Toshio Hakoshima

Gibberellin-induced DELLA recognition by the gibberellin receptor GID1

Gibberellins control a range of growth and developmental processes in higher plants and have been widely used in the agricultural industry. By binding to a nuclear receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins, including GIBBERELLIN INSENSITIVE (GAI). The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. Here we present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the amino-terminal DELLA domain of GAI. In this complex, GID1A occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor that is distinct from the mechanism of the hormone perception and effector recognition of the known auxin receptors.

Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain

Radixin is a member of the ezrin/radixin/moesin (ERM) family of proteins, which play a role in the formation of the membrane‐associated cytoskeleton by linking actin filaments and adhesion proteins. This cross‐linking activity is regulated by phosphoinositides such as phosphatidylinositol 4,5‐bisphosphate (PIP2) in the downstream of the small G protein Rho. The X‐ray crystal structures of the radixin FERM domain, which is responsible for membrane binding, and its complex with inositol‐(1,4,5)‐trisphosphate (IP3) have been determined. The domain consists of three subdomains featuring a ubiquitin‐like fold, a four‐helix bundle and a phosphotyrosine‐binding‐like domain, respectively. These subdomains are organized by intimate interdomain interactions to form characteristic grooves and clefts. One such groove is negatively charged and so is thought to interact with basic juxta‐membrane regions of adhesion proteins. IP3 binds a basic cleft that is distinct from those of pleckstrin homology domains and is located on a positively charged flat molecular surface, suggesting an electrostatic mechanism of plasma membrane targeting. Based on the structural changes associated with IP3 binding, a possible unmasking mechanism of ERM proteins by PIP2 is proposed.

Crystal structure of an IRF‐DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences

There has been growing interest in the role of the IRF (interferon regulatory factor) family of transcription factors in the regulation of immune responses, cytokine signaling, and oncogenesis. These members are characterized by their well‐conserved DNA binding domains at the N‐terminal regions. Here we report the 2.2 Å resolution crystal structure of the DNA binding domain of one such family member, IRF‐2, bound to DNA. The structure reveals its recognition sequence, AANNGAAA (here, recognized bases are underlined and in bold, and N indicates any base), and its cooperative binding to a tandem repeat of the GAAA core sequence induced by DNA structure distortions. These facts explain well the diverse binding properties of the IRF family members, which bind to both single and tandemly repeated sequences. Furthermore, we also identified the ‘helix‐hairpin‐strand motif’ at the C terminus of the recognition helix as a metal binding site that is commonly found in certain classes of DNA‐interactive proteins. Our results provide new insights into the structure and function of this family of transcription factors.

Insights into multistep phosphorelay from the crystal structure of the C-terminal HPt domain of ArcB.

The histidine-containing phosphotransfer (HPt) domain is a novel protein module with an active histidine residue that mediates phosphotransfer reactions in the two-component signaling systems. A multistep phosphorelay involving the HPt domain has been suggested for these signaling pathways. The crystal structure of the HPt domain of the anaerobic sensor kinase ArcB has been determined at 2.06 A resolution. The domain consists of six alpha helices containing a four-helix bundle-folding. The pattern of sequence similarity of the HPt domains of ArcB and components in other signaling systems can be interpreted in light of the three-dimensional structure and supports the conclusion that the HPt domains have a common structural motif both in prokaryotes and eukaryotes.

CRYSTAL STRUCTURE OF PHO4 BHLH DOMAIN-DNA COMPLEX : FLANKING BASE RECOGNITION

The crystal structure of a DNA‐binding domain of PHO4 complexed with DNA at 2.8 Å resolution revealed that the domain folds into a basic–helix–loop–helix (bHLH) motif with a long but compact loop that contains a short α‐helical segment. This helical structure positions a tryptophan residue into an aromatic cluster so as to make the loop compact. PHO4 binds to DNA as a homodimer with direct reading of both the core E‐box sequence CACGTG and its 3′‐flanking bases. The 3′‐flanking bases GG are recognized by Arg2 and His5. The residues involved in the E‐box recognition are His5, Glu9 and Arg13, as already reported for bHLH/Zip proteins MAX and USF, and are different from those recognized by bHLH proteins MyoD and E47, although PHO4 is a bHLH protein.

Structural basis of adhesion-molecule recognition by ERM proteins revealed by the crystal structure of the radixin-ICAM-2 complex

ERM (ezrin/radixin/moesin) proteins recognize the cytoplasmic domains of adhesion molecules in the formation of the membrane‐associated cytoskeleton. Here we report the crystal structure of the radixin FERM (4.1 and ERM) domain complexed with the ICAM‐2 cytoplasmic peptide. The non‐polar region of the ICAM‐2 peptide contains the RxxTYxVxxA sequence motif to form a β‐strand followed by a short 310‐helix. It binds the groove of the phosphotyrosine‐binding (PTB)‐like subdomain C mediated by a β—β association and several side‐chain interactions. The binding mode of the ICAM‐2 peptide to the FERM domain is distinct from that of the NPxY motif‐containing peptide binding to the canonical PTB domain. Mutation analyses based on the crystal structure reveal the determinant elements of recognition and provide the first insights into the physical link between adhesion molecules and ERM proteins.

Structural basis for the diversity of DNA recognition by bZIP transcription factors.

The basic region leucine zipper (bZIP) proteins form one of the largest families of transcription factors in eukaryotic cells. Despite relatively high homology between the amino acid sequences of the bZIP motifs, these proteins recognize diverse DNA sequences. Here we report the 2.0 Å resolution crystal structure of the bZIP motif of one such transcription factor, PAP1, a fission yeast AP-1-like transcription factor that binds DNA containing the novel consensus sequence TTACGTAA. The structure reveals how the Pap1-specific residues of the bZIP basic region recognize the target sequence and shows that the side chain of the invariant Asn in the bZIP motif adopts an alternative conformation in Pap1. This conformation, which is stabilized by a Pap1-specific residue and its associated water molecule, recognizes a different base in the target sequence from that in other bZIP subfamilies.

Structures of D14 and D14L in the strigolactone and karrikin signaling pathways

Strigolactones (SLs) are plant hormones that inhibit shoot branching. DWARF14 (D14) inhibits rice tillering and is an SL receptor candidate in the branching inhibition pathway, whereas the close homologue DWARF14‐LIKE (D14L) participates in the signaling pathway of karrikins (KARs), which are derived from burnt vegetation as smoke stimulants of seed germination. We provide the first evidence for direct binding of the bioactive SL analogue GR24 to D14. Isothermal titration calorimetry measurements show a D14–GR24 binding affinity in the sub‐micromolar range. Similarly, bioactive KAR1 directly binds D14L in the micromolar range. The crystal structure of rice D14 shows a compact α‐/β‐fold hydrolase domain forming a deep ligand‐binding pocket capable of accommodating GR24. Insertion of four α‐helices between β6 strand and αD helix forms the helical cap of the pocket, although the pocket is open to the solvent. The pocket contains the conserved catalytic triad Ser‐His‐Asp aligned with the oxyanion hole, suggesting hydrolase activity. Although these structural characteristics are conserved in D14L, the D14L pocket is smaller than that of D14. The KAR‐insensitive mutation kai2‐1 is located at the prominent long β6‐αD1 loop, which is characteristic in D14 and D14L, but not in related α‐/β‐fold hydrolases.

AT base pairs are less stable than GC base pairs in Z-DNA: The crystal structure of d(m5CGTAm5CG)

Two hexanucleoside pentaphosphates , 5-methyl and 5-bromo cytosine derivatives of d( CpGpTpApCpG ) have been synthesized, crystallized, and their three-dimensional structure solved. They both form left-handed Z-DNA and the methylated derivative has been refined to 1.2 A resolution. These are the first crystal Z-DNA structures that contain AT base pairs. The overall form of the molecule is very similar to that of the unmethylated or the fully methylated (dC-dG)3 hexamer although there are slight changes in base stacking. However, significant differences are found in the hydration of the helical groove. When GC base pairs are present, the helical groove is systematically filled with two water molecules per base pair hydrogen bonded to the bases. Both of these water molecules are not seen in the electron density map in the segments of the helix containing AT base pairs, probably because of solvent disorder. This could be one of the features that makes AT base pairs form Z-DNA less readily than GC base pairs.

The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of PKN/PRK1.

The small G protein Rho has emerged as a key regulator of cellular events involving cytoskeletal reorganization. Here we report the 2.2 A crystal structure of RhoA bound to an effector domain of protein kinase PKN/PRK1. The structure reveals the antiparallel coiled-coil finger (ACC finger) fold of the effector domain that binds to the Rho specificity-determining regions containing switch I, beta strands B2 and B3, and the C-terminal alpha helix A5, predominantly by specific hydrogen bonds. The ACC finger fold is distinct from those for other small G proteins and provides evidence for the diverse ways of effector recognition. Sequence analysis based on the structure suggests that the ACC finger fold is widespread in Rho effector proteins.