Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

CBMs (carbohydrate-binding modules) function independently to assist carbohydrate-active enzymes. Family 21 CBMs contain approx. 100 amino acid residues, and some members have starchbinding functions or glycogen-binding activities. We report here the first structure of a family 21 CBM from the SBD (starch-binding domain) of Rhizopus oryzae glucoamylase (RoCBM21) determined by NMR spectroscopy. This CBM has a β-sandwich fold with an immunoglobulin-like structure. Ligand-binding properties of RoCBM21 were analysed by chemical-shift perturbations and automated docking. Structural comparisons with previously reported SBDs revealed two types of topologies, namely type I and type II, with CBM20, CBM25, CBM26 and CBM41 showing type I topology, with CBM21 and CBM34 showing type II topology. According to the chemical-shift perturbations, RoCBM21 contains two ligand-binding sites. Residues in site II are similar to those found in the family 20 CBM from Aspergillus niger glucoamylase (AnCBM20). Site I, however, is embedded in a region with unique sequence motifs only found in some members of CBM21s. Additionally, docking of β-cyclodextrin and malto-oligosaccharides highlights that side chains of Y83 and W47 (one-letter amino acid code) form the central part of the conserved binding platform in the SBD. The structure of RoCBM21 provides the first direct evidence of the structural features and the basis for protein–carbohydrate recognition from an SBD of CBM21.

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The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Journal of Biological Chemistry ◽

10.1074/jbc.m109142200 ◽

2001 ◽

Vol 276 (51) ◽

pp. 48580-48587 ◽

Cited By ~ 78

Author(s):

Mirjam Czjzek ◽

David N. Bolam ◽

Amor Mosbah ◽

Julie Allouch ◽

Carlos M. G. A. Fontes ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Carbohydrate Binding ◽

Ligand Binding Site ◽

Carbohydrate Binding Modules ◽

Common Sequence

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Determination of Amino Acid Residues that are Accessible from the Ligand Binding Crevice in the Seventh Transmembrane-Spanning Region of the Human A1 Adenosine Receptor

Molecular Pharmacology ◽

10.1124/mol.59.5.1187 ◽

2001 ◽

Vol 59 (5) ◽

pp. 1187-1195 ◽

Cited By ~ 13

Author(s):

Eric S. Dawson ◽

Jack N. Wells

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Adenosine Receptor ◽

Amino Acid Residues ◽

A1 Adenosine Receptor

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A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽

10.1182/blood.v93.3.918 ◽

1999 ◽

Vol 93 (3) ◽

pp. 918-924 ◽

Cited By ~ 19

Author(s):

Eileen Collins Tozer ◽

Elizabeth K. Baker ◽

Mark H. Ginsberg ◽

Joseph C. Loftus

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Single Amino Acid ◽

Chimeric Receptor ◽

Amino Acid Residues ◽

Genetic Approach ◽

Specific Amino Acid ◽

Transfected Cells ◽

Binding Function ◽

A Cell

Abstract An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

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Identification of Amino Acid Residues That Determine pH Dependence of Ligand Binding to the Asialoglycoprotein Receptor during Endocytosis

Journal of Biological Chemistry ◽

10.1074/jbc.274.50.35400 ◽

1999 ◽

Vol 274 (50) ◽

pp. 35400-35406 ◽

Cited By ~ 36

Author(s):

Stephanie Wragg ◽

Kurt Drickamer

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Ph Dependence ◽

Asialoglycoprotein Receptor ◽

Amino Acid Residues

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The Differential Binding Affinities of the Luteinizing Hormone (LH)/Choriogonadotropin Receptor for LH and Choriogonadotropin Are Dictated by Different Extracellular Domain Residues

Molecular Endocrinology ◽

10.1210/me.2004-0410 ◽

2005 ◽

Vol 19 (5) ◽

pp. 1263-1276 ◽

Cited By ~ 19

Author(s):

Colette Galet ◽

Mario Ascoli

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Binding Affinity ◽

Extracellular Domain ◽

Binding Affinities ◽

Amino Acid Residues ◽

Identify Amino Acid ◽

Β Sheets ◽

Leucine Rich Repeats ◽

High Degree

Abstract The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the β-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the β-sheets of LRR1–9 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the β-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.

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