Abstract
G-protein-coupled receptors (GPCRs)-the largest family of cell-surface membrane proteins-mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Angiotensin II / chemistry
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Angiotensin II / metabolism
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Animals
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Antibodies, Monoclonal / chemistry*
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Antibodies, Monoclonal / genetics
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Antibodies, Monoclonal / isolation & purification
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Antibodies, Monoclonal / metabolism
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Baculoviridae / genetics
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Baculoviridae / metabolism
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Binding Sites
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Cloning, Molecular
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Crystallography, X-Ray
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Cytochrome b Group / chemistry*
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Cytochrome b Group / genetics
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Cytochrome b Group / metabolism
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Ergotamine / chemistry
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Ergotamine / metabolism
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Escherichia coli / chemistry
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Humans
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Immunoglobulin Fab Fragments / chemistry*
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Immunoglobulin Fab Fragments / genetics
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Immunoglobulin Fab Fragments / isolation & purification
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Immunoglobulin Fab Fragments / metabolism
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Mice
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Models, Molecular
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Protein Binding
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Protein Conformation, alpha-Helical
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Receptor, Angiotensin, Type 2 / chemistry*
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Receptor, Angiotensin, Type 2 / genetics
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Receptor, Angiotensin, Type 2 / metabolism
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Receptor, Serotonin, 5-HT1B / chemistry*
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Receptor, Serotonin, 5-HT1B / genetics
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Receptor, Serotonin, 5-HT1B / metabolism
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Recombinant Fusion Proteins / chemistry*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Sf9 Cells
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Spodoptera
Substances
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Antibodies, Monoclonal
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Cytochrome b Group
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Escherichia coli Proteins
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Immunoglobulin Fab Fragments
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Receptor, Angiotensin, Type 2
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Receptor, Serotonin, 5-HT1B
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Recombinant Fusion Proteins
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Angiotensin II
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cytochrome b562, E coli
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Ergotamine