Nobuhiro Suzuki

Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB Activation

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

Complexity in influenza virus targeted drug design: interaction with human sialidases

With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.

Structural basis of ubiquitin recognition by mammalian Eap45 GLUE domain.

ESCRT-II, a complex that sorts ubiquitinated membrane proteins to lysosomes, localizes to endosomes through interaction between the Vps36 subunits GLUE domain and phosphatidylinositides (PIs). In yeast, a ubiquitin (Ub)-interacting NZF domain is inserted in Vps36 GLUE, whereas its mammalian counterpart, Eap45 GLUE, lacks the NZF domain. In the Eap45 GLUE–Ub complex structure, Ub binds far from the proposed PI-binding site of Eap45 GLUE, suggesting their independent binding.

Crystallization of small proteins assisted by green fluorescent protein.

The generation of crystal lattice contacts by proteinaceous tags fused to target proteins is an attractive approach to aid in the crystallization of otherwise intractable proteins. Here, the use of green fluorescent protein (GFP) fusions for this purpose is demonstrated, using ubiquitin and the ubiquitin-binding motif (UBM) of Y-family polymerase ι as examples. The structure of the GFP-ubiquitin fusion protein revealed that the crystal lattice was formed by GFP moieties. Ubiquitin was accommodated in the lattice through interactions with the peripheral loops of GFP. However, in the GFP-UBM fusion crystal UBM formed extensive interactions with GFP and these interactions, together with UBM dimerization, mediated the crystal packing. Interestingly, the tyrosine residues that are involved in mediating crystal contacts in both GFP-ubiquitin and GFP-UBM crystals are arranged in a belt on the surface of the β-barrel structure of GFP. Therefore, it is likely that GFP can assist in the crystallization of small proteins and of protein domains in general.

Differential Mechanisms for SHP2 Binding and Activation Are Exploited by Geographically Distinct Helicobacter pylori CagA Oncoproteins

Helicobacter pylori East Asian CagA is more closely associated with gastric cancer than Western CagA. Here we show that, upon tyrosine phosphorylation, the East Asian CagA-specific EPIYA-D segment binds to the N-SH2 domain of pro-oncogenic SHP2 phosphatase two orders of magnitude greater than Western CagA-specific EPIYA-C. This high-affinity binding is achieved via cryptic interaction between Phe at thexa0+5 position from phosphotyrosine in EPIYA-D and a hollow on the N-SH2 phosphopeptide-binding floor. Also, duplication of EPIYA-C in Western CagA, which increases gastric cancer risk, enables divalent high-affinity binding with SHP2 via N-SH2 and C-SH2. These strong CagA bindings enforce enzymatic activation of SHP2, which endows cells with neoplastic traits. Mechanistically, N-SH2 inxa0SHP2 is in an equilibrium between stimulatory relaxed and inhibitory squeezed states, which is fixed upon high-affinity CagA binding to the relaxed state that stimulates SHP2. Accordingly, East Asian CagA and Western CagA exploit distinct mechanisms for SHP2 deregulation.

A novel mode of ubiquitin recognition by the ubiquitin‐binding zinc finger domain of WRNIP1

The ubiquitin‐binding zinc finger (UBZ) is a type of zinc‐coordinating β‐β‐α fold domain found mainly in proteins involved in DNA repair and transcriptional regulation. Here, we report the crystal structure of the UBZ domain of Y‐family DNA polymerase (pol) η and the crystal structure of the complex between the UBZ domain of Werner helicase‐interacting protein 1 (WRNIP1) and ubiquitin, crystallized using the GFP fusion technique. In contrast to the pol η UBZ, which has been proposed to bind ubiquitin via its C‐terminal α‐helix, ubiquitin binds to a novel surface of WRNIP1 UBZ composed of the first β‐strand and the C‐terminal α‐helix. In addition, we report the structure of the tandem UBZ domains of Tax1‐binding protein 1 (TAX1BP1) and show that the second UBZ of TAX1BP1 binds ubiquitin, presumably in a manner similar to that of WRNIP1 UBZ. We propose that UBZ domains can be divided into at least two different types in terms of the ubiquitin‐binding surfaces: the pol η type and the WRNIP1 type.

Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity

Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of glycoside hydrolase family 66 and it produces cycloisomaltooligosaccharides (CIs). Small CIs (CI-7-9) and large CIs (CI-≥10) are designated as oligosaccharide-type CIs (oligo-CIs) and megalosaccharide-type CIs (megalo-CIs) respectively. CITase from Bacillus circulans T-3040 (BcCITase) produces mainly CI-8 with little megalo-CIs. It has two family 35 carbohydrate-binding modules (BcCBM35-1 and BcCBM35-2). BcCBM35-1 is inserted in a catalytic domain of BcCITase and BcCBM35-2 is located at the C-terminal region. Our previous studies suggested that BcCBM35-1 has two substrate-binding sites (B-1 and B-2) [Suzuki et al. (2014) J. Biol. Chem. 289, 12040-12051]. We implemented site-directed mutagenesis of BcCITase to explore the preference for product size on the basis of the 3D structure of BcCITase. Mutational studies provided evidence that B-1 and B-2 contribute to recruiting substrate and maintaining product size respectively. A mutant (mutant-R) with four mutations (F268V, D469Y, A513V and Y515S) produced three times as much megalo-CIs (CI-10-12) and 1.5xa0times as much total CIs (CI-7-12) as compared with the wild-type (WT) BcCITase. The 3D structure of the substrate-enzyme complex of mutant-R suggested that the modified product size specificity was attributable to the construction of novel substrate-binding sites in the B-2 site of BcCBM35-1 and reactivity was improved by mutation on subsite -3 on the catalytic domain.

Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1 → 4)-glucan

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1xa0→xa06)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1xa0→xa06)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1xa0→xa04)->xa0α-(1xa0→xa02)-xa0>xa0α-(1xa0→xa03)-xa0>xa0α-(1xa0→xa06)-glucobiose and maltopentaosexa0>xa0maltotetraosexa0>xa0maltotriosexa0>xa0maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by 13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1xa0→xa04) to α-(1xa0→xa06) transglucosylation activity. Ps6GT31A elongated α-(1xa0→xa06)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1xa0→xa06)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

Cation-driven Optical Properties of Artificial Luciferases.

The present study demonstrates cation-driven optical properties of artificial luciferases (ALucs) from copepod luciferases, as an optical readout for bioanalysis. An assignment of the supersecondary structure code (SSC) of ALucs revealed that ALucs carry a helix-loop-helix structure, which appears at the same sites of the EF-hands of typical Ca(2+)-binding proteins. A mutagenesis study shows that the EF-hand-like structure is a pivotal site for enzymatic activity. The effects of 20 kinds of mono- and multivalent cations on ALuc activities were estimated with column-purified ALuc16. High pH values boost the ALuc activities with both the native coelenterazine and an analog called 6-pi-OH-CTZ. Multivalent cations, Ca(II), Mg(II), and Cr(VI), elevate and prolong the ALuc activities, whereas Co(II), Cu(II) and Pb(II) greatly hamper the ALuc activities. Ca(II) greatly prolongs the optical intensities, suggesting a contribution to the structural robustness of ALucs. The inhibitory effect of multivalent cations on the ALuc activities was utilized for creating dose-response curves. The intrinsic cation-driven selectivity and optical intensity of ALucs enable researchers to constitute de novo biosensors for multivalent cations.

Structure determination of NEMO (NF-κB essential modulator) UBAN domain

Structure determination of NEMO(NF-;κB essential modulator) UBAN domain Simin Rahighi, Masato Akutsu, Nobuhiro Suzuki, Masato Kawasaki, Ryuichi Kato, Ivan Dikic, Soichi Wakatsuki High Energy Accelerator Research organization (KEK), Materilas Structure Science, 1-1 Oho, Tsukuba, Ibaraki, 305-0801, Japan, Department of Materials Structure Science, The Graduate University for Advanced Studies (SOKENDAI), Japan, Institute for Biochemistry II, Goethe University Medical School, Frankfurt, Germany, E-mail:simin@ post.kek.jp