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Loop B is a major structural component of the 5-HT3 receptor.

Thompson AJ, Lochner M, Lummis SC - Biophys. J. (2008)

Bottom Line: Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor.The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein.Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, United Kingdom.

ABSTRACT
The 5-HT(3) receptor belongs to a family of therapeutically important neurotransmitter-gated receptors whose ligand binding sites are formed by the convergence of six peptide loops (A-F). Here we have mutated 15 amino acid residues in and around loop B of the 5-HT(3) receptor (Ser-177 to Asn-191) to Ala or a residue with similar chemical properties. Changes in [3H]granisetron binding affinity (K(d)) and 5-HT EC(50) were determined using receptors expressed in human embryonic kidney 293 cells. Substitutions at all but one residue (Thr-181) altered or eliminated binding for one or both mutants. Receptors were nonfunctional or EC(50) values were altered for all but two mutants (S182T, I190L). Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor. The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein. Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

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Location of the amino acid residues examined in the current study. (A) Two adjacent subunits (principal and complementary), showing the positions of the six binding loops A-F. (B) The amino acid sequence for the extracellular domain of the murine 5-HT3A receptor (accession No. Q6J1J7), aligned with AChBP isolated from Lymnaea stagnalis (P58154) and the nACh receptor α1 subunit (P02710). The residues examined are highlighted as white text on a black background. Loop F residues that we have reported on previously are highlighted as white text on a gray background (25). The six binding loops are indicated by black lines above the text. The positions of β-sheets are shown by gray lines beneath the text. Numbering of residues and structural features are taken from the AChBP protein crystal structure (3).
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fig1: Location of the amino acid residues examined in the current study. (A) Two adjacent subunits (principal and complementary), showing the positions of the six binding loops A-F. (B) The amino acid sequence for the extracellular domain of the murine 5-HT3A receptor (accession No. Q6J1J7), aligned with AChBP isolated from Lymnaea stagnalis (P58154) and the nACh receptor α1 subunit (P02710). The residues examined are highlighted as white text on a black background. Loop F residues that we have reported on previously are highlighted as white text on a gray background (25). The six binding loops are indicated by black lines above the text. The positions of β-sheets are shown by gray lines beneath the text. Numbering of residues and structural features are taken from the AChBP protein crystal structure (3).

Mentions: All 15 amino acids within a contiguous sequence of the 5-HT3A receptor subunit (Fig. 1) were mutated to either Ala or an amino acid with properties similar to the wild-type amino acid (subsequently referred to as a conserved amino acid change). Mutant receptors were characterized using [3H]granisetron binding to explore changes in the ligand binding site (Table 1, Fig. 2), and 5-HT induced changes in membrane potential were measured using a voltage-sensitive fluorescent dye (Table 2, Fig. 2). Wild-type receptors had a [3H]granisetron binding affinity (Kd) of 0.5 nM (n = 11) and 5-HT EC50 of 0.24 μM (n = 12), values that are similar to those previously published using the same techniques (15,24,25).


Loop B is a major structural component of the 5-HT3 receptor.

Thompson AJ, Lochner M, Lummis SC - Biophys. J. (2008)

Location of the amino acid residues examined in the current study. (A) Two adjacent subunits (principal and complementary), showing the positions of the six binding loops A-F. (B) The amino acid sequence for the extracellular domain of the murine 5-HT3A receptor (accession No. Q6J1J7), aligned with AChBP isolated from Lymnaea stagnalis (P58154) and the nACh receptor α1 subunit (P02710). The residues examined are highlighted as white text on a black background. Loop F residues that we have reported on previously are highlighted as white text on a gray background (25). The six binding loops are indicated by black lines above the text. The positions of β-sheets are shown by gray lines beneath the text. Numbering of residues and structural features are taken from the AChBP protein crystal structure (3).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2599823&req=5

fig1: Location of the amino acid residues examined in the current study. (A) Two adjacent subunits (principal and complementary), showing the positions of the six binding loops A-F. (B) The amino acid sequence for the extracellular domain of the murine 5-HT3A receptor (accession No. Q6J1J7), aligned with AChBP isolated from Lymnaea stagnalis (P58154) and the nACh receptor α1 subunit (P02710). The residues examined are highlighted as white text on a black background. Loop F residues that we have reported on previously are highlighted as white text on a gray background (25). The six binding loops are indicated by black lines above the text. The positions of β-sheets are shown by gray lines beneath the text. Numbering of residues and structural features are taken from the AChBP protein crystal structure (3).
Mentions: All 15 amino acids within a contiguous sequence of the 5-HT3A receptor subunit (Fig. 1) were mutated to either Ala or an amino acid with properties similar to the wild-type amino acid (subsequently referred to as a conserved amino acid change). Mutant receptors were characterized using [3H]granisetron binding to explore changes in the ligand binding site (Table 1, Fig. 2), and 5-HT induced changes in membrane potential were measured using a voltage-sensitive fluorescent dye (Table 2, Fig. 2). Wild-type receptors had a [3H]granisetron binding affinity (Kd) of 0.5 nM (n = 11) and 5-HT EC50 of 0.24 μM (n = 12), values that are similar to those previously published using the same techniques (15,24,25).

Bottom Line: Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor.The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein.Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, United Kingdom.

ABSTRACT
The 5-HT(3) receptor belongs to a family of therapeutically important neurotransmitter-gated receptors whose ligand binding sites are formed by the convergence of six peptide loops (A-F). Here we have mutated 15 amino acid residues in and around loop B of the 5-HT(3) receptor (Ser-177 to Asn-191) to Ala or a residue with similar chemical properties. Changes in [3H]granisetron binding affinity (K(d)) and 5-HT EC(50) were determined using receptors expressed in human embryonic kidney 293 cells. Substitutions at all but one residue (Thr-181) altered or eliminated binding for one or both mutants. Receptors were nonfunctional or EC(50) values were altered for all but two mutants (S182T, I190L). Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor. The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein. Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

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