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Updated: Mar 16, 2026
Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
Published on: October 2, 2017
The 1.1 Å resolution structure of a periplasmic phosphate-binding protein from Stenotrophomonas maltophilia: a
Ronan Keegan1, David G Waterman1, David J Hopper2
1STFC, Rutherford Appleton Laboratory, Harwell Oxford, Didcot OX11 0FA, England.
Abstract:
During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) from Alcaligenes sp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement using MOLREP in which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in the MOLREP peak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. A BLAST search then indicated that the molecule was most probably a phosphate-binding protein from Stenotrophomonas maltophilia (UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of the S. maltophilia protein has been refined to an R factor of 10.15% and an Rfree of 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles.
Insights
Crystallization efforts for a dioxygenase enzyme unexpectedly yielded crystals of a phosphate-binding protein from Stenotrophomonas maltophilia. This protein is linked to bacterial virulence and biofilm formation, revealing new insights into pathogenic mechanisms.
Area of Science:
- Structural Biology
- Biochemistry
- Microbiology
Background:
- Initial attempts to crystallize 2,4-dihydroxyacetophenone dioxygenase (DAD) from Alcaligenes sp. 4HAP resulted in high-resolution crystals.
- These crystals were initially presumed to be DAD but failed molecular replacement analysis with known DAD structures.
- Further investigation ruled out common crystallization contaminants as the source of the crystals.
Purpose of the Study:
- To identify the protein responsible for the unexpected crystal formation.
- To determine the structure of the unknown protein and elucidate its function.
- To explore the potential implications of the identified protein in bacterial pathogenesis.
Main Methods:
- Systematic molecular replacement using a script that tested all PDB chains against the diffraction data.
- High-resolution (1.1 Å) electron density map calculation and de novo model building.
- Sequence identification via BLAST search and sequence fitting to the atomic resolution map.
- Structure refinement to R-factor 10.15% and R-free 12.46% at 1.1 Å resolution.
Main Results:
- The crystals were identified as a phosphate-binding protein from Stenotrophomonas maltophilia (UniProt ID B4SL31).
- The refined structure reveals a type II periplasmic binding protein fold with extensive loop regions.
- A dehydrated phosphate anion is tightly bound between the protein's two domains, interacting with conserved residues and helix dipoles.
Conclusions:
- The study successfully identified an unexpected protein contaminant in enzyme crystallization experiments.
- The high-resolution structure of the Stenotrophomonas maltophilia phosphate-binding protein provides detailed insights into its ligand-binding mechanism.
- This protein family's association with virulence and biofilm formation in antibiotic-resistant bacteria highlights its significance in pathogenic microbiology.
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