structural homology search with the DALI algorithm indicated that the folding of PsbP is very similar to that of Mog1p, a regulatory protein for the nuclear transport of Ran GTPase. The structure of PsbP provides insight into its novel function in GTP-regulated metabolism in PS II.

Crystallization and preliminary crystallographic studies on the extrinsic 23 kDa protein in the oxygen-evolving complex of photosystem II.

The extrinsic 23 kDa protein in the oxygen-evolving complex (OEC23 or PsbP) of photosystem II (PS II) helps to retain calcium and chloride ions, which are essential cofactors for the photosynthetic evolution of oxygen. OEC23 is found in the PS II complex from higher plants and green algae, but not in that from cyanobacteria or other ancestral photosynthetic organisms. In order to elucidate the three-dimensional structure and its molecular evolution, OEC23 was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as a precipitant. Diffraction data to a resolution of 2.0 A were collected from a native crystal belonging to space group P2(1)2(1)2, with unit-cell parameters a = 91.36, b = 74.15, c = 52.16 A.

Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin.

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS.

Escherichia coli B gamma-glutamylcysteine synthetase: modification, purification, crystallization and preliminary crystallographic analysis.

Escherichia coli B gamma-glutamylcysteine synthetase (gammaGCS) catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form the glutathione precursor gamma-L-Glu-Cys and is a target for development of potential therapeutic agents. By introducing four point mutations of surface-exposed cysteine residues to serine, the gammaGCS was purified to homogeneity; single crystals have been obtained using the hanging-drop vapour-diffusion method with sodium formate. The gammaGCS crystal diffracted to 2.8 A and belongs to space group R3, with unit-cell parameters a = b = 326.7, c = 103.9 A.