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The recent crystal structure determinations of druggable class A G protein-coupled receptors (GPCRs) have opened up excellent opportunities in structure-based ligand discovery for this pharmaceutically important protein family. We have developed and validated a customized structure-based virtual fragment screening protocol against the recently determined human histamine H(1) receptor (H(1)R) crystal structure. The method combines molecular docking simulations with a protein-ligand interaction fingerprint (IFP) scoring method. The optimized in silico screening approach was successfully applied to identify a chemically diverse set of novel fragment-like (≤22 heavy atoms) H(1)R ligands with an exceptionally high hit rate of 73%. Of the 26 tested fragments, 19 compounds had affinities ranging from 10 μM to 6 nM. The current study shows the potential of in silico screening against GPCR crystal structures to explore novel, fragment-like GPCR ligand space.

G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H(1) receptor (H(1)R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H(1)R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination., (Copyright © 2011 Elsevier Inc. All rights reserved.)

CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4(+) T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Anion exchangers are membrane proteins that have been identified in a wide variety of species, where they transport Cl(-) and HCO3(-)across the cell membrane. In this study, we cloned an anion-exchange protein from the genome of the basidiomycete Phanerochaete chrysosporium (PcAEP). PcAEP is a 618-amino acid protein that is homologous to the human anion exchanger (AE1) with 22.9% identity and 40.3% similarity. PcAEP was overexpressed by introducing the PcAEP gene into the genome of Pichia pastoris. As a result, PcAEP localized in the membrane of P. pastoris and was solubilized successfully by n-dodecyl-β-D-maltoside. His-tagged PcAEP was purified as a single band on SDS-PAGE using immobilized metal affinity chromatography and gel filtration chromatography. Purified PcAEP was found to bind to SITS, an inhibitor of the AE family, suggesting that the purified protein is folded properly. PcAEP expressed and purified using the present system could be useful for biological and structural studies of the anion exchange family of proteins., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H(1) receptor (H(1)R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H(1)R complex with doxepin, a first-generation H(1)R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp 428(6.48), a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H(1)R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys 191(5.39) and/or Lys 179(ECL2), both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H(1)R antagonist specificity against H(1)R., (©2011 Macmillan Publishers Limited. All rights reserved)

Background: Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies., Results: The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells., Conclusion: Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.

Purpose of Work: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.

Background: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown., Methods: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed., Results: As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL., Conclusions: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL., General Significance: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The effective xylooligosaccharides (XOs) production from the waste medium after mushroom cultivation (WM) was investigated. The WM contains rich nutrients (protein, etc.) which induce Maillard reaction with reducing sugars under hydrothermal conditions. To improve the productivity of XOs, the suitable pretreatment combined with washing and grinding was investigated, and subsequently hydrothermal treatment was demonstrated with batch type and continuous flow type reactor. The washing pretreatment with hot water of 60 degrees C was effective to remove nutrients from the WM, and it led to prevent brownish discoloration on the hydrothermal treatment. On the basis of experimental data, industrial XOs production processes consisting of the pretreatment, hydrothermal treatment and purification step was designed. During the designed process, 2.3 kg-dry of the purified XOs was produced from 30 kg-wet of the WM (15% yield as dry basis weight). Theoretical yield of XOs attained to 48% as xylan weight in the WM., ((c) 2010 Elsevier Ltd. All rights reserved.)

Objective: The objective of our study was to evaluate the changes in quantitative diffusion tensor (DT) metrics and normalized T2-signal intensity (nT2-SI) values of normal-appearing cortical gray matter (CGM) and deep gray matter (DGM) in patients with multiple sclerosis (MS)., Methods: Fifty patients with MS and 25 patients with no MS matched on sex/age were selected as controls. Conventional magnetic resonance imaging and DT imaging were performed. Fractional anisotropy (FA)/mean diffusivity (MD) and nT2-SI values of CGM and DGM were measured. Analyses of variance between the 2 groups were analyzed; Pearson correlations between DT metrics and nT2-SI values and brain parenchymal fraction (BPF) and T2 lesion volumes (LVs) were used., Results: Patients with MS showed larger MD/smaller FA values in the CGM region compared with controls (P < 0.05). However, MD/FA values were not statistically significant in the DGM between MS and healthy control group. In DGM of MS patients, a significant decrease of nT2-SI values were observed when compared with controls (P < 0.05), but nT2-SI values in the CGM of MS patients showed no significant decrease. In CGM, only MD values of frontal lobes in MS patients were significantly (negatively) correlated with BPF(right: P = 0.009, left: P = 0.036) or T2 LVs (right: P = 0.002, left: P = 0.047). Normalized T2-SI values in all DGM regions of MS patients were significantly correlated with BPF (r = 0.282-0.504, P < 0.05) except for the left thalamus and bilateral red nucleus. There was no correlation between nT2-SI in all DGM regions and T2 LVs of MS patients., Conclusion: In CGM, the change in quantitative DT metrics of MS patients and the association with BPF and T2 LVs suggest the existence of microstructural destruction corresponding to inflammation, demyelination, or wallerian degeneration, but the changes in CGM were independent of the concomitant changes in BPF and T2 lesion. In DGM, a decrease of nT2-SI in MS patients and the correlation of nT2-SI values with BPF (brain atrophy) suggest excessive iron deposition related to chronic destruction. Our investigation indicates the possibility of different mechanism of pathological change in CGM and DGM.

Purpose: To evaluate retrospectively quantitative diffusion tensor imaging (DTI) values of hyperintense lesions on nonenhanced T1-weighted magnetic resonance (MR) images in patients with multiple sclerosis (MS) to elucidate the degree of demyelination or remyelination associated with T1 hyperintense lesions and study their relationship to MR markers of tissue damage (brain atrophy)., Materials and Methods: Institutional review board approval was obtained; informed consent was waived for this HIPAA-compliant study, including 76 patients with MS and 20 healthy control subjects without evidence of MS clinically or on imaging. T1 lesions were compared with normal white matter on nonenhanced images and judged to be hyperintense. Quantitative DTI metrics of T1 hyperintense lesions were examined, and the relationship between DTI parameters and brain atrophy were investigated in this study., Results: At least one T1 hyperintense lesion was found in 16 patients (total, 28 lesions). Hyperintense lesions on T1-weighted imaging (T1WI) had lower mean diffusion (MD) than others signal intensity lesions on T1WI but higher MD than normal white matter (F = 3.931; P < 0.001); Fractional anisotropy (FA; F = 3.24; P < 0.001) and volume ratio (VR; F = 1.664; P < 0.001) were higher in hyperintense lesions on T1WI than hypointense/isointense lesions on T1WI, but were lower than normal-appearing white matter (NAWM) and normal white matter in controls. There was correlation between FA and VR (r = 0.678; P < 0.001) and inverse correlation between FA and MD (r = -0.437; P = 0.02), MD and VR (r = -0.423; P 0.025) for T1 hyperintense lesion. The MD values of T1 hyperintense lesions (r = -0.304; P < 0.001) and the VR values of T1 hyperintense lesions (r = 0.096; P = 0.042) were significantly (negative) correlated with Brain parenchymal fraction (BPF; higher BPF score); the FA values of T1 hyperintense lesions (r = -0.111; P = 0.018), the MD values of T1 hyperintense lesions (r = 0.379; P < 0.001) and the VR values of T1 hyperintense lesions (r = -0.142; P = 0.003) were significantly correlated with third ventricular width (lower width). However, the FA value of T1 hyperintense lesions was not significantly associated with BPF(r = 0.083; P = 0.08)., Conclusion: The quantitative DTI values of T1 hyperintense MS plaques were between hypo-/isointense lesions and NAWM or normal white matter, and correlated with BPF and third ventricular width. Our results supports the notion that axonal remyelination may be the reason for T1 hyperintense lesions., ((c) 2010 Wiley-Liss, Inc.)

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-beta-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.

N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand-receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1-10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B(max) value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.

Human leukocyte antigen-G (HLA-G) is a nonclassical MHC class I (MHCI) molecule that is expressed mainly on placenta trophoblast cells. Leukocyte Ig-like receptor B2 (LILRB2) is a human inhibitory immune receptor that recognizes HLA-G with a higher affinity than any other MHCI although this interaction is only in the microM range. The interaction between HLA-G and LILRB2 seems to play a dominant role in the escape of the fetus from the maternal immune response. Here we report the crystallization and x-ray analysis of the LILRB2/HLA-G complex. The extracellular domains of HLA-G and LILRB2 were expressed in Escherichia coli, refolded and purified. The initial crystallization trials using novel PEG-based screening sets provided crystals of the LILRB2/HLA-G complex with 40-50% PEG400 as the precipitant. These crystals belong to space group P3(1)21 (a=b=81.4 A, c=186.7 A, gamma=120 degrees ). Dehydration of the crystals by soaking them in a solution containing a higher concentration of PEG400 dramatically improved the resolution and also the mosaicity.

Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

The decolorization of industrial dyes was investigated using extracellular enzymes produced by 21 basidiomycetes, mainly edible mushrooms. Among the 27 dyes used in this study, nine were decolorized by over 40%. Most fungi decolorized Acid Orange 20, but they showed different specificities in the case of the other dyes. Determination of activity staining by native polyacrylamide gel electrophoresis revealed that all the decolorization activities corresponded to 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation activities.

Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein-protein interactions have been performed, the very weak interactions generally observed in cell-cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (K(d) greater than muM range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLA-Cw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 muM (HLA-Cw4) and 5.7 muM (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell-cell recognition receptors involved in immunologically relevant events.

Tyrosine is an important amino acid in protein-protein interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein interactions, we performed crystallographic studies and thermodynamic analyses of the interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the antigen-antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the antigen-antibody interaction through a diverse set of structural and thermodynamic interactions.

Purpose: PD173074, a small molecule inhibitor of VEGF-RII and FGF-RI, targets neoangiogenesis and mitogenesis. This study aimed to analyze a single-compound-driven inhibition of FGF and VEGF receptors in pancreatic cancer., Experimental Design: RT-PCR and Western blots were performed to quantify protein expression and phosphorylation. Anchorage dependent and independent growth assays were used to study cell growth. With flow cytometry, cell cycle analysis and apoptosis were studied. In vivo HPAF-II and MIA PaCa-2 cells were xenografted. Animals were treated daily for 10 weeks. Immunohistochemistry was used to quantify microvessel density and apoptosis., Results: Highest levels of FGF-RI were detectable in MIA PaCa-2 cells, lowest in HPAF-II cells. PD173074 inhibited cell growth most prominently in cells expressing high levels of FGF-RI. Cell cycle progression was inhibited by blocking transition in the G(0)/G(1) phase, and consequently, apoptosis was increased. In vivo significant inhibition of orthotopic tumor growth was achieved by a combination effect of inhibition of mitogenesis, induction of apoptosis, and reduction of angiogenesis in PD173074-treated animals., Conclusions: These data highlight VEGF-RII and FGF-RI as therapeutic targets and suggest a potential role for the combined use of tyrosine kinase inhibitors in the management of inoperable pancreatic cancer patients.

HLA-G is a nonclassical MHC class I (MHCI) molecule that can suppress a wide range of immune responses in the maternal-fetal interface. The human inhibitory immune receptors leukocyte Ig-like receptor (LILR) B1 [also called LIR1, Ig-like transcript 2 (ILT2), or CD85j] and LILRB2 (LIR2/ILT4/CD85d) preferentially recognize HLA-G. HLA-G inherently exhibits various forms, including beta(2)-microglobulin (beta(2)m)-free and disulfide-linked dimer forms. Notably, LILRB1 cannot recognize the beta(2)m-free form of HLA-G or HLA-B27, but LILRB2 can recognize the beta(2)m-free form of HLA-B27. To date, the structural basis for HLA-G/LILR recognition remains to be examined. Here, we report the 2.5-A resolution crystal structure of the LILRB2/HLA-G complex. LILRB2 exhibits an overlapping but distinct MHCI recognition mode compared with LILRB1 and dominantly recognizes the hydrophobic site of the HLA-G alpha3 domain. NMR binding studies also confirmed these LILR recognition differences on both conformed (heavy chain/peptide/beta(2)m) and free forms of beta(2)m. Binding studies using beta(2)m-free MHCIs revealed differential beta(2)m-dependent LILR-binding specificities. These results suggest that subtle structural differences between LILRB family members cause the distinct binding specificities to various forms of HLA-G and other MHCIs, which may in turn regulate immune suppression.

Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group 2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and Fc alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.

HLA-G is a non-classical MHC class I, which binds to inhibitory receptors, such as Leukocyte Ig-like receptors, to induce a wide range of tolerogenic immunological effects. HLA-G can be expressed as a disulfide-liked dimer both in solution and at the cell surface. However, the three-dimensional structure of the HLA-G dimer is unknown. Here, we report the crystallization of the disulfide-linked dimer form of HLA-G by adding dithiothreitol (DTT), enabling a 3.2-A data set to be collected. We also show that DTT promotes disulfide bond exchange of refolded HLA-G, whose free cysteine was protected, thus facilitating its dimerization. This technique could also be applied for disulfide-mediated dimer/multimer formation of refolded proteins harbouring free cysteines.

HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys(42)-Cys(42) disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1:2 (HLA-G dimer:receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.

The human inhibitory receptor, leukocyte immunoglobulin (Ig)-like receptor B1 (also called Ig-like transcript (ILT) 2, CD85j), is broadly expressed on leukocytes. LILRB1 binds to a wide range of major histocompatibility complex class I molecules (MHCIs) and transduces negative signals that can, for example, prevent killing of MHCI-expressing cells. Here we report the kinetic, thermodynamic, NMR and crystallographic analyses of MHCI recognition by LILRB1. Kinetic studies demonstrated that LILRB1 binds to MHCIs with fast association and dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses showed that LILRB1-MHCI interactions are entropically driven (-TdeltaS = -9.4 approximately -6.6 kcal mol(-1)) with low heat capacity changes (deltaC(p) = -0.22 approximately -0.10 kcal mol(-1) K(-1)). The crystal structures of LILRB1 in the different crystal forms exhibited variation in the elbow angle between the two N-terminal Ig-like domains, indicating interdomain flexibility. Consistently, NMR analysis provided the direct evidence of the conformational changes of LILRB1 upon the MHCI binding. These findings suggest that LILRB1-MHCI interactions, while involving some conformational adjustment, are not accompanied by a very large reduction in conformational flexibility at the binding interface. This mode of binding is distinct from "induced-fit" binding, which is associated with large reductions in conformational flexibility, and would be suitable for rapid engagement of MHCIs to enable fast monitoring of the expression level of MHCIs on target cells.

Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1/LIR1/ILT2) is an inhibitory receptor broadly expressed on leukocytes and recognizes HLA-class I and human cytomegalovirus UL18. LILRB1 is encoded within the leukocyte receptor complex on 19q13.4, previously implicated to be a susceptibility region to systemic lupus erythematosus (SLE). In this study, we screened for polymorphisms of LILRB1 and examined their association with SLE and rheumatoid arthritis (RA). In the 5' portion of LILRB1, three haplotypes containing four non-synonymous substitutions within the ligand-binding domains and two single nucleotide polymorphisms within the promoter region were identified and designated as PE01-03. In the 3' portion, two haplotypes (CY01, 02) containing a non-synonymous substitution of the cytoplasmic region were identified. CY01 and 02 did not co-segregate with PE01-03. Significant association with susceptibility to SLE or RA was not observed; however, among the subjects not carrying RA-associated HLA-DRB1 shared epitope (SE), LILRB1.PE01/01 diplotype was significantly associated with RA (odds ratio 2.05, P = 0.019 and Pc = 0.038). Gross difference was not observed in the crystal structures, thermostabilities and binding affinities to HLA-class I ligands among LILRB1.PE01-03 haplotype products; however, surface expression of LILRB1 was significantly decreased in lymphocytes and monocytes from the carriers of PE01 haplotype. These findings demonstrated that LILRB1 is highly polymorphic and is associated with susceptibility to RA in HLA-DRB1 SE negative subjects, possibly by insufficient inhibitory signaling in leukocytes. In addition, these observations suggested that the polymorphisms of LILR family members may be substantially involved in the diversity of human immune responses.

Novel, cell-based assays, based on bioluminescence resonance energy transfer, have been developed for FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling at receptor-proximal steps. In a stable transfectant of the HEK-293 cell line expressing human FcepsilonRIalpha, FcepsilonRIbeta, and FcRgamma-GFP2 and Syk(1-265)-Rluc fusion proteins, FcepsilonRI cross-linking markedly increased BRET2 ratios, which are the ratios of GFP2 emission to Rluc emission. These ratios reflect the FcRgamma-GFP2-Syk(1-265)-Rluc interaction in living cells. The signals are specifically inhibited by the Src-family kinase inhibitor PP2. Separately, in transient transfectants expressing GPVI, FcRgamma-GFP2, and Syk(1-265)-Rluc, the GPVI-specific ligand convulxin induced a two-fold increase in the BRET2 ratio and this increase was also inhibited by PP2. Finally, a differential assay was developed which permits the measurement of FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling in the same cell. These assays provide useful methods for monitoring FcRgamma-Syk interaction in real time in living cells and may contribute to the understanding of signal regulation through FcRgamma-containing receptors.

Cyclin-dependent kinase 2 (cdk2) activation requires phosphorylation of Thr160 and dissociation from cyclin A. The T-loop of cdk2 contains a regulatory phosphorylation site at Thr160. An interaction between cdc-associated phosphatase (KAP) and cdk2 compromises the interaction between cdk2 and cyclin A, which permits access of KAP, a Thr160-directed phosphatase, to its substrate, cdk2. We have reported that KAP is bound and activated by a nuclear membrane protein, HTm4. Here, we present in vitro data showing the direct interaction between the HTm4 C terminus and KAP Tyr141. We show that this interaction not only facilitates access of KAP to Thr160 and accelerates KAP kinetics, but also forces exclusion of cyclin A from the KAP.cdk2 complex.

Human cytomegalovirus carries a gene, UL18, that is homologous to cellular major histocompatibility complex (MHC) class I genes. Like MHC class I molecules, the protein product of the UL18 gene associates with beta2-microglobulin, and the stability of this complex depends on peptide loading. UL18 protein binds to ILT2 (CD85j), an inhibitory receptor present on B cells, monocytes, dendritic cells, T cells, and NK cells that also recognizes classical and nonclassical MHC molecules. These observations suggest that UL18 may play a role in viral immune evasion, but its real function is unclear. Since this molecule has similarity with polymorphic MHC proteins, we explored whether the UL18 gene varied between virus isolates. We report here that the UL18 gene varies significantly between virus isolates: amino acid substitutions were found in the predicted alpha1, alpha2, and alpha3 domains of the UL18 protein molecule. We also studied the ability of several variant UL18 proteins to bind to the ILT2 receptor. All of the variants tested bound to ILT2, but there were marked differences in the affinity of binding to this receptor. These differences were reflected in functional assays measuring inhibition of the cytotoxic capacity of NK cells via interaction with ILT2. In addition, the variants did not bind other members of the CD85 family. The implications of these data are discussed.

PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.

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 microM), 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.

We exactly evaluate the third-neighbor correlator S(z)(j)S(z)(j+3) and all the possible nonzero correlators S(alpha)(j)S(beta)(j+1)S(gamma;)(j+2)S(delta)(j+3) of the one-dimensional spin-1/2 Heisenberg XXX antiferromagnet in the ground state without magnetic field. All the correlators are expressed in terms of certain combinations of logarithm ln 2, the Riemann zeta function zeta(3), zeta(5) with rational coefficients. The results accurately coincide with the numerical ones obtained by the density-matrix renormalization group method and the numerical diagonalization.

To study the role of hydrogen bonding via interfacial water molecules in protein-protein interactions, we examined the interaction between hen egg white lysozyme (HEL) and its HyHEL-10 variable domain fragment (Fv) antibody. We constructed three antibody mutants (l-Y50F, l-S91A, and l-S93A) and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the mutations significantly decreased the negative enthalpy change (8-25 kJ mol(-1)), despite some offset by a favorable entropy change. X-ray crystallography demonstrated that the complexes had nearly identical structures, including the positions of the interfacial water molecules. Taken together, the isothermal titration calorimetric and x-ray crystallographic results indicate that hydrogen bonding via interfacial water enthalpically contributes to the Fv-HEL interaction despite the partial offset because of entropy loss, suggesting that hydrogen bonding stiffens the antigen-antibody complex.

Recently a new integral equation describing the thermodynamics of the 1D Heisenberg model was discovered by Takahashi. Using the integral equation we have succeeded in obtaining the high-temperature expansion of the specific heat and the magnetic susceptibility up to O[(J/T)(100)]. This is much higher than those obtained so far by the standard methods such as the linked-cluster algorithm. Our results will be useful to examine various approximation methods to extrapolate the high-temperature expansion to the low temperature region.

A structural and thermodynamic study of the entropic contribution of salt bridge formation to the interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wild-type Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the antigen-antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein antigen-antibody interaction through reduction of entropy loss due to dehydration and structural changes.

Suspension-cultured poplar (Populus alba) cells produce two distinct endo-1,4-beta-glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wall-bound endo-1, 4-beta-glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo-1,4-beta-glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello-oligosaccharides. Cello-oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello-oligosaccharides by the extracellular enzyme by 90% and that by the wall-bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo-1,4-beta-glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension-cultured poplar cells.

The introduction of open-heart surgery more than 4 decades ago signaled a new era in medicine. For the 1st time, previously untreatable cardiac anomalies became amenable to surgical therapy. The use of the heart-lung machine seemed to grant the surgeon unlimited time in which to operate inside the heart. Still frustrated by poor operating conditions and the threat of air embolism, Denis Melrose introduced elective cardiac arrest in 1955. His use of a potassium citrate solution seemed to offer a safe method to effect a quiet, bloodless field. However, a few years after its inception, numerous reports began to question the safety of this approach, and the Melrose technique was abandoned in the early 1960s. Nearly 15 years elapsed before potassium-based cardioplegia regained popularity. During this period, topical hypothermia, coronary perfusion with intermittent aortic occlusion, and normothermic ischemia were evaluated and discarded. A few European investigators like Hoelscher, Bretschneider, and Kirsch had maintained their interest in chemical cardioplegia, and it was through their efforts that future researchers like Hearse and Gay spearheaded the return to potassium-based cardioplegia, which today forms the core of the cardiac surgeon's myocardial protective armamentarium and has contributed towards lowering operative mortality rates.

To investigate the fine substrate specificities of four highly purified exo-type cellulases (Exo-A from Aspergillus niger, CBHI and CBHII from Trichoderma reesei, and Ex-1 from Irpex lacteus), water-soluble substrates such as barley glucan, xyloglucan from tamarind (Tamarindus indica L.), and their oligosaccharides were employed. Four exo-type cellulases immediately hydrolyzed 3-O-beta-D-cellotriosylglucose to produce cellobiose and laminaribiose. In contrast, CBHII showed no hydrolytic activity towards 3(2)-O-beta-D-cello-biosylcellobiose, which was hydrolyzed to cellobiose by the other exo-type cellulases. These cellulases hydrolyzed the internal linkages of barley glucan and lichenan in an endo-type fashion to produce cellobiose and mix-linked oligosaccharides as main products. The DP-lowering activities of the four exo-type cellulases on barley glucan were in the order of Ex-1, CBHII, Exo-A, and CBHI. Based on gel permeation chromatography analysis of the hydrolysates, Ex-1 seemed to attack the internal cellobiosyl unit adjacent to beta-1,3-glucosidic linkages in barley glucan molecule more frequently than did the other cellulases. Xyloglucan was hydrolyzed only by CBHI and CBHII, and produced hepta-, octa-, and nona-saccharides. In addition, a xyloglucan tetradecasaccharide (XG14) was split only to heptasaccharide (XG7) by CBHI and CBHII.