Grip and slip of L1-CAM on adhesive substrates direct growth cone haptotaxis.

Chemical cues presented on the adhesive substrate direct cell migration, a process termed haptotaxis. To migrate, cells must generate traction forces upon the substrate. However, how cells probe substrate-bound cues and generate directional forces for migration remains unclear. Here, we show that the cell adhesion molecule (CAM) L1-CAM is involved in laminin-induced haptotaxis of axonal growth cones. L1-CAM underwent grip and slip on the substrate. The ratio of the grip state was higher on laminin than on the control substrate polylysine; this was accompanied by an increase in the traction force upon laminin. Our data suggest that the directional force for laminin-induced growth cone haptotaxis is generated by the grip and slip of L1-CAM on the substrates, which occur asymmetrically under the growth cone. This mechanism is distinct from the conventional cell signaling models for directional cell migration. We further show that this mechanism is disrupted in a human patient with L1-CAM syndrome, suffering corpus callosum agenesis and corticospinal tract hypoplasia.

MT1-MMP recognition by ERM proteins and its implication in CD44 shedding.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a key enzyme involved in tumor cell invasion by shedding their cell-surface receptor CD44 anchored with F-actin through ezrin/radixin/moesin (ERM) proteins. We found the cytoplasmic tail of MT1-MMP directly binds the FERM domain of radixin, suggesting F-actin-based recruitment of MT1-MMP to CD44 for invasion. Our crystal structure shows that the central region of the MT1-MMP cytoplasmic tail binds subdomain A of the FERM domain, and makes an antiparallel ß-ß interaction with ß2A-strand. This binding mode is distinct from the previously determined binding mode of CD44 to subdomain C. We showed that radixin simultaneously binds both MT1-MMP and CD44, indicating ERM protein-mediated colocalization of MT1-MMP and its substrate CD44 and anchoring to F-actin. Our study implies that ERM proteins contribute toward accelerated CD44 shedding by MT1-MMP through ERM protein-mediated interactions between their cytoplasmic tails.

Structural basis of DDB1-and-Cullin 4-associated Factor 1 (DCAF1) recognition by merlin/NF2 and its implication in tumorigenesis by CD44-mediated inhibition of merlin suppression of DCAF1 function.

Merlin, a tumor suppressor encoded by the neurofibromatosis type 2 gene, has been shown to suppress tumorigenesis by inhibiting the Cullin 4-RING E3 ubiquitin ligase CRL4(DCAF) (1) in the nucleus. This inhibition is mediated by direct binding of merlin to DDB1-and-Cullin 4-associated Factor 1 (DCAF1), yet the binding mode of merlin to DCAF1 is not well defined. Here, we report structural and biophysical studies of the merlin binding to DCAF1 and its interference with CD44 binding. The crystal structure of the merlin FERM domain bound to the DCAF1 C-terminal acidic tail reveals that the hydrophobic IILXLN motif located at the C-terminal end of DCAF1 binds subdomain C of the FERM domain by forming a ß-strand. The binding site and mode resemble that of merlin binding to the CD44 cytoplasmic tail. Competition binding assay showed that CD44 and DCAF1 compete for binding to the merlin FERM domain in solution. The CD44 cytoplasmic tail is known to be cleaved for nuclear translocation by regulated intra-membrane proteolysis (RIP). Our structure implies that, in the nucleus, the CD44 cytoplasmic tail cleaved by RIP could release DCAF1 from merlin by competing for binding to the merlin FERM domain, which results in the inhibition of merlin-mediated suppression of tumorigenesis.

Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR.

End-binding protein 1 (EB1) is one of the best studied plus-end tracking proteins. It is known that EB1 specifically binds the plus ends of microtubules (MTs) and promotes MT growth. EB1 activity is thought to be autoinhibited by an intramolecular interaction. Recent cryo-EM analyses showed that the CH domain of Mal3p (Schizosaccharomyces pombe EB1 homolog) binds to GMPCPP-MT (Sandblad, L. Cell 127 (2006) 1415-24), and strongly binds GTPγS-MT which is proposed to mimic MT plus ends better than GMPCPP-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Here, we report on the MT binding sites of the CH domain of EB1 as revealed by NMR using the transferred cross-saturation method. In this study, we used GMPCPP-MT and found that the MT binding sites are very similar to the binding site for GTPγS-MT as suggested by cryo-EM (Maurer S.P. et al. Cell 149 (2012) 371-82). Notably, the N-terminal tip of helix α6 of the CH domain did not make contact with GMPCPP-MT, in contrast to the cryo-EM study which showed that it is closely located to a putative switch region of ß-tubulin in GTPγS-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Further, we found that the intramolecular interaction site of EB1 overlaps the MT binding sites, indicating that the MT binding sites are masked by interaction with the C-terminal domain. We propose a structural view of autoinhibition and its release mechanism through competition binding with binding partners such as adenomatous polyposis coli protein.

The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module.

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide-exchange factors that possess the PH-CC-Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH-CC-Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three-helical globular Ex subdomain. The PH subdomain resembles the beta-spectrin PH domain, suggesting non-canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2-interacting proteins for binding to PHCCEx domain, Motif-I in CD44 and ephrinB's and the NMDA receptor, and Motif-II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.

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-/ß-fold hydrolase domain forming a deep ligand-binding pocket capable of accommodating GR24. Insertion of four α-helices between ß6 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 ß6-αD1 loop, which is characteristic in D14 and D14L, but not in related α-/ß-fold hydrolases.

W27 IgA suppresses growth of Escherichia in an in vitro model of the human intestinal microbiota.

W27 monoclonal immunoglobulin A (IgA) suppresses pathogenic Escherichia coli cell growth; however, its effect on the human intestine remains unclear. We aimed to determine how W27 IgA affects the human colonic microbiota using the in vitro microbiota model. This model was established using fecal samples collected from 12 healthy volunteers; after anaerobic cultivation, each model was found to retain the genera found in the original human fecal samples. After pre-incubating W27 IgA with the respective fecal sample under aerobic conditions, the mixture of W27 IgA (final concentration, 0.5 µg/mL) and each fecal sample was added to the in vitro microbiota model and cultured under anaerobic conditions. Next-generation sequencing of the bacterial 16S rRNA gene revealed that W27 IgA significantly decreased the relative abundance of bacteria related to the genus Escherichia in the model. Additionally, at a final concentration of 5 µg/mL, W27 IgA delayed growth in the pure culture of Escherichia coli isolated from human fecal samples. Our study thus revealed the suppressive effect of W27 IgA on the genus Escherichia at relatively low-concentrations and the usefulness of an in vitro microbiota model to evaluate the effect of IgA as a gut microbiota regulator.

Crystal structure of N-terminal degron-truncated human glutamine synthetase.

Glutamine synthetase (GS) is a decameric enzyme that plays a key role in nitrogen metabolism. Acetylation of the N-terminal degron (N-degron) of GS is essential for ubiquitylation and subsequent GS degradation. The full-length GS structure showed that the N-degron is buried inside the GS decamer and is inaccessible to the acetyltransferase. The structure of N-degron-truncated GS reported here reveals that the N-degron is not essential for GS decamer formation. It is also shown that the N-degron can be exposed to a solvent region through a series of conformational adjustments upon ligand binding. In summary, this study elucidated the dynamic movement of the N-degron and the possible effect of glutamine in enhancing the acetylation process.

A GH13 α-glucosidase from Weissella cibaria uncommonly acts on short-chain maltooligosaccharides.

α-Glucosidase (EC 3.2.1.20) is a carbohydrate-hydrolyzing enzyme which generally cleaves α-1,4-glycosidic bonds of oligosaccharides and starch from the nonreducing ends. In this study, the novel α-glucosidase from Weissella cibaria BBK-1 (WcAG) was biochemically and structurally characterized. WcAG belongs to glycoside hydrolase family 13 (GH13) and to the neopullanase subfamily. It exhibits distinct hydrolytic activity towards the α-1,4 linkages of short-chain oligosaccharides from the reducing end. The enzyme prefers to hydrolyse maltotriose and acarbose, while it cannot hydrolyse cyclic oligosaccharides and polysaccharides. In addition, WcAG can cleave pullulan hydrolysates and strongly exhibits transglycosylation activity in the presence of maltose. Size-exclusion chromatography and X-ray crystal structures revealed that WcAG forms a homodimer in which the N-terminal domain of one monomer is orientated in proximity to the catalytic domain of another, creating the substrate-binding groove. Crystal structures of WcAG in complexes with maltose, maltotriose and acarbose revealed a remarkable enzyme active site with accessible +2, +1 and -1 subsites, along with an Arg-Glu gate (Arg176-Glu296) in front of the active site. The -2 and -3 subsites were blocked by Met119 and Asn120 from the N-terminal domain of a different subunit, resulting in an extremely restricted substrate preference.

Structural insights into vesicle amine transport-1 (VAT-1) as a member of the NADPH-dependent quinone oxidoreductase family.

Vesicle amine transport protein-1 (VAT-1) has been implicated in the regulation of vesicular transport, mitochondrial fusion, phospholipid transport and cell migration, and is a potential target of anticancer drugs. Little is known about the molecular function of VAT-1. The amino acid sequence indicates that VAT-1 belongs to the quinone oxidoreductase subfamily, suggesting that VAT-1 may possess enzymatic activity in unknown redox processes. To clarify the molecular function of VAT-1, we determined the three-dimensional structure of human VAT-1 in the free state at 2.3 Å resolution and found that VAT-1 forms a dimer with the conserved NADPH-binding cleft on each protomer. We also determined the structure of VAT-1 in the NADP-bound state at 2.6 Å resolution and found that NADP binds the binding cleft to create a putative active site with the nicotine ring. Substrate screening suggested that VAT-1 possesses oxidoreductase activity against quinones such as 1,2-naphthoquinone and 9,10-phenanthrenequinone.

Asymmetric Open-Closed Dimer Mechanism of Polyhydroxyalkanoate Synthase PhaC.

Biodegradable polyester polyhydroxyalkanoate (PHA) is a promising bioplastic material for industrial use as a replacement for petroleum-based plastics. PHA synthase PhaC forms an active dimer to polymerize acyl moieties from the substrate acyl-coenzyme A (CoA) into PHA polymers. Here we present the crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, bound to CoA. The structure reveals an asymmetric dimer, in which one protomer adopts an open conformation bound to CoA, whereas the other adopts a closed conformation in a CoA-free form. The open conformation is stabilized by the asymmetric dimerization and enables PhaC to accommodate CoA and also to create the product egress path. The bound CoA molecule has its ß-mercaptoethanolamine moiety extended into the active site with the terminal SH group close to active center Cys291, enabling formation of the reaction intermediate by acylation of Cys291.

Crystal structure of a YeeE/YedE family protein engaged in thiosulfate uptake.

We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Structural basis of thalidomide enantiomer binding to cereblon.

Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. However, the precise mechanism by which the two isomers exert their different activities remains poorly understood. Here, we present structural and biochemical studies of (S)- and (R)-enantiomers bound to the primary target of thalidomide, cereblon (CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress optical isomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bind the tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish compared to the (R)-enantiomer. This study has established a mechanism by which thalidomide exerts its effects in a stereospecific manner at the atomic level.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44.

CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell-cell and cell-matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane-cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 A, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 A.

Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics.

Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaC Cs -CAT. The structure shows that PhaC Cs -CAT forms an α/ß hydrolase fold comprising α/ß core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaC Cn -CAT). Structural comparison showed PhaC Cn -CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaC Cs -CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaC Cn -CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.

Molecular Characterization of Viral Responsive Protein 15 and Its Possible Role in Nuclear Export of Virus in Black Tiger Shrimp Penaeus monodon.

A viral responsive protein 15 from Penaeus monodon (PmVRP15) has been reported to be important for white spot syndrome virus (WSSV) infection in vivo. This work aims to characterize PmVRP15 and investigate its possible role in nuclear import/export of the virus. Circular dichroism spectra showed that PmVRP15 contains high helical contents (82%). Analytical ultracentrifugation suggested that PmVRP15 could possibly form oligomers in solution. A subcellular fractionation study showed that PmVRP15 was found in heavy and light membrane fractions, indicating that PmVRP15 may be associated with endoplasmic reticulum. Double-stranded RNAi-mediated knockdown of PmVRP15 gene expression in vitro showed no effect on WSSV copy number in whole hemocyte cells. However, PmVRP15 silencing resulted in an accumulation of WSSV DNA in the nucleus of PmVRP15-silenced hemocytes. Immunofluorescence confocal microscopy showed that PmVRP15 knockdown hemocytes had a much lower level of VP28 (WSSV envelope protein), in comparison to that in the control. It is likely that PmVRP15 may play a role in viral nuclear egress.

Contribution of indirect action to radiation-induced mammalian cell inactivation: dependence on photon energy and heavy-ion LET.

The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/microm to 440 keV/microm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/microm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.

Structural basis for autoinhibition and its relief of MOB1 in the Hippo pathway.

MOB1 protein is a key regulator of large tumor suppressor 1/2 (LATS1/2) kinases in the Hippo pathway. MOB1 is present in an autoinhibited form and is activated by MST1/2-mediated phosphorylation, although the precise mechanisms responsible for autoinhibition and activation are unknown due to lack of an autoinhibited MOB1 structure. Here, we report on the crystal structure of full-length MOB1B in the autoinhibited form and a complex between the MOB1B core domain and the N-terminal regulation (NTR) domain of LATS1. The structure of full-length MOB1B shows that the N-terminal extension forms a short ß-strand, the SN strand, followed by a long conformationally flexible positively-charged linker and α-helix, the Switch helix, which blocks the LATS1 binding surface of MOB1B. The Switch helix is stabilized by ß-sheet formation of the SN strand with the S2 strand of the MOB1 core domain. Phosphorylation of Thr12 and Thr35 residues structurally accelerates dissociation of the Switch helix from the LATS1-binding surface by the "pull-the-string" mechanism, thereby enabling LATS1 binding.

Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs.

The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.