Yasuo Shirakihara

Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G

Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on myelomonocytic cells, whereas ILT2 (also known as leukocyte Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range of immune cells including subsets of natural killer and T cells. Both ILTs contain immunoreceptor tyrosine-based inhibitory receptor motifs in their cytoplasmic tails that inhibit cellular responses by recruiting phosphatases such as SHP-1 (Src homology 2 domain containing tyrosine phosphatase 1). Although these ILTs have been shown to recognize a broad range of classical and nonclassical human MHC class I molecules (MHCIs), their precise binding properties remain controversial. We have used surface plasmon resonance to analyze the interaction of soluble forms of ILT4 and ILT2 with several MHCIs. Although the range of affinities measured was quite broad (Kd = 2–45 μM), some interesting differences were observed. ILT2 generally bound with a 2- to 3-fold higher affinity than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3- to 4-fold higher affinity than to classical MHCIs, suggesting that ILT/HLA-G recognition may play a dominant role in the regulation of natural killer, T, and myelomonocytic cell activation. Finally, we show that ILT2 and ILT4 effectively compete with CD8 for MHCI binding, raising the possibility that ILT2 modulates CD8+ T cell activation by blocking the CD8 binding as well as by recruiting inhibitory molecules through its immunoreceptor tyrosine-based inhibitory receptor motif.

The crystal structure of the nucleotide-free α3β3 subcomplex of F1-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer

Abstract Background: F 1 -ATPase, an oligomeric assembly with subunit stoichiometry α 3 β 3 γ δ ϵ, is the catalytic component of the ATP synthase complex, which plays a central role in energy transduction in bacteria, chloroplasts and mitochondria. The crystal structure of bovine mitochondrial F 1 -ATPase displays a marked asymmetry in the conformation and nucleotide content of the catalytic β subunits. The α 3 β 3 subcomplex of F 1 -ATPase has been assembled from subunits of the moderately thermophilic Bacillus PS3 made in Escherichia coli , and the subcomplex is active but does not show the catalytic cooperativity of intact F 1 -ATPase. The structure of this subcomplex should provide new information on the conformational variability of F 1 -ATPase and may provide insights into the unusual catalytic mechanism employed by this enzyme. Results: The crystal structure of the nucleotide-free bacterial α 3 β 3 subcomplex of F 1 -ATPase, determined at 3.2 A resolution, shows that the oligomer has exact threefold symmetry. The bacterial β subunits adopt a conformation essentially identical to that of the nucleotide-free β subunit in mitochondrial F 1 -ATPase; the α subunits have similar conformations in both structures. Conclusions: The structures of the bacterial F 1 -ATPase α and β subunits are very similar to their counterparts in the mitochondrial enzyme, suggesting a common catalytic mechanism. The study presented here allows an analysis of the different conformations adopted by the α and β subunits and may ultimately further our understanding of this mechanism.

Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T

Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 Å resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 Å resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing against TM-C. In the C-terminal region of TM-C, a crucial water in the coiled-coil core broke the local 2-fold symmetry and helps generate a kink on one helix. In the presence of a TnT fragment, the asymmetry in TM-C facilitates formation of a 4-helix bundle containing two TM-C chains and one chain each of TM-N and TnT. Mutating the residues that generate the asymmetry in TM-C caused a marked decrease in the affinity of troponin for actin-tropomyosin filaments. The highly conserved region of TnT, in which most cardiomyopathy mutations reside, is crucial for interacting with tropomyosin. The structure of the ternary complex also explains why the skeletal- and cardiac-muscle specific C-terminal region is required to bind TnT and why tropomyosin homodimers bind only a single TnT. On actin filaments, the head-to-tail junction can function as a molecular swivel to accommodate irregularities in the coiled-coil path between successive tropomyosins enabling each to interact equivalently with the actin helix.

Crystal Structures of the Multidrug Binding Repressor Corynebacterium glutamicum CgmR in Complex with Inducers and with an Operator

CgmR (CGL2612) from Corynebacterium glutamicum is a multidrug-resistance-related transcription factor belonging to the TetR family, which is a protein family of widespread bacterial transcription factors typically involved in environmental response. Here, we report the crystal structures of CgmR homodimeric repressor in complex with two distinct inducers (1.95 and 1.4 Å resolution) and with an operator (2.5 Å resolution). The CgmR-operator complex showed that two CgmR dimers bound to the operator, and each half-site of the palindromic operator was asymmetrically recognized by two DNA-binding domains from different dimers on the opposite sides of the DNA. The inducer complexes demonstrated that both bound inducers act as a wedge to alter the operator-binding conformation of the repressor by steric inhibition. As steric hindrance is used, various drugs should act as inducers if they have sufficient volume for the conformation change and if their bindings sufficiently reduce free energy. The comparative structural study of CgmR free protein, in complex with operator, and with inducers, implies the other mechanism that might contribute to multidrug response of the repressor.

Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism

F1‐ATPase (F1) is the catalytic sector in FoF1‐ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β‐subunits undergo nucleotide occupancy‐dependent and concerted open–close conformational changes that are accompanied by rotation of the γ‐subunit. Bacterial and chloroplast F1 are inhibited by their own ε‐subunit. In the ε‐inhibited Escherichia coli F1 structure, the ε‐subunit stabilizes the overall conformation (half‐closed, closed, open) of the β‐subunits by inserting its C‐terminal helix into the α3β3 cavity. The structure of ε‐inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε‐subunit, but the thermophilic ε‐subunit stabilizes another unique overall conformation (open, closed, open) of the β‐subunits. The ε‐C‐terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε‐C‐terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε‐inhibition differently. For inhibition, the ε‐subunit contacts the second catches of some of the β‐ and α‐subunits, the N‐ and C‐terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β‐ and α‐ second catches in ε‐inhibition‐specific positions, and prevent rotation of the γ‐subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1.

The quaternary structure of the eukaryotic DNA replication proteins Sld7 and Sld3.

The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomes. Yeast Sld3 and its metazoan counterpart treslin are the hub proteins mediating protein associations critical for formation of the helicase. The Sld7 protein interacts with Sld3, and the complex formed is thought to regulate the function of Sld3. Although Sld7 is a non-essential DNA replication protein that is found in only a limited range of yeasts, its depletion slowed the growth of cells and caused a delay in the S phase. Recently, the Mdm2-binding protein was found to bind to treslin in humans, and its depletion causes defects in cells similar to the depletion of Sld7 in yeast, suggesting their functional relatedness and importance during the initiation step of DNA replication. Here, the crystal structure of Sld7 in complex with Sld3 is presented. Sld7 comprises two structural domains. The N-terminal domain of Sld7 binds to Sld3, and the C-terminal domains connect two Sld7 molecules in an antiparallel manner. The quaternary structure of the Sld3-Sld7 complex shown from the crystal structures appears to be suitable to activate two helicase molecules loaded onto replication origins in a head-to-head manner.

Crystal Structure of the Homology Domain of the Eukaryotic DNA Replication Proteins Sld3/Treslin

The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomal DNA. Yeast Sld3 and its metazoan counterpart Treslin are the hub proteins mediating protein associations critical for the helicase formation. Here, we show the crystal structure of the central domain of Sld3 that is conserved in Sld3/Treslin family of proteins. The domain consists of two segments with 12 helices and is sufficient to bind to Cdc45, the essential helicase component. The structure model of the Sld3-Cdc45 complex, which is crucial for the formation of the active helicase, is proposed.

Crystal structure of salt-tolerant glutaminase from Micrococcus luteus K-3 in the presence and absence of its product l-glutamate and its activator Tris

Glutaminase from Micrococcus luteus K‐3 [Micrococcus glutaminase (Mglu); 456 amino acid residues (aa); 48 kDa] is a salt‐tolerant enzyme. Our previous study determined the structure of its major 42‐kDa fragment. Here, using new crystallization conditions, we determined the structures of the intact enzyme in the presence and absence of its product l‐glutamate and its activator Tris, which activates the enzyme by sixfold. With the exception of a ‘lid’ part (26‐29 aa) and a few other short stretches, the structures were all very similar over the entire polypeptide chain. However, the presence of the ligands significantly reduced the length of the disordered regions: 41 aa in the unliganded structure (N), 21 aa for l‐glutamate (G), 8 aa for Tris (T) and 6 aa for both l‐glutamate and Tris (TG). l‐Glutamate was identified in both the G and TG structures, whereas Tris was only identified in the TG structure. Comparison of the glutamate‐binding site between Mglu and salt‐labile glutaminase (YbgJ) from Bacillus subtilis showed significantly smaller structural changes of the protein part in Mglu. A comparison of the substrate‐binding pocket of Mglu, which is highly specific for l‐glutamine, with that of Erwinia carotovora asparaginase, which has substrates other than l‐glutamine, shows that Mglu has a larger substrate‐binding pocket that prevents the binding of l‐asparagine with proper interactions.

Draft Genome Sequence of a Thermophilic Member of the Bacillaceae, Anoxybacillus flavithermus Strain Kn10, Isolated from the Kan-nawa Hot Spring in Japan

ABSTRACT Here, we report the draft genome sequence of the Anoxybacillus flavithermus Kn10 strain (NBRC 109594), isolated from a water drain of the Kan-nawa Hot Spring in Japan. The draft genome sequence is composed of 90 contigs for 2,772,624 bp with 41.6% G+C content and contains 2,883 protein-coding genes and 80 tRNA genes.

Crystallization and preliminary X-ray crystallographic studies of salt-tolerant glutaminase from Micrococcus luteus K-3.

Glutaminase from the marine bacterium Micrococcus luteus K-3 (Micrococcus glutaminase) is a salt-tolerant protein which shows equivalent activities both in the absence and the presence of 3 M sodium chloride and is distinct from halophilic proteins, which are inactivated in the absence of salt. To investigate the mechanisms of the salt-tolerant adaptation of Micrococcus glutaminase, the glutaminase and its major fragment containing about 80% of the protein were crystallized using the hanging-drop vapour-diffusion method. The glutaminase crystals belong to space group P622, with unit-cell parameters a = b = 111.4, c = 210.9 A, alpha = beta = 90, gamma = 120 degrees, and diffract to 2.6 A resolution. The fragment crystals belong to space group F222, with unit-cell parameters a = 115.7, b = 116.4, c = 144.9 A, alpha = beta = gamma = 90 degrees, and diffract to 2.4 A resolution. Data from selenomethionine (SeMet) substituted glutaminase crystals and from SeMet-substituted fragment crystals were collected to 2.6 and 2.4 A resolution, respectively. Structural analyses of the glutaminase and its fragment are currently being attempted using the multiwavelength anomalous diffraction (MAD) phasing method.