The phytohormone abscisic acid (ABA) mediates the adaptation of plants to environmental stresses such as drought and regulates developmental signals such as seed maturation. Within plants, the PYR/PYL/RCAR family of START proteins receives ABA to inhibit the phosphatase activity of the group-A protein phosphatases 2C (PP2Cs), which are major negative regulators in ABA signalling. Here we present the crystal structures of the ABA receptor PYL1 bound with (+)-ABA, and the complex formed by the further binding of (+)-ABA-bound PYL1 with the PP2C protein ABI1. PYL1 binds (+)-ABA using the START-protein-specific ligand-binding site, thereby forming a hydrophobic pocket on the surface of the closed lid. (+)-ABA-bound PYL1 tightly interacts with a PP2C domain of ABI1 by using the hydrophobic pocket to cover the active site of ABI1 like a plug. Our results reveal the structural basis of the mechanism of (+)-ABA-dependent inhibition of ABI1 by PYL1 in ABA signalling.

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Escherichia coli is a widely used host organism for recombinant technology, and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.ΔA grew as vigorously as BL21(DE3) in rich medium at 25–42°C, and its derivative B-95.ΔAΔfabR was better adapted to low temperatures and minimal media than B-95.ΔA. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.ΔA strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.

SummaryThe involvement of kinesin motor proteins in both cell-tip growth and cell-shape determination has been well characterized in various organisms. However, the functions of kinesins during cell morphogenesis in higher plants remain largely unknown. In the current study, we demonstrate that an armadillo repeat-containing kinesin-related protein, ARMADILLO REPEAT KINESIN1 (ARK1), is involved in root-hair morphogenesis. Microtubule polymers are more abundant in ark1 null allele root hairs, but analysis shows that these extra microtubules are concentrated in the endoplasm, and not in the cortical array, suggesting that ARK1 regulates tip growth by limiting the assembly and distribution of endoplasmic microtubules. The ARK1 gene has two homologues in the Arabidopsis genome, ARK2 and ARK3, and our results show that ARK2 is involved in rootcell morphogenesis. We further reveal that a NIMA-related protein kinase, NEK6, binds to the ARK family proteins and has pleiotropic effects on epidermal-cell morphogenesis, suggesting that NEK6 is involved in cell morphogenesis in Arabidopsis via microtubule functions associated with these armadillo repeat-containing kinesins. We discuss the function of NIMA-related protein kinases and armadillo repeat-containing kinesins in the cell morphogenesis of eukaryotes.

Ca 2؉ -ATPase of skeletal muscle sarcoplasmic reticulum is an ATPdriven Ca 2؉ pump consisting of three cytoplasmic domains and 10 transmembrane helices. In the absence of Ca 2؉ , the three cytoplasmic domains gather to form a compact headpiece, but the ATPase is unstable without an inhibitor. Here we describe the crystal structures of Ca 2؉ -ATPase in the absence of Ca 2؉ stabilized with cyclopiazonic acid alone and in combination with other inhibitors. Cyclopiazonic acid is located in the transmembrane region of the protein near the cytoplasmic surface. The binding site partially overlaps with that of 2,5-di-tert-butyl-1,4-dihydroxybenzene but is separate from that of thapsigargin. The overall structure is significantly different from that stabilized with thapsigargin: The cytoplasmic headpiece is more upright, and the transmembrane helices M1-M4 are rearranged. Cyclopiazonic acid primarily alters the position of the M1 helix and thereby M2 and M4 and then M5. Because M5 is integrated into the phosphorylation domain, the whole cytoplasmic headpiece moves. These structural changes show how an event in the transmembrane domain can be transmitted to the cytoplasmic domain despite flexible links between them. They also reveal that Ca 2؉ -ATPase has considerable plasticity even when fixed by a transmembrane inhibitor, presumably to accommodate thermal fluctuations.Ca 2ϩ -ATPase ͉ ion pump ͉ membrane protein C a 2ϩ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) is structurally and functionally the best studied member of the P-type ion-translocating ATPases (1, 2). Located in the sarcoplasmic reticulum membrane, SERCA1 pumps Ca 2ϩ released into muscle cells during contraction and thereby causes relaxation. The reaction cycle consists of at least eight intermediates, and crystal structures have been determined for six of them (3-9). These crystal structures show that SERCA1 consists of three cytoplasmic domains (A, actuator; N, nucleotide binding; and P, phosphorylation) and 10 (M1-M10) transmembrane helices (3). They also demonstrate that ATPase undergoes large and global conformational changes during the reaction cycle. In conventional E1/E2 theory (10-12), active transport of Ca 2ϩ is achieved by alternating the affinity and accessibility of the transmembrane Ca 2ϩ -binding sites. That is, in E1, the binding sites have high affinity and face the cytoplasm; in E2, the binding sites have low affinity to Ca 2ϩ and face the lumen of the sarcoplasmic reticulum. Although H ϩ and water, instead of Ca 2ϩ , stabilize the binding sites in E2 (13), this form of the ATPase is unstable, particularly when solubilized (14). Therefore, so far all successful crystallization experiments in the absence of Ca 2ϩ used inhibitors that stabilize the transmembrane domain.Several inhibitors specific to SERCA1 are available (15-17). By far the best known and most potent inhibitor is thapsigargin (TG), a plant sesquiterpene lactone (18), with the dissociation constant in the subnanomolar range (15). Another commonly used inhibito...