Identification of the Distance between the Homologous Halves of P-glycoprotein That Triggers the High/Low ATPase Activity Switch
| Autor: | Tip W. Loo, David M. Clarke |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Protein Conformation ATPase ATP-binding cassette transporter Biochemistry Mice chemistry.chemical_compound Adenosine Triphosphate Protein structure Membrane Biology Animals Humans Point Mutation ATP Binding Cassette Transporter Subfamily B Member 1 Molecular Biology P-glycoprotein biology Cell Biology Transmembrane protein Transmembrane domain Cross-Linking Reagents Membrane protein chemistry biology.protein Biophysics Adenosine triphosphate |
| Zdroj: | Journal of Biological Chemistry. 289:8484-8492 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m114.552075 |
| Popis: | P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. Each homologous half contains a transmembrane domain with six transmembrane segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the transmembrane domain and NBDs, respectively. Drug binding activates ATPase activity by an unknown mechanism. There is no high resolution structure of human P-gp, but homology models based on the crystal structures of bacterial, mouse, and Caenorhabditis elegans ATP-binding cassette drug pumps yield both open (NBDs apart) and closed (NBDs together) conformations. Molecular dynamics simulations predict that the NBDs can be separated over a range of distances (over 20 Å). To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 Å (α-carbons). We observed that ATPase activity increased over 10-fold when the cysteines were cross-linked at distances between 6 and 19 Å, although cross-linking at distances greater than 20 Å yielded basal levels of activity. The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 Å, respectively. We predict that the high/low ATPase activity switch may occur at a distance where the NBDs are predicted in molecular dynamic simulations to undergo pronounced twisting as they approach each other (Wise, J. G. (2012) Biochemistry 51, 5125-5141). |
| Databáze: | OpenAIRE |
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