Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli*
| Autor: | Erwin P. P. Vos, Jaap Broos, Ben H. Hesp, Milena Opacic |
|---|---|
| Přispěvatelé: | X-ray Crystallography |
| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Monosaccharide Transport Proteins
CYTOPLASMIC B-DOMAIN Plasma protein binding Substrate analog medicine.disease_cause 2ND-ORDER RATE CONSTANTS Biochemistry Models Biological chemistry.chemical_compound Membrane Biology medicine Fluorescence Resonance Energy Transfer ENZYME-IIMTL Mannitol Binding site Phosphorylation Phosphoenolpyruvate Sugar Phosphotransferase System Molecular Biology Escherichia coli Binding Sites biology Chemistry ACTIVE-SITE Escherichia coli Proteins Active site Cell Biology FLUORESCENCE DECAY Förster resonance energy transfer Spectrometry Fluorescence Membrane protein DEPENDENT PHOSPHOTRANSFERASE SYSTEM TRYPTOPHAN PHOSPHORESCENCE SPECTROSCOPY Membrane topology NMR-SPECTROSCOPY Biophysics biology.protein Protein Multimerization MEMBRANE TOPOLOGY TRANSIENT STATE KINETICS Protein Binding |
| Zdroj: | The Journal of Biological Chemistry, 285(33), 25324-25331. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC |
| ISSN: | 0021-9258 |
| Popis: | The mannitol transporter from Escherichia coli, EII(mtl), belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII(mtl) is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC(mtl)). To localize this binding site, 19 single Trp-containing mutants of EII(mtl) were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IIC(mtl). Combined with the available two-dimensional projection maps of IIC(mtl), it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EII(mtl) becomes phosphorylated at Cys(384) in the cytoplasmic B domain. Stably phosphorylated EII(mtl) mutants were constructed, and FRET experiments showed that the position of mannitol in IIC(mtl) remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|