scholarly journals The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding

2006 ◽  
Vol 396 (3) ◽  
pp. 469-477 ◽  
Author(s):  
Wei-I Chou ◽  
Tun-Wen Pai ◽  
Shi-Hwei Liu ◽  
Bor-Kai Hsiung ◽  
Margaret D.-T. Chang
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Catalytic Domain ◽  
Rhizopus Oryzae ◽  
Structural Information ◽  
Molecular Model ◽  
Site Directed Mutagenesis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Ligand Binding Sites

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.

scholarly journals The Family 6 Carbohydrate Binding ModuleCmCBM6-2 Contains Two Ligand-binding Sites with Distinct Specificities

2004 ◽  
Vol 279 (20) ◽  
pp. 21552-21559 ◽  
Author(s):  
Joanna L. Henshaw ◽  
David N. Bolam ◽  
Virgínia M. R. Pires ◽  
Mirjam Czjzek ◽  
Bernard Henrissat ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Carbohydrate Binding ◽  
The Family ◽  
Ligand Binding Sites

Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path

2008 ◽  
Vol 416 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Jung-Yu Tung ◽  
Margaret Dah-Tsyr Chang ◽  
Wei-I Chou ◽  
Yen-Yi Liu ◽  
Yi-Hung Yeh ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Hydrophobic Interaction ◽  
Binding Sites ◽  
Catalytic Domain ◽  
Rhizopus Oryzae ◽  
Binding Domain ◽  
Scatchard Analysis ◽  
Carbohydrate Binding ◽  
Binding Isotherm ◽  
Long Chain

GA (glucoamylase) hydrolyses starch and polysaccharides to β-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with β-cyclodextrin (SBD–βCD) and maltoheptaose (SBD–G7) were determined. Two carbohydrate binding sites, I (Trp47) and II (Tyr32), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr32 was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.

New opportunities for protease ligand-binding site comparisons using SitesBase

2007 ◽  
Vol 35 (3) ◽  
pp. 561-565 ◽  
Author(s):  
N.D. Gold ◽  
K. Deville ◽  
R.M. Jackson
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Structural Information ◽  
Cross Reactivity ◽  
Rapid Expansion ◽  
Structure Based Drug Design ◽  
Structure Database ◽  
Ligand Binding Sites ◽  
Source Of Information

The rapid expansion of structural information for protein ligand-binding sites is potentially an important source of information in structure-based drug design and in understanding ligand cross-reactivity and toxicity. We have developed SitesBase, a comprehensive database of ligand-binding sites extracted automatically from the Macromolecular Structure Database. SitesBase is an easily accessible database which is simple to use and holds pre-calculated information about structural similarities between known ligand-binding sites. These similarities are presented to the wider community enabling binding-site comparisons for therapeutically interesting protein families, such as the proteases and for new proteins to enable the discovery of interesting new structure–function relationships. The database is available from http://www.modelling.leeds.ac.uk/sb/.

scholarly journals Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jothi K. Yuvaraj ◽  
Rebecca E. Roberts ◽  
Yonathan Sonntag ◽  
Xiao-Qing Hou ◽  
Ewald Grosse-Wilde ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Bark Beetle ◽  
Pest Control ◽  
Binding Sites ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Odorant Receptors ◽  
Functional Evolution ◽  
General Importance ◽  
Insight Into

Abstract Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

scholarly journals Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus

2015 ◽  
Vol 471 (3) ◽  
pp. 403-414 ◽  
Author(s):  
M. Florencia Rey-Burusco ◽  
Marina Ibáñez-Shimabukuro ◽  
Mads Gabrielsen ◽  
Gisela R. Franchini ◽  
Andrew J. Roe ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Ligand Binding ◽  
Tissue Distribution ◽  
Binding Sites ◽  
Binding Proteins ◽  
Retinol Binding Protein ◽  
Internal Cavity ◽  
Binding Characteristics ◽  
Necator Americanus ◽  
Ligand Binding Sites

Necator americanus fatty acid and retinol-binding protein-1 (Na-FAR-1) is an abundantly expressed FAR from a parasitic hookworm. The present work describes its tissue distribution, structure and ligand-binding characteristics and shows that Na-FAR-1 expands to transport multiple FA molecules in its internal cavity.

scholarly journals Adenosine Receptors of Cerebral Microvessels and Choroid Plexus

1986 ◽  
Vol 6 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Rajesh N. Kalaria ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Ligand Binding ◽  
Choroid Plexus ◽  
Adenosine Receptors ◽  
Binding Sites ◽  
Specific Binding ◽  
Cerebral Microvessels ◽  
High Affinity ◽  
Receptor Sites ◽  
Ligand Binding Sites

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

A naturally occurring Tyr143HisαIIb mutation abolishes αIIbβ3 function for soluble ligands but retains its ability for mediating cell adhesion and clot retraction: comparison with other mutations causing ligand-binding defects

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3485-3491 ◽  
Author(s):  
Teruo Kiyoi ◽  
Yoshiaki Tomiyama ◽  
Shigenori Honda ◽  
Seiji Tadokoro ◽  
Morio Arai ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Binding Sites ◽  
Clot Retraction ◽  
Naturally Occurring ◽  
Binding Function ◽  
293 Cells ◽  
Ligand Binding Sites ◽  
Functional Defect ◽  
And Function

The molecular basis for the interaction between a prototypic non–I-domain integrin, αIIbβ3, and its ligands remains to be determined. In this study, we have characterized a novel missense mutation (Tyr143His) in αIIb associated with a variant of Glanzmann thrombasthenia. Osaka-12 platelets expressed a substantial amount of αIIbβ3(36%-41% of control) but failed to bind soluble ligands, including a high-affinity αIIbβ3-specific peptidomimetic antagonist. Sequence analysis revealed that Osaka-12 is a compound heterozygote for a single 521T>C substitution leading to a Tyr143His substitution in αIIb and for the null expression of αIIb mRNA from the maternal allele. Given that Tyr143 is located in the W3 4-1 loop of the β-propeller domain of αIIb, we examined the effects of Tyr143His or Tyr143Ala substitution on the expression and function of αIIbβ3 and compared them with KO (Arg-Thr insertion between 160 and 161 residues of αIIb) and with the Asp163Ala mutation located in the same loop by using 293 cells. Each of them abolished the binding function of αIIbβ3 for soluble ligands without disturbing αIIbβ3 expression. Because immobilized fibrinogen and fibrin are higher affinity/avidity ligands for αIIbβ3, we performed cell adhesion and clot retraction assays. In sharp contrast to KO mutation and Asp163AlaαIIbβ3, Tyr143HisαIIbβ3-expressing cells still had some ability for cell adhesion and clot retraction. Thus, the functional defect induced by Tyr143HisαIIb is likely caused by its allosteric effect rather than by a defect in the ligand-binding site itself. These detailed structure–function analyses provide better understanding of the ligand-binding sites in integrins.

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