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Structure determination of OppA at 2.3 A resolution using multiple-wavelength anomalous dispersion methods
I D Glover1, R C Denny, N D Nguti
1Department of Physics, Keele University, Staffordshire, England.
Acta Crystallographica. Section D, Biological Crystallography
|January 1, 1995
Summary
The bacterial peptide transporter OppA (Oligopeptide Permease A) structure was determined using X-ray crystallography. This reveals its bilobal, beta-stranded, three-domain architecture and peptide-binding site.
Area of Science:
- Structural biology
- Bacterial physiology
- Protein crystallography
Background:
- OppA is a bacterial transport protein crucial for peptide uptake across the cytoplasmic membrane.
- It exhibits broad specificity, binding peptides of 2-5 residues.
- Understanding OppA's structure is key to elucidating its transport mechanism.
Purpose of the Study:
- To determine the high-resolution crystal structure of OppA from Salmonella typhimurium.
- To visualize the peptide-binding site within the OppA protein structure.
Main Methods:
- Cloning and expression of OppA in E. coli.
- Cocrystallization with uranyl acetate to obtain two crystal forms.
- Multiple-wavelength anomalous diffraction (MAD) data collection at the uranium L(III) edge.
- Maximum-likelihood phasing for phase determination.
- X-ray crystallography at 2.3 A resolution.
Main Results:
- Successfully determined the electron-density map of OppA at 2.3 A resolution.
- Revealed OppA as a bilobal protein composed primarily of beta-strands across three domains.
- Identified the tri-lysine ligand clearly situated within the peptide-binding pocket between the two lobes.
Conclusions:
- The determined structure provides unprecedented insight into the architecture of OppA.
- The visualization of the bound tri-lysine ligand confirms the peptide-binding site's location and accessibility.
- This structural information will facilitate further studies on the mechanism of bacterial peptide transport.