Thalidomide and its derivatives exert not only therapeutic effects as immunomodulatory drugs (IMiDs) but also adverse effects such as teratogenicity, which are due in part to different C2H2 zinc-finger (ZF) transcription factors, IKZF1 (or IKZF3) and SALL4, respectively. Here, we report the structural bases for the SALL4-specific proteasomal degradation induced by 5-hydroxythalidomide, a primary thalidomide metabolite generated by the enzymatic activity of cytochrome P450 isozymes, through the interaction with cereblon (CRBN). The crystal structure of the metabolite-mediated human SALL4-CRBN complex and mutagenesis studies elucidate the complex formation enhanced by the interaction between CRBN and an additional hydroxy group of (S)-5-hydroxythalidomide and the variation in the second residue of β-hairpin structure that underlies the C2H2 ZF-type neo-morphic substrate (neosubstrate) selectivity of 5-hydroxythalidomide. These findings deepen our understanding of the pharmaceutical action of IMiDs and provide structural evidence that the glue-type E3 ligase modulators cause altered neosubstrate specificities through their metabolism.

FlAlyA is an endolytic enzyme with a preference for polymannuronate. The crystal structure and mutagenesis studies elucidated that the structural variations at outer uronate-binding subsites +2, +3 and -2 control the enzymatic properties of PL-7 family enzymes. Lys158 mutations changed the pH dependency and enhanced the production of mono-and disaccharides.

Alginate is an abundant algal polysaccharide, composed of beta-D-mannuronate and its C5 epimer alpha-L-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate lyase (EC 4.2.2.3) from Aplysia kurodai, AkAly30, is a eukaryotic member of the polysaccharide lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a beta-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 angstrom resolution and the putative substrate-binding model show that the enzyme adopts a beta-jelly roll fold at the core of the structure and that Lys-99, Tyr-140, and Tyr-142 form catalytic residues in the active site. Their arrangements allow the carboxyl group of mannuronate residues at subsite +1 to form ionic bonds with Lys-99. The coupled tyrosine forms a hydrogen bond network with the glycosidic bond, and the hydroxy group of Tyr-140 is located near the C5 atom of the mannuronate residue. These interactions could promote the beta-elimination of the mannuronate residue at subsite +1. More interestingly, Gly-118 and the disulfide bond formed by Cys-115 and Cys-124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite -1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly-118 or the Cys-115-Cys-124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate recognition mode explaining the polymannuronate specificity of PL-14 alginate lyases.

Human leukocyte cell-derived chemotaxin 2 (LECT2), which is predominantly expressed in the liver, is a multifunctional protein. LECT2 is becoming a potential therapeutic target for several diseases of worldwide concern such as rheumatoid arthritis, hepatocellular carcinoma, and obesity. Here, we present the crystal structure of LECT2, the first mammalian protein whose structure contains an M23 metalloendopeptidase fold. The LECT2 structure adopts a conserved Zn(II) coordination configuration but lacks a proposed catalytic histidine residue, and its potential substrate-binding groove is blocked in the vicinity of the Zn(II)-binding site by an additional intrachain loop at the N terminus. Consistent with these structural features, LECT2 was found to be catalytically inactive as a metalloendopeptidase against various types of peptide sequences, including pentaglycine. In addition, a surface plasmon resonance analysis demonstrated that LECT2 bound to the c-Met receptor with micromolar affinity. These results indicate that LECT2 likely plays its critical roles by acting as a ligand for the corresponding protein receptors rather than as an enzymatically active peptidase. The intrachain loop together with the pseudo-active site groove in LECT2 structure may be specific for interactions between LECT2 and receptors. Our study reveals a mechanistic basis for the functional evolution of a mammalian protein with an M23 metalloendopeptidase fold and potentially broadens the implications for the biological importance of noncatalytic peptidases in the M23 family.

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca2+-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2.

Strigolactones (SLs) are phytohormones that inhibit shoot branching and function in the rhizospheric communication with symbiotic fungi and parasitic weeds. An α/β-hydrolase protein, DWARF14 (D14), has been recognized to be an essential component of plant SL signalling, although its precise function remains unknown. Here we present the SL-dependent interaction of D14 with a gibberellin signalling repressor SLR1 and a possible mechanism of phytohormone perception in D14-mediated SL signalling. D14 functions as a cleavage enzyme of SLs, and the cleavage reaction induces the interaction with SLR1. The crystal structure of D14 shows that 5-hydroxy-3-methylbutenolide (D-OH), which is a reaction product of SLs, is trapped in the catalytic cavity of D14 to form an altered surface. The D14 residues recognizing D-OH are critical for the SL-dependent D14−SLR1 interaction. These results provide new insight into crosstalk between gibberellin and SL signalling pathways. Both strigolactone and DELLA plant signalling pathways have a role in shoot branching. In this study, Nakamura et al.show that DWARF14 cleaves strigolactones creating a binding surface for the DELLA protein SLR1, thereby providing a mechanism for pathway crosstalk.

Flavin reductase HpaCSt catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaCSt, which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaCSt was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS–PAGE. The purified refolded HpaCSt did not contain FMN, unlike the same enzyme expressed as a soluble protein. After the addition of FMN to the protein solution, the refolded enzyme showed a higher activity than the enzyme expressed as the soluble protein. Crystals of the refolded enzyme were obtained by adding FMN, FAD, or riboflavin to the protein solution and without the addition of flavin compound.

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.

TTHA0303 (TT2238) is a 19-kDa neutral protein (number of amino acids = 169, theoretical pI = 6.99) from an extremely thermophilic Gram-negative eubacterium Thermus thermophilus HB8, which is capable of growing at 85°C. TTHA0303-like proteins are found only in several bacterial genera such as Thermus, Deinococcus, Geobacillus, and Bacillus. The crystal structure of YfiT (17% sequence identical to TTHA0303, PDB: 1RXQ)1 is the only structure reported so far for TTHA0303-like proteins. Here, we report the 2.0-Å crystal structure of TTHA0303. TTHA0303 and YfiT commonly have a four-helix bundle fold with an unusual down-up-up-down topology, and a characteristic histidine triad (HT) on the molecular surface. We have revealed some unique features of TTHA0303 (1) it has a cleavable loop between two parallel helices α2 and α3, (2) its HT does not coordinate any metal ion, even in the presence of 100 mM Zn2+, (3) it is monomeric in solution, unlike homodimeric YfiT. SeMet-labeled TTHA0303 protein was expressed in E. coli B834(DE3)pLysS cells (Novagen) harboring a pET-11a (Novagen)-based expression plasmid. The expressed protein was purified by heat treatment (70°C, 10 min) and the following six steps of column chromatographies: HiPrep 26/10 Desalting, Super Q Toyopearl 650M, Resource S, Hydroxyapatite CHT20, HiLoad 16/60 Superdex 75 pg, and HiPrep26/10 Desalting (GE Healthcare, Tosoh, and Bio-Rad). Crystallization was carried out by a microbatch method, where 0.5 μL of 13 mg/mL SeMet-TTHA0303 solution and 0.5 μL of crystallization buffer consisting of 20% (w/v) PEG 1000, 100 mM Tris-HCl (pH 7.0), and 200 mM Zn acetate were mixed to form a drop in 15 μL of oil (paraffin:silicon = 7:3, v/v) and kept at 18°C. Rod-shaped crystals with approximate dimensions of 80 μm × 30 μm × 30 μm were obtained in a month. The crystal diffracted X-rays beyond 2.0-Å resolution. Se-MAD data were obtained at SPring-8 (Harima, Japan) BL26B2 at wavelengths of 0.97914 Å (peak), 0.97950 Å (edge), and 0.90000 Å (remote). The diffraction data were processed with HKL2000,2 the phase problem was solved, the initial model was built with SOLVE/RESOLVE,3, 4 and the protein structure was refined with REFMAC5 and COOT6 and verified with PROCHECK.7 Gel filtration analysis was performed with a Superdex 75 10/30 HR column (GE Healthcare), a buffer of 50 mM Tris-HCl (pH 7.0), 100 mM Zn acetate, and 150 mM NaCl on an AKTA purifier (GE Healthcare) at a flow rate of 0.5 mL/min at 20°C. MALDI-TOF-MS analysis was carried out with an AXIMA CFRplus mass spectrometer (Shimadzu). TTHA0303 protein was crystallized in the space group P41212 with two protein molecules in an asymmetric unit. The crystal structure of TTHA0303 (169 amino acid residues) was solved by the Se-MAD method at 2.0-Å resolution and was refined to Rwork and Rfree factors of 20.4% and 25.3%, respectively. Diffraction data and refinement statistics are given in Table I. The two monomers in the asymmetric unit adopt almost identical conformations, with an atomic RMS difference of 0.3 Å for 120 aligned Cα atoms. TTHA0303 protein has a four-helix bundle structure with a topology of α1 (10–30) ↓- α2 (47–72) ↑- α3 (106–126) ↑- α4 (145–165) ↓ (Fig. 1). The protein structure does not include the N- and C-termini (1–8 and 167/168–169) and a ∼30-residue loop between the two parallel helices α2 and α3 (73/74–103) because of very weak or unobserved electron densities (the residue numbers appearing in this article are those of TTHA0303, as shown in Fig. 3). The DALI8 structural database search showed that the most structurally similar protein to TTHA0303 is YfiT from Bacillus subtilis (PDB code, 1RXQ).1 These proteins share 25% sequence identity and a 2.2-Å RMS difference for 123 aligned Cα atoms. They commonly have a histidine triad (HT) formed by conserved His53, His155, and His159 on the molecular surface (Fig. 2). But these molecules have different features as well. The HT of YfiT coordinates a Ni2+ ion together with three H2O molecules, but the HT of TTHA0303 coordinates no metal ion, even in the presence of 100 mM Zn2+ in the crystallization drop. Since the geometry of the HT, metal ion, and Glu81 in YfiT is similar to that of active-site residues in some zinc proteases such as thermolysin, a possible hydrolase activity of YfiT has been proposed.1 In contrast, TTHA0303, which has Pro81 instead of Glu, cannot form a YfiT-like putative active site of metalloprotease. Furthermore, TTHA0303 was found to be monomeric in solution, as revealed by gel filtration chromatography (data not shown), in contrast to YfiT, which exists as a homodimer in solution.1 The overall structure of monomeric TTHA0303 (A; PDB: 2YQY) and its comparison with homodimeric YfiT (B; PDB: 1RXQ)1 drawn with PyMOL.14 Four α helices forming the four-helix bundle fold are labeled as α1–α4 from the N-terminus to the C-terminus. Arrows show the directions of the helices: ↑up and ↓down. The YfiT protomer aligned with TTHA0303 is colored, while the other protomer is shown in gray. The histidine triads (HTs) of TTHA0303 (A) and YfiT (B) drawn with PyMOL.14 The HT of YfiT coordinates a Ni2+ ion together with three H2O molecules, whereas the HT of TTHA0303 does not coordinate a Zn2+ ion, even in the presence of 100 mM Zn2+. Structure-based sequence alignment of TTHA0303 and YfiT based on the results of DALI structural comparison.8 YuaE is aligned accordingly. Gaps (----) are inserted for the best alignment. The residue numbers are those of the TTHA0303 protein. The secondary structure elements of TTHA0303 and YfiT are shown above the TTHA0303 sequence and below the YfiT sequence, respectively. @@@@ and → represent an α helix and a β strand, respectively. * shows a His residue involved in the histidine triad (HT). #### indicates the proteolytically removed region, as revealed by MALDI-TOF-MS analysis. Gray characters indicate the regions not modeled because of very weak or unobserved electron densities. BLAST9 sequence database search showed that TTHA0303-like proteins with conserved His53, His155, and His159 are found only in several bacterial genera such as Thermus, Deinococcus, Geobacillus, and Bacillus. Genome databases show that T. thermophilus has no other TTHA0303-like protein,10, 11 while B. subtilis has two TTHA0303-like proteins, YfiT and YuaE, both of which show 17% sequence identity to TTHA0303.12 Sequence alignment shows that YuaE is more similar to TTHA0303 than to YfiT (Fig. 3). In fact, YuaE has Pro81 (as in TTHA0303) instead of Glu81 (as in YfiT). The loop between α2 and α3 of TTHA0303/YuaE is longer than the corresponding loop of YfiT by 11–12 residues. The SeMet-TTHA0303 protein we used for crystallization migrated as a single band on SDS-PAGE just after the purification, but the protein was cleaved at several positions in the loop between α2 and α3 after storage at 4°C for several months (see Fig. 3). The resulting TTHA0303 protein lacked the long loop between α2 and α3, as revealed by MALDI-TOF-MS analysis. This observation is consistent with no electron densities for the loop between α2 and α3 being observed and there being no space to accommodate the missing loop in the crystal due to the crystal packing. Therefore, the loop between α2 and α3 must also have been removed in the crystallization process of TTHA0303 at 18°C. In contrast, the short loop between α2 and α3 in YfiT was not cleaved.1 Since the TTHA0303 samples were purified by a heat treatment (70°C, 10 min) followed by six steps of column chromatographies, the removal of the loop occurred probably by potential proteolytic activity of TTHA0303, not by contaminated E. coli proteases. The possible metalloprotease activity of TTHA0303, particularly its self-processing activity to remove the long loop between α2 and α3, is being investigated. Very recently, the crystal structure of mycothiol-dependent maleylpyruvate isomerase (MDMPI) from the Gram-positive bacterium Corynebacterium glutamicum (PDB code, 2NSF) has been reported.13 MDMPI consists of an N-terminal metal-binding domain and a C-terminal domain with a novel folding pattern. The core of the N-terminal metal-binding domain consists of five α-helices, and is very similar to TTHA0303 and YfiT with atomic RMS differences of 2.2 Å for 123 Cα atoms and 2.8 Å and 146 Cα atoms, respectively, although the metal ligands in MDMPI are His, Glu, and His instead of an HT. Its putative active pocket includes the metal ion (Zn2+), the metal binding residues, and other conserved residues located between the two domains, and both the N- and C-terminal domains are necessary for its enzyme activity. In a similar manner, TTHA0303 and YfiT may represent one part of heterodimeric enzymes whose partners have yet to be identified. The atomic coordinates and experimental structure factors of TTHA0303 have been deposited to PDB under the code 2YQY. This work was performed under the Structural-Biological Whole Cell Project led by Dr. Seiki Kuramitsu at RIKEN SPring-8 Center. The synchrotron-radiation experiments were performed at BL26B2 in SPring-8 (Harima, Japan). This work was supported by Targeted Proteins Research Program (TPRP) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Bickerstaff's brainstem encephalitis (BBE), Miller Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS) are thought to be closely related and to form a continuous spectrum. However, chronic polyneuropathy in BBE has not been reported. We report the temporal profile of anti-ganglioside antibody titer in a case of BBE-like brainstem encephalitis complicated with chronic polyneuropathy. A 71-year-old Japanese woman presented with drowsiness and cerebellar ataxia in addition to mild weakness in distal limb muscles. Anti-GalNAc-GD1a IgG and anti-GalNAc-GM1b IgG antibodies were positive in her serum. Brain magnetic resonance imaging revealed high-intensity signals in the midbrain, pons, and middle cerebellar peduncles on T2-weighted images. Central nervous system manifestations improved after immunomodulating therapy that included prednisolone, plasmapheresis and intravenous immunoglobulin. Nevertheless, the distal muscle weakness was exacerbated when the anti-GalNAc-GD1a IgG titer was elevated. Nerve conduction study indicated motor and sensory neuropathy which was developed motor dominant axonal damage. These findings suggest that anti-ganglioside antibodies, including anti-GalNAc-GD1a IgG, may be involved in a common autoimmune mechanism in BBE-like brainstem encephalitis and chronic motor dominant axonal neuropathy. However, the fact that the latter manifestation exacerbated after the improvement of former one possibly indicates different thresholds of neurologic symptoms mediated by anti-ganglioside antibodies in the present patient.

Epstein-Barr virus (EBV) infection causes a wide range of neurologic and hematologic manifestations. We report a 72-year-old Japanese male patient with severe chronic active EBV infection syndrome (SCAEBV) who presented with Guillain-Barré syndrome (GBS) and developed hemophagocytic lymphohistiocytosis (HLH) several months after the onset of GBS. He showed acute onset of distal muscle weakness, ophthalmoplegia and bulbar palsy. Results of nerve conduction study revealed acute motor-sensory axonal neuropathy (AMSAN). His serum was positive for anti-LM1 IgG and anti-GM1b IgM. Titers of antibodies to EBV-related antigens indicated chronic reactivated EBV infection. Treatment with IVIg resolved the acute ophthalmoplegia, but there was no notable improvement in the AMSAN and bulbar palsy despite repeated. Finally, he developed refractory HLH resulting in a fatal outcome. In the present patient, it seems that SCAEBV was associated with the development of GBS and fatal HLH via parainfectious autoimmunity and direct infectious immune mechanisms, respectively.

We investigated the localization of GalNAc-GD1a biochemically in the human and bovine peripheral nervous system (PNS). The high-performance thin-layer chromatography (HPTLC)-overlay method with rabbit IgG polyclonal antibody against GalNAc-GD1a (anti-GalNAc-GD1a antibody) revealed expression of GalNAc-GD1a in the ventral spinal nerve roots (VRs) but not in the dorsal spinal nerve roots (DRs) of both species. The amount of GalNAc-GD1a in the human and bovine VRs was 2.22 +/- 0.35 microg/g wet tissue and 7.71 +/- 0.49 microg/g wet tissue, respectively. These results suggest that IgG anti-GalNAc-GD1a antibody may be involved in disturbance of peripheral motor nerves and in the pathogenesis of pure motor neuropathy.

We describe seven patients with isolated cranial neuropathy in whom serum anti-glycolipid antibodies were detected. Trigeminal sensory neuropathy was found in four patients, who had exhibited symptoms for 2 months to 4 years. The other three patients showed facial nerve palsy with or without ophthalmoparesis. Temporal profile analysis of anti-glycolipid antibodies revealed that titers of anti-glycolipid IgM antibodies against GM2 and LM1 gradually decreased in patients having chronic trigeminal sensory neuropathy. In patients with acute trigeminal sensory neuropathy, elevation of anti-LM1 antibody titers continued over 12 months although anti-GalNAc-GD1a antibody disappeared. On the other hand, titers of anti-glycolipid antibodies rapidly decreased in patients with acute facial nerve palsy with or without ophthalmoparesis. We conclude that anti-glycolipid antibodies may play an important role in the development of isolated cranial neuropathy in some patients.

We report a 27-year-old man with Guillain-Barré syndrome (GBS) preceded by cytomegalovirus infection. He was admitted to our hospital because of distal dominant weakness and sensory disturbance 5 days after fever. Double filtration plasmapheresis (DFPP) was performed and clinical symptoms temporary but dramatically improved. However facial nerve palsy, difficulty in swallowing food, weakness, dysautonomia and respiratory failure rapidly progressed within 5 days after the onset. Repeated DFPP failed to improve his symptoms. Two months after the onset, he did not improve at all. On T1-weighted MRI, nerve roots were still enhanced with gadolinium, and CSF examination revealed 1,324 mg/dl of protein. These findings suggested us the existence of continuous inflammation on nerve roots. We gave steroid-pulse therapy. He dramatically improved after this treatment. We repeated steroid-pulse therapy for seven times. He was discharged without any major complication 6 months after the onset. Steroid-pulse therapy should be considered in GBS patients associated with CMV infection when other conventional treatments are ineffective.

Since plasma exchange (PE) and intravenous immunoglobulin (IVIg) have been widely used in treatment for Guillain–Barré syndrome (GBS), early relapse and treatment-related fluctuation have been a potential problem, but little is known about the mechanism of relapse and fluctuation. We describe a patient who had GBS with treatment-related fluctuation. A 37-year-old Japanese man exhibited acute distal-dominant weakness in upper limbs after upper respiratory infection. His cranial nerve system was normal and muscle weakness was limited to upper limbs. Anti-GT1a IgG was strongly positive and anti-GQ1b IgG was also detected in his serum. Muscle weakness responded well to double-filtration plasmapheresis (DFPP) followed by IVIg, but relapsed 45 days after the initial treatment. Although repeated treatments were effective, the patient showed additional minor deterioration twice. Motor nerve conduction velocities (MCVs) corresponded to the muscle weakness, but elevated level of cerebrospinal fluid (CSF) protein remained and anti-ganglioside antibody titers steadily decreased throughout the clinical course. These findings indicate that the clinical fluctuation was not due to changes in the production of anti-ganglioside antibodies but presumably to the transient beneficial effects of DFPP/IVIg and the outlasting inflammatory response in peripheral nerves.