Structure, function, and evolution of biogenic amine-binding proteins in soft ticks

Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to the lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.     

NMR solution structure of lipocalin-type prostaglandin D synthase: evidence for partial overlapping of catalytic pocket and retinoic acid-binding pocket within the central cavity

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2), a common precursor of various prostanoids, to produce PGD(2), an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD(2)-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel beta-barrel and a long alpha-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH(2) analog, revealed that PGH(2) almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH(2) binding mode. These results indicated that the two binding sites for PGH(2) and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.     

Tick histamine-binding proteins: isolation, cloning, and three-dimensional structure.

High-affinity histamine-binding proteins (HBPs) were discovered in the saliva of Rhipicephalus appendiculatus ticks. Their ability to outcompete histamine receptors indicates that they suppress inflammation during blood feeding. The crystal structure of a histamine-bound HBP, determined at 1.25 A resolution, reveals a lipocalin fold novel in containing two binding sites for the same ligand. The sites are orthogonally arranged and highly rigid and form an internal surface of unusual polar character that complements the physicochemical properties of histamine. As soluble receptors of histamine, HBPs offer a new strategy for controlling histamine-based diseases.     

<Emphasis Type="Italic">In Silico</Emphasis> Study on Retinoid-binding Modes in Human RBP and ApoD Lipocalins

Lipocalins are proteins with highly homologous structures but diverse sequences that are potential candidates for scaffold protein engineering with novel ligand-binding functions. Numerous crystal structures of lipocalin-ligand complexes have been identified and used in the study of their binding modes. On the other hand, crystallization studies cannot meet the increasing demand for novel lipocalin-ligand complexes in scaffold engineering, which requires rapid computational analyses of their binding modes in parallel. Human retinol-binding protein (RBP) and apolipoprotein D (apoD) are sequentially very distant proteins, but they show tight binding against retinoids, such as retinol and retinoic acid. In the present study, complexes of the two lipocalins with retinol and retinoic acid were modeled computationally by a molecular docking simulation, and their ligand-binding modes were analyzed at a molecular level. The models identified the crucial residues of lipocalins that interact with the ligands and revealed the similarities and differences in their retinoid-binding modes as well as in the specific interactions of the retinoid species within the same lipocalin. An analysis of the amino acid propensity of the retinoid-binding residues suggested that the evolutionary preference of the residues is restricted to the binding pocket rather than the entire protein. The distribution of charged residues around the terminus of retinoic acid showed a huge difference between RBP and ApoD, which might be a factor for the different binding affinities of lipocalins against retinoic acid. This in silico study is expected to be applied to scaffold protein engineering for novel retinoid-binding lipocalins.     

Structural and active site analysis of plasmepsins of Plasmodium falciparum: potential anti-malarial targets

Comparative protein modeling, active site analysis and binding site specificity for the homologous series of plasmepsins (PM's), present in food vacuole of Plasmodium falciparum, are carried out. Four loops (L1, L2, L3 and L4), which show maximum structural deviations irrespective of type of inhibitor, have been identified. Comparison of the crystal structures of ligand complexes reveal that residues belonging to these loops have negligible coulomb and VDW interactions with the inhibitor but play major role in determining the openness of the binding cavity. The coulomb and VDW interactions between the PMII subsite pockets and inhibitors, which play a major role in determining the inhibition constants, are delineated. Besides small displacements, the catalytic residues D32 of PMII undergoes rotation around the Cgamma-Cbeta single bond to assist catalysis whereas side chain conformational deviations are not observed in D214 on plasmepsin activation. The mutant S79D of PMII (and the corresponding residues of PMI and PMIV) which helps in recognizing and cleaving substrates containing lysine at P1 position is surrounded by highly polar atmosphere stabilized by lysine. However, in PMIII significantly lower polar atmosphere around the mutant A78S/A78D is observed. Large buried side chain area of residues located at M15 and I289 of PMII (and corresponding residues of PMI and PMIV) corroborates well with increase in specificity constant for hydrophobic substrates.     

Comparative analysis of different competitive antagonists interaction with NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor

The antagonist-bound conformation of the NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor are modeled using the crystal structure of the DCKA (5,7-dichloro-kynurenic acid)-bound form of the NR1 subunit ligand-binding core (S1S2). Five different competitive NMDA receptor antagonists [(1) DL-AP5; (2) DL-AP7; (3) CGP-37847; (4) CGP 39551; (5) (RS)-CPP] have been docked into both NR2A and NR2B subunits. Experimental studies report NR2A and NR2B subunits having dissimilar interactions and affinities towards the antagonists. However, the molecular mechanism of this difference remains unexplored. The distinctive features in the antagonist's interaction with these two different but closely related (approximately 80% sequence identity at this region) subunits are analyzed from the patterns of their hydrogen bonding. The regions directly involved in the antagonist binding have been classified into seven different interaction sites. Two conserved hydrophilic pockets located at both the S1 and S2 domains are found to be crucial for antagonist binding. The positively charged (Lys) residues present at the second interaction site and the invariant residue (Arg) located at the fourth interaction site are seen to influence ligand binding. The geometry of the binding pockets of NR2A and NR2B subunits have been determined from the distance between the C-alpha atoms in the residues interacting with the ligands. The binding pockets are found to be different for NR2A and NR2B. There are gross dissimilarities in competitive antagonist binding between these two subunits. The binding pocket geometry identified in this study may have the potential for future development of selective antagonists for the NR2A or NR2B subunit.     

Exon-intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family

All tick proteins assigned to the lipocalin family lack the structural conserved regions (SCRs) that are characteristic of the kernel lipocalins and can thus be classified as outliers. These tick proteins have been assigned to the tick lipocalin family based on database searches that indicated homology between tick sequences and the fact that the histamine binding protein (HBP2) from the hard tick Rhipicephalus appendiculatus (Ixodidae) shows structural similarity to the lipocalin fold. Sequence identity between kernel and outlier lipocalins falls below 20% and the question raised is whether the outlier and kernel lipocalins are truly homologous. More specifically in the case of the tick lipocalins, whether their structural fold is derived from the lipocalin fold or whether convergent evolution resulted in the generation of the basic lipocalin-like fold which consists of an eight stranded continuous anti-parallel beta-barrel terminated by a C-terminal alpha-helix that lies parallel to the barrel. The current study determined the gene structure for HBP2 and TSGP1, TSGP2 and TSGP4, lipocalins identified from the soft tick Ornithodoros savignyi (Argasidae). All tick lipocalins have four introns (A-D) with conserved positions and phases within the tick lipocalin sequence alignment. The positions and phase information are also conserved with regard to the rest of the lipocalin family. Phylogenetic analysis using this information shows conclusively that tick lipocalins are evolutionary related to the rest of the lipocalin family. Tick lipocalins are grouped within a monophyletic clade that indicates a monophyletic origin within the tick lineage and also group with the other arthropod lipocalins in a larger clade. Phylogenetic analysis of sequence alignments based on conserved secondary structure of the lipocalin fold support the conclusions from the gene structure trees. These results indicate that exon-intron arrangement can be useful for the inclusion of outlier lipocalins within the larger lipocalin family.     

1H and 31P nuclear magnetic resonance investigation of the interaction between 2,3-diphosphoglycerate and human normal adult hemoglobin

High-resolution 1H and 31P nuclear magnetic resonance spectroscopy has been used to investigate the binding of 2,3-diphosphoglycerate to human normal adult hemoglobin and the molecular interactions involved in the allosteric effect of the 2,3-diphosphoglycerate molecule on hemoglobin. Individual hydrogen ion NMR titration curves have been obtained for 22-26 histidyl residues of hemoglobin and for each phosphate group of 2,3-diphosphoglycerate with hemoglobin in both the deoxy and carbonmonoxy forms. The results indicate that 2,3-diphosphoglycerate binds to deoxyhemoglobin at the central cavity between the two beta chains and the binding involves the beta 2-histidyl residues. Moreover, the results suggest that the binding site of 2,3-diphosphoglycerate to carbonmonoxyhemoglobin contains the same (or at least some of the same) amino acid residues responsible for binding in the deoxy form. As a result of the specific interactions with 2,3-diphosphoglycerate, the beta 2-histidyl residues make a significant contribution to the alkaline Bohr effect under these experimental conditions (up to 0.5 proton/Hb tetramer). 2,3-Diphosphoglycerate also affects the individual hydrogen ion equilibria of several histidyl residues located away from the binding site on the surface of the hemoglobin molecule, and, possibly, in the heme pockets. These results give the first experimental demonstration that long-range electrostatic and/or conformational effects of the binding could play an important role in the allosteric effect of 2,3-diphosphoglycerate on hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbonic anhydrase activators: kinetic and X-ray crystallographic study for the interaction of D- and L-tryptophan with the mammalian isoforms I-XIV

An activation study of mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms I-XIV with D- and L-tryptophan has been performed both by means of kinetic and X-ray crystallographic techniques. These compounds show a time dependent activity against isozyme CA II, with activation constants of 1.13 microM for L-Trp and 0.37 microM for D-Trp, respectively, after 24 h of incubation between enzyme and activator. The high resolution X-ray crystal structure of the hCA II-D-Trp adduct revealed the activator to bind in a totally unprecedented way to the enzyme active site as compared to histamine, L-/D-Phe, L-/D-His or L-adrenaline. D-Trp is anchored at the edge of the CA II active site entrance, strongly interacting with amino acid residues Asp130, Phe131 and Gly132 as well as with a loop of a second symmetry related protein molecule from the asymmetric unit, by means of hydrogen bonds and several weak van der Waals interactions involving Glu234, Gly235, Glu236 and Glu238. Thus, a second activator binding site (B) within the CA II cavity has been detected, where only D-Trp was shown so far to bind, in addition to the activator binding site A, in which histamine, L-/D-Phe, and L-/D-His are bound. These findings explain the strong affinity of D-Trp for CA II and may be useful for designing novel classes of CA activators by using this compound as lead molecule.     

A method for localizing ligand binding pockets in protein structures

The accurate identification of ligand binding sites in protein structures can be valuable in determining protein function. Once the binding site is known, it becomes easier to perform in silico and experimental procedures that may allow the ligand type and the protein function to be determined. For example, binding pocket shape analysis relies heavily on the correct localization of the ligand binding site. We have developed SURFNET-ConSurf, a modular, two-stage method for identifying the location and shape of potential ligand binding pockets in protein structures. In the first stage, the SURFNET program identifies clefts in the protein surface that are potential binding sites. In the second stage, these clefts are trimmed in size by cutting away regions distant from highly conserved residues, as defined by the ConSurf-HSSP database. The largest clefts that remain tend to be those where ligands bind. To test the approach, we analyzed a nonredundant set of 244 protein structures from the PDB and found that SURFNET-ConSurf identifies a ligand binding pocket in 75% of them. The trimming procedure reduces the original cleft volumes by 30% on average, while still encompassing an average 87% of the ligand volume. From the analysis of the results we conclude that for those cases in which the ligands are found in large, highly conserved clefts, the combined SURFNET-ConSurf method gives pockets that are a better match to the ligand shape and location. We also show that this approach works better for enzymes than for nonenzyme proteins.     

Catalytic site modifications of TAP1 and TAP2 and their functional consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.     

Mechanism of membrane binding of the phospholipase D1 PX domain

Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphosphate (PtdIns-(3,4,5)P(3)) > phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate > other phosphoinositides. The PtdIns(3,4,5)P(3) binding was facilitated by the cationic residues (Lys(119), Lys(121), and Arg(179)) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P(3) to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P(3) and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1.     

The interaction of folylpolyglutamates with deoxyhemoglobin. Identification of the binding site

Previous studies have shown that pteroylheptaglutamate (PteGlu7) can form a 1:1 complex with deoxyhemoglobin. The solution and crystallographic studies reported in this paper delineate the nature of the PteGlu7 binding site. We find that the three structural elements of PteGlu7 (the pteridine moiety, the p-aminobenzoyl portion, and the glutamate groups) each contribute to the binding energy by interacting with residues in the central cavity between the beta subunits and with residues at the alpha 1 beta 1 interface. Identification of the 2,3-diphosphoglycerate (DPG) binding site as part of the PteGlu7 binding site was accomplished in two ways; first by the demonstration of reduced PteGlu7 binding to hemoglobin selectively modified by pyridoxylation at this site, and second by the finding that DPG and PteGlu7 bind to deoxyhemoglobin in a competitive manner. In addition, since analogs of PteGlu7 in which the pteridine moiety is modified display reduced binding, it can be concluded that the pteridine group also contributes significantly to the binding energy. The crystallographic studies are completely consistent with the results determined in solution. A difference electron density image at 4.3 A resolution shows that the pteridine and p-aminobenzoyl groups are nestled against an interior edge of the alpha 1 beta 1 interface with the pteridine ring interacting with Phe 36 alpha 1 and the p-aminobenzoyl group positioned against a portion of the H helix between residues Lys 132 beta 1 and Ala 135 beta 1. The difference density for the glutamate residues is less well resolved (for reasons described in the text), but it is clear that some of the carboxylate side chains must interact with residues at the DPG binding site.     

Role of binding pockets for amino-terminal peptide residues in HLA-B27 allorecognition.

The peptide binding site of HLA-B27 and other class I Ag consists of a series of pockets that bind peptide side chains. Two of these pockets interact with the amino-terminal peptide residue (pocket A) and with the highly conserved second residue (pocket B). In this study, the role of pockets A and B in HLA-B27-specific T cell allorecognition has been analyzed. Four HLA-B27 mutants with single or double changes in pocket B (24T----A, 45E----M, 67C----V, and 24,67T,C----A,V) and three mutants with single changes in pocket A (163E----T, 167W----S, and 171Y----H) were constructed by site-directed mutagenesis and expressed in HMy2.C1R cells after DNA-mediated gene transfer. These transfectants were used as target cells in cytotoxicity assays with a series of HLA-B27-specific CTL. All the mutations analyzed affected allorecognition by a significant proportion of the CTL tested, but no single change abrogated recognition by all CTL. The global effects of each mutation on allorecognition were comparable to one another, except for the effect of the change at position 67, which was smaller. The behavior of individual CTL with the mutants was very diverse, ranging from CTL that did not recognize most of the mutants to CTL recognizing all of them. Thus, some alloreactive CTL can withstand drastic alterations in pockets A and B. Two CTL showed heteroclytic effects towards the V67 and M45 mutants. CTL behavior with the H171 mutant was closely parallel to that with the B*2703 subtype, having a single Y----H change at position 59. This parallelism correlates with the similar role of Tyr59 and Tyr171 in establishing hydrogen bonds with the amino termini of HLA-B27-bound peptides. The results demonstrate that altering the structure of pockets that interact with the amino-terminal first and second residues of HLA-B27-bound peptides significantly affects recognition by alloreactive CTL, and they strongly suggest widespread peptide involvement in HLA-B27 allorecognition.     

Molecular basis for the anti-sickling activity of aromatic amino acids and related compounds: a proton nuclear magnetic resonance investigation

High-resolution proton nuclear magnetic resonance spectroscopy and relaxation techniques have been used to investigate the interactions of sickle cell hemoglobin (Hb S) and human normal adult hemoglobin (Hb A) with p-bromobenzyl alcohol, L-phenylalanine, L-tryptophan, and L-valine. With the exception of valine, all these compounds inhibit the polymerization of deoxy-Hb S [Noguchi, C. T., & Schechter, A. N. (1978) Biochemistry 17, 5455)). Using transferred nuclear Overhauser effects among the proton resonances of the compound of interest and the corresponding longitudinal relaxation rates (T1(-1], we have shown that the binding of each of the compounds investigated to deoxy-Hb S is comparable to that to deoxy-Hb A. Intermolecular transferred nuclear Overhauser effects have been observed between proton resonances of the anti-sickling compounds and specific protons situated in the heme pockets of Hb. On the basis of these results, we suggest that one binding site, common to all compounds with anti-sickling activity, is at or near the heme pockets in the alpha and beta chains of both deoxy-HB S and deoxy-Hb A. The proton T1(-1) values of the histidyl residues situated over the surface of the hemoglobin molecule indicate that a second binding site is located at or near the beta 6 position, containing the mutation in Hb S (beta 6Glu----Val). The binding of the compounds investigated to the latter site induces conformational changes in the amino-terminal domains of the beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

The crystal structure of N(10)-formyltetrahydrofolate synthetase from Moorella thermoacetica

The structure was solved at 2.5 A resolution using multiwavelength anomalous dispersion (MAD) scattering by Se-Met residues. The subunit of N(10)-formyltetrahydrofolate synthetase is composed of three domains organized around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modeling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha-helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half-barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface.     

Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes

Porcine odorant binding protein (pOBP) is a monomer of 157 amino acid residues, purified in abundance from pig nasal mucosa. In contrast to the observation on lipocalins as retinol binding protein (RBP), major urinary protein (MUP) or bovine odorant binding protein (bOBP), no naturally occurring ligand was found in the beta-barrel cavity of pOBP. Porcine OBP was therefore chosen as a simple model for structure/function studies with odorant molecules. In competition experiments with tritiated pyrazine, the affinity of pOBP towards several odorant molecules belonging to different chemical classes has been found to be of the micromolar order, with a 1:1 stoichiometry. The X-ray structures of pOBP complexed to these molecules were determined at resolution between 2.15 and 1.4 A. As expected, the electron density of the odorant molecules was observed into the hydrophobic beta-barrel of the lipocalin. Inside this cavity, very few specific interactions were established between the odorant molecule and the amino acid side-chains, which did not undergo significant conformational change. The high B-factors observed for the odorant molecules as well as the existence of alternative conformations reveal a non-specific mode of binding of the odorant molecules in the cavity.     

Immunocalins: a lipocalin subfamily that modulates immune and inflammatory responses

A subset of the lipocalins, notably alpha(1)-acid glycoprotein, alpha(1)-microglobulin, and glycodelin, exert significant immunomodulatory effects in vitro. Interestingly, all three are encoded from the q32-34 region of human chromosome 9, together with at least four other lipocalins (neutrophil gelatinase-associated lipocalin, complement factor gamma-subunit, tear prealbumin, and prostaglandin D synthase) that also may have anti-inflammatory and/or antimicrobial activity. This review addresses important features of this genetically linked subfamily of lipocalins (involvement with the acute phase response, immunomodulatory and anti-inflammatory properties, the tissue localization, complex formation with other proteins and receptors, etc.). It is likely that these proteins have evolved to be an integrated part of the body's defense system as part of the extended cytokine network. Its members exert a regulatory, dampening influence on the inflammatory cascade, thereby protecting against tissue damage from excessive inflammation. That most major mammalian allergens are lipocalins may reflect this connection of lipocalins with the immune system. We propose that this immunologically active lipocalin subset be named the 'immunocalins', signifying not only the structural homology and close genetic linkage of its members, but also their protective involvement with immunological and inflammatory processes. As immune mediators, immunocalins appear to use at least three interactive sites: the lipocalin 'pocket', binding sites for other plasma proteins, and binding sites for cell surface receptors.     

Computational selection, identification and structural analysis of ω-aminotransferases with various substrate specificities from the genome sequence of Mesorhizobium loti MAFF303099

ω-Aminotransferase (ω-AT) is an important class of enzymes for the synthesis of chiral amines or β-amino acids. Family profile analysis was applied to screen putative ω-ATs from Mesorhizobium loti MAFF303099, a nitrogen fixation bacterium that has a larger number of ATs than other microorganisms. By family profile analysis, we selected 10 putative ω-ATs according to E-value. The functions of the putative ω-ATs were investigated by examining activities towards amines and/or β-amino acids. 10 putative proteins were found to have ω-AT activity with narrow or broad substrate specificity. Structure analysis using crystal structure of mll7127 and homology models of mll1632 and mll3663 indicated that the structures of active sites of the enzymes were very similar and highly conserved, but their substrate specificities appeared to be determined by residues positioned at the entrance region of the active site binding pockets.