Atsushi Kodan

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

Significance P-glycoprotein exports various hydrophobic chemicals in an ATP-dependent manner, determines their absorption and distribution in the body, and is involved in multidrug resistance (MDR) in tumors. Understanding the mechanism of the multidrug transport is important for designing drugs of good bioavailability and efficient cancer chemotherapy. We determined the high-resolution crystal structures of a eukaryotic P-glycoprotein homolog and revealed the detailed architecture of its transmembrane domains, which contain an exit gate for substrates that opens to the extracellular side and two entrance gates that open to the intramembranous region and the cytosolic side. We propose a motion of the transmembrane domains powered by the association of two nucleotide-binding domains on ATP binding that is different from other transporters. 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.

A stilbene synthase from Japanese red pine (Pinus densiflora): implications for phytoalexin accumulation and down-regulation of flavonoid biosynthesis.

Stilbene synthase (STS) and chalcone synthase (CHS) are plant-specific polyketide synthases that play key roles in the stilbenoid and flavonoid biosyntheses, respectively. We have recently isolated from Pinus densiflora three STS cDNAs (PDSTS1, PDSTS2, and PDSTS3) and one CHS cDNA (PDCHSX). We then heterologously expressed these cDNAs in Escherichia coli and characterized their properties. An unusual STS isozyme, PDSTS3, lacks the common C-terminal extension of STS because of a frame-shift mutation and shows the highest pinosylvin-forming activity among the STSs tested. Pinosylvin was shown to be a potent inhibitor of PDCHSX (Ki = 6 μM) as well as PDSTS2 (Ki = 13 μM), which presumably maintains the balance between the stilbenoid and flavonoid biosyntheses. PDSTS3 was insensitive to product inhibition. We identified PDSTS3 in the pine seedlings as well as full-length STS. The data provide evidence that PDSTS3 is involved in the potential regulation of the stilbenoid and flavonoid biosynthetic pathways in pine trees.

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

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.

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 ∼ 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 kcat values both with substrate (ksub) and without substrate (kbasal), but decreased the ksub/kbasal 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 [ksub/Km(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.

Improved expression and purification of human multidrug resistance protein MDR1 from baculovirus-infected insect cells.

Multidrug resistance protein MDR1 (P-glycoprotein/ABCB1) is an ATP-dependent efflux pump for various cytotoxic agents, and is implicated in the resistance of human tumors to chemotherapeutic drugs. To achieve the three-dimensional structural analysis for its mechanistic implications, large amounts of high-quality and homogeneous MDR1 protein are essential. Here we report a cost-effective method for large-scale expression of human MDR1 using a baculovirus/insect expressSF+ cell system and an alterative purification method to maintain MDR1 in a monodispersed state. After extensively optimizing the detergent, pH, and additives, a high yield (2.8 mg/L) of purified MDR1 was obtained by immobilized metal chelate affinity and size-exclusion chromatographies with 49% recovery. The purified MDR1 exhibited specific ATP hydrolase activity (1.7 micromol/min/mg) in the presence of a substrate, verapamil. This value was 14-fold greater than the basal activity without the drug. Size-exclusion chromatography analysis of purified MDR1 showed a monodispersed elution profile. The present purification method provides suitable material for structural and functional studies on human MDR1.

Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography

High-energy X-rays are essential for de novo structure determination with strong anomalous scattering from selenium or mercury. Single-wavelength anomalous diffraction phasing using selenomethionine-derivatization and mercury-soaking techniques has been successfully applied to serial femtosecond crystallography with 13.0 keV or 12.6 keV X-rays produced at SACLA.