Closure of the Cytoplasmic Gate Formed by TM5 and TM11 during Transport in the Oxalate/Formate Exchanger from Oxalobacter formigenes
| Autor: | Osigbemhe Iyalomhe, Peter C. Maloney, David S. Cafiso, Dawn Z. Herrick |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Cytoplasm Protein Conformation Oxalobacter formigenes Biochemistry Article Oxalate 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Protein structure Bacterial Proteins Protein Interaction Domains and Motifs Formate 030304 developmental biology Mesylates 0303 health sciences biology Chemistry Membrane transport protein Oxalic Acid Electron Spin Resonance Spectroscopy Membrane Transport Proteins Biological Transport Periplasmic space biology.organism_classification Recombinant Proteins Major facilitator superfamily Kinetics Crystallography Cross-Linking Reagents Amino Acid Substitution Liposomes Periplasm Helix biology.protein Indicators and Reagents Mutant Proteins Spin Labels 030217 neurology & neurosurgery |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi5012173 |
| Popis: | OxlT, the oxalate/formate exchanger of Oxalobacter formigenes, is a member of the major facilitator superfamily of transporters. In the present work, substrate (oxalate) was found to enhance the reactivity of the cysteine mutant S336C on the cytoplasmic end of helix 11 to methanethiosulfonate ethyl carboxylate. In addition, S336C is found to spontaneously cross-link to S143C in TM5 in either native or reconstituted membranes under conditions that support transport. Continuous wave EPR measurements are consistent with this result and indicate that positions 143 and 336 are in close proximity in the presence of substrate. These two residues are localized within helix interacting GxxxG-like motifs (G₁₄₀LASG₁₄₄ and S₃₃₆DIFG₃₄₀) at the cytoplasmic poles of TM5 and TM11. Pulse EPR measurements were used to determine distances and distance distributions across the cytoplasmic or periplasmic ends of OxlT and were compared with the predictions of an inside-open homology model. The data indicate that a significant population of transporter is in an outside-open configuration in the presence of substrate; however, each end of the transporter exhibits significant conformational heterogeneity, where both inside-open and outside-open configurations are present. These data indicate that TM5 and TM11, which form part of the transport pathway, transiently close during transport and that there is a conformational equilibrium between inside-open and outside-open states of OxlT in the presence of substrate. |
| Databáze: | OpenAIRE |
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