Seizo Koshiba

Phosphatidylinositol monophosphate-binding interface in the oomycete RXLR effector AVR3a is required for its stability in host cells to modulate plant immunity.

The oomycete pathogen Phytophthora infestans causes potato late blight, one of the most economically damaging plant diseases worldwide. P. infestans produces AVR3a, an essential modular virulence effector with an N-terminal RXLR domain that is required for host-cell entry. In host cells, AVR3a stabilizes and inhibits the function of the E3 ubiquitin ligase CMPG1, a key factor in host immune responses including cell death triggered by the pathogen-derived elicitor protein INF1 elicitin. To elucidate the molecular basis of AVR3a effector function, we determined the structure of Phytophthora capsici AVR3a4, a close homolog of P. infestans AVR3a. Our structural and functional analyses reveal that the effector domain of AVR3a contains a conserved, positively charged patch and that this region, rather than the RXLR domain, is required for binding to phosphatidylinositol monophosphates (PIPs) in vitro. Mutations affecting PIP binding do not abolish AVR3a recognition by the resistance protein R3a but reduce its ability to suppress INF1-triggered cell death in planta. Similarly, stabilization of CMPG1 in planta is diminished by these mutations. The steady-state levels of non–PIP-binding mutant proteins in planta are reduced greatly, although these proteins are stable in vitro. Furthermore, overexpression of a phosphatidylinositol phosphate 5-kinase results in reduction of AVR3a levels in planta. Our results suggest that the PIP-binding ability of the AVR3a effector domain is essential for its accumulation inside host cells to suppress CMPG1-dependent immunity.

Solution Structure of the SEA Domain from the Murine Homologue of Ovarian Cancer Antigen CA125 (MUC16)

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique α/β sandwich fold composed of two α helices and four antiparallel β strands and has a characteristic turn named the TY-turn between α1 and α2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the β sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the β2 and β3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting β2 and β3.

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the α‐subunit of the only translation initiation factor (eIF2α) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N‐terminus, which is termed the RWD domain after three major RWD‐containing proteins: RING finger‐containing proteins, WD‐repeat‐containing proteins, and yeast DEAD (DEXD)‐like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an α + β sandwich fold consisting of two layers: a four‐stranded antiparallel β‐sheet, and three side‐by‐side α‐helices, with an αββββαα topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive β‐turns that overlap with each other by two residues (triple β‐turn). As putative binding sites with GCN1, a structure‐based alignment allowed the identification of several surface residues in α‐helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin‐conjugating enzymes (E2s), with most of the structural differences in the region connecting β‐strand 4 and α‐helix 3. The structural architecture, including the triple β‐turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein‐binding that plays specific roles in individual RWD‐containing proteins.

Solution structure of a BolA‐like protein from Mus musculus

The BolA‐like proteins are widely conserved from prokaryotes to eukaryotes. The BolA‐like proteins seem to be involved in cell proliferation or cell‐cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA‐like protein. The overall topology is αββααβα, in which β1 and β2 are antiparallel, and β3 is parallel to β2. This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA‐like proteins are assembled on the one side of the protein.

Solution structure of the PWWP domain of the hepatoma-derived growth factor family

Among the many PWWP‐containing proteins, the largest group of homologous proteins is related to hepatoma‐derived growth factor (HDGF). Within a well‐conserved region at the extreme N‐terminus, HDGF and five HDGF‐related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin‐associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF‐related protein‐3 (HRP‐3) by NMR spectroscopy. The structure consists of a five‐stranded β‐barrel with a PWWP‐specific long loop connecting β2 and β3 (PR‐loop), followed by a helical region including two α‐helices. Its structure was found to have a characteristic solvent‐exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the β1/β2 loop (β‐β arch) and the β3/β4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

Characterization of acyl-CoA-binding protein (ACBP) in the pheromone gland of the silkworm, Bombyx mori.

Various fatty acyl-CoAs are involved as intermediates or precursors of sex pheromone components in the biosynthetic pathway of the pheromones in many lepidopteran insects. We have purified a 10-kDa protein from the cytosolic fraction of Bombyx mori pheromone glands by using affinity chromatography with a palmitoyl-CoA-agarose column and reversed-phase HPLC. Amino acid sequence analysis of the fragment peptides obtained from the purified protein, and a homology search, revealed that this protein was a member of acyl-CoA-binding proteins (ACBPs). MALDI-TOF mass spectral analysis of the purified protein and cloning of the gene from a pheromone gland cDNA library confirmed B. mori ACBP to be a 90 amino acid protein with 78.9% identity to that of Manduca sexta ACBP. The secondary structure of the recombinant B. mori ACBP was determined by NMR spectroscopy. Northern blot analysis demonstrated that B. mori ACBP was predominantly expressed in the pheromone gland and the corresponding transcript was expressed from the day before adult eclosion. Present results suggest that ACBP plays a significant role in the production of sex pheromones regulated by the neurohormone, pheromone biosynthesis activating neuropeptide (PBAN).

Solution structure of the catalytic domain of the mitochondrial protein ICT1 that is essential for cell vitality

The ICT1 protein was recently reported to be a component of the human mitoribosome and to have codon-independent peptidyl-tRNA hydrolysis activity via its conserved GGQ motif, although little is known about the detailed mechanism. Here, using NMR spectroscopy, we determined the solution structure of the catalytic domain of the mouse ICT1 protein that lacks an N-terminal mitochondrial targeting signal and an unstructured C-terminal basic-residue-rich extension, and we examined the effect of ICT1 knockdown (mediated by small interfering RNA) on mitochondria in HeLa cells using flow cytometry. The catalytic domain comprising residues 69-162 of the 206-residue full-length protein forms a structure with a β1-β2-α1-β3-α2 topology and a structural framework that resembles the structure of GGQ-containing domain 3 of class 1 release factors (RFs). Half of the structure, including the GGQ-containing loop, has essentially the same sequence and structure as those in RFs, consistent with the peptidyl-tRNA hydrolysis activity of ICT1 on the mitoribosome, which is analogous to RFs. However, the other half of the structure differs in shape from the corresponding part of RF domain 3 in that in ICT1, an α-helix (α1), instead of a β-turn, is inserted between strand β2 and strand β3. A characteristic groove formed between α1 and the three-stranded antiparallel β-sheet was identified as a putative ICT1-specific functional site by a structure-based alignment. In addition, the structured domain that recognizes stop codons in RFs is replaced in ICT1 by a C-terminal basic-residue-rich extension. It appears that these differences are linked to a specific function of ICT1 other than the translation termination mediated by RFs. Flow cytometry analysis showed that the knockdown of ICT1 results in apoptotic cell death with a decrease in mitochondrial membrane potential and mass. In addition, cytochrome c oxidase activity in ICT1 knockdown cells was decreased by 35% compared to that in control cells. These results indicate that ICT1 function is essential for cell vitality and mitochondrial function.

Solution structure of the kinase-associated domain 1 of mouse microtubule-associated protein/microtubule affinity-regulating kinase 3.

Microtubule‐associated protein/microtubule affinity‐regulating kinases (MARKs)/PAR‐1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C‐terminal domain, which is termed the kinase‐associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that ∼50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact α+β structure with a βαββββα topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu‐Leu‐Lys‐Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.

Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1 from their autoinhibited forms

DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide exchange factor, controls lymphocyte migration through ras-related C3 botulinum toxin substrate (Rac) activation. Dedicator of cytokinesis 2–engulfment and cell motility protein 1 (DOCK2•ELMO1) complex formation is required for DOCK2-mediated Rac signaling. In this study, we identified the N-terminal 177-residue fragment and the C-terminal 196-residue fragment of human DOCK2 and ELMO1, respectively, as the mutual binding regions, and solved the crystal structure of their complex at 2.1-Å resolution. The C-terminal Pro-rich tail of ELMO1 winds around the Src-homology 3 domain of DOCK2, and an intermolecular five-helix bundle is formed. Overall, the entire regions of both DOCK2 and ELMO1 assemble to create a rigid structure, which is required for the DOCK2•ELMO1 binding, as revealed by mutagenesis. Intriguingly, the DOCK2•ELMO1 interface hydrophobically buries a residue which, when mutated, reportedly relieves DOCK180 from autoinhibition. We demonstrated that the ELMO-interacting region and the DOCK-homology region 2 guanine nucleotide exchange factor domain of DOCK2 associate with each other for the autoinhibition, and that the assembly with ELMO1 weakens the interaction, relieving DOCK2 from the autoinhibition. The interactions between the N- and C-terminal regions of ELMO1 reportedly cause its autoinhibition, and binding with a DOCK protein relieves the autoinhibition for ras homolog gene family, member G binding and membrane localization. In fact, the DOCK2•ELMO1 interface also buries the ELMO1 residues required for the autoinhibition within the hydrophobic core of the helix bundle. Therefore, the present complex structure reveals the structural basis by which DOCK2 and ELMO1 mutually relieve their autoinhibition for the activation of Rac1 for lymphocyte chemotaxis.

NMR solution structures of actin depolymerizing factor homology domains

Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin‐depolymerizing factor homology (ADF‐H) domains. ADF‐H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best‐characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF‐H domain of human HIP‐55‐drebrin‐like protein, the first published structure of a drebrin‐like domain (mammalian), and the first published structure of GMF β (mouse). We also determined the structures of mouse GMF γ, the mouse coactosin‐like domain and the C‐terminal ADF‐H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F‐actin) and globular actin (G‐actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F‐actin binding sites of mouse GMF β and GMF γ contain two additional β‐strands not seen in other ADF‐H structures. The G‐actin binding site of the ADF‐H domain of human HIP‐55‐drebrin‐like protein is absent and distorted in mouse GMF β and GMF γ.