CusF-Ag(I) Structure Reveals Unusual Cu(I)/Ag(I) Coordination
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The document reports on research into the E. coli protein CusF. Key findings include: 1) The crystal structure of CusF bound to Ag(I) was determined to 1.0 angstrom resolution, revealing Ag(I) is coordinated by methionines M47 and M49 and histidine H36 in a beta barrel structure similar to the apo structure. 2) EXAFS was used to study CusF bound to Cu(I) since crystals could not be grown, showing Cu(I) is in a three-coordinate environment involving the same ligands. 3) Together the structures provide insight into how CusF sequesters toxic Cu(I) and Ag
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CusF-Ag(I) Structure Reveals Unusual Cu(I)/Ag(I) Coordination
- 2. Rudy Alvarado Jordan Davis Jennifer Jones Raul Soto
- 3. Unusual Cu(I) / Ag(I) coordination of Escherichia coli CusF as revealed by atomic resolution crystallography and X- ray absorption spectroscopy Isabell R Loftin, Sylvia Franke, Ninian J Blackburn, Megan M McEvoy Protein Science (2007), 16:2287 – 2293.
- 4. 2QCP in RCSB Protein Data Bank Periplasmic copper/silver-binding protein Protein structure: 5-stranded beta-barrel Key part of the CusCFBA system in E.Coli, responsible for Cu/Ag tolerance Paper reports the X-Ray structure for the CusF-Ag(I) complex, and the XAS data for the CusF-Cu(I) complex.
- 5. Copper and silver are micronutrients, needed in only tiny amounts Both metals have wide ranging implications Antibiotics Chemotherapy
- 6. Prevent efflux of drugs Reducing amount needed Decreasing side effects Less risk of toxicity
- 7. Found only in Cu/Ag resistance systems Binds Cu(I) under anaerobic conditions Studied here at CSUCI
- 8. Proton-substrate antiporter Operon is up-regulated when metal levels rise Mutation studies show CusF to be critical Homologues present in all Cu/Ag tolerance systems
- 9. Focused on CusF Studying folding kinetics Folds slower than anticipated Mutagenesis to determine cause N- and C-terminal deletions Proline to Alanine substitutions
- 10. Preparation of CusF for crystallization PCR for gene amplification Gene encoding CusF with a N-terminal methionine was cloned into pPR-IBA1 Helps to facilitate crystallization by eliminating nine N- terminal residues Unstructured in solution and no role in metal binding E. coli BL21-DE3 cells with plasmid encoding CusF grown on LB medium Pure protein: treated with 10mM EDTA, dialyzed against 20mM HEPES at pH 7.5,with a concentration of 20 mg/mL
- 11. AgNO3 added to CusF twofold molar excess of silver to CusF Hanging-drop vapor diffusion: Drops set up by mixing 10μL protein solution with 10μL reservoir solution 100mM sodium acetate trihydrate pH 4.6, 2.3M ammonium sulfate, and 2mM silver nitrate Equilibrated against 1mL reservoir solution at room temp.
- 12. Crystals grew as clusters of long rods Dimensions: 0.4mm x 0.4mm x 0.8mm Orthorhombic Space Group: P212121
- 13. Crystals transferred to solutions enriched to 2.6M, 3.1M, and 3.3M ammonium sulfate flash frozen in liquid nitrogen Data: 100 K Stanford Synchrotron Radiation lab beamline 9-2m Wavelength = 0.97946 Å CrystalClear to process Scaled with SCALA in CCP4 Merges many observations of reflections into an average intensity
- 14. Molecular replacement MOLEREP in CCP4 using apo-CusF coordinates, pdb code IZEQ REFMAC5 used to refine structure, combined with manual rebuilding using COOT PROCHECK used to generate a Ramachandran plot 97.1% of residues in favored regions 2.9% in allowed regions Block diagonal least squares refinement used for the final models with SHELX-97 with Ag(I) PDB: accession code 2QCP
- 15. XAS= X-ray absorption spectroscopy E.coli cells having the plasmid with the gene that encodes CusF grown in LB medium. Over-expression and purification Pure protein: treated with 10mM EDTA, dialyzed against 20mM MOPS, pH7.0 Anaerobic chamber solutions oxygen free 0.5M ascorbate, buffered with 20mM MOPS added to protein sample Final concentration = 50mM
- 16. CuCl2 added to protein solution to 80% of CusF concentration to prevent excess of Cu(I) into different copper forms Protein sample dialyzed against 20mM MOPS and 10mM ascorbate 25% ethylene glycol added Sample transferred into EXAFS cells, flash frozen in liquid nitrogen EXAFS = extended x-ray absorption fine structure XANES = x-ray absorption near-edge structure CusF-Cu(I) final concentration = 0.5mM
- 17. EXAFS and XANES data collected at Stanford Synchrotron Radiation Lab. 3 GeV, 50-10 mA, beamline 9-3 Collected in fluorescence mode Scans of sample with only sample buffer was collected and averaged to create a baseline Samples measured as aqueous glasses (>20% ethylene glycol) at 10 K
- 18. continued… EXAFSPAK program: data reduction and background subtraction EXCURVE 9.2 program: spectral simulation EXAFS data simulated with mixed-shell model Imidazole and methionine coordination Shell refinement, maintaining ideal ring geometry Continued refinement, constraints lifted Allow outer shells of imidazole rings to move within 10% of ideal positions
- 19. continued… Conditions refined in the fit: shell occupancy (N) Cu-scatter distance (R) Debye-Waller factor (2σ2) threshold energy for photoelectron ionization (E0)
- 20. Structure of CusF–Ag(I) was determined to 1.0 Å resolution using X-Ray crystallography. Construct used lacks the N-terminal 9 residues, includes the natural C terminus. Structure of CusF-Ag(I) is very similar to the structure of the apo-CusF (pdb code IZEQ)
- 21. Backbone overlay apo-CusF [blue] CusF-Ag(I) [green] Similar to reported structure of apo-CusF (pdb: 1ZEQ) Beta-barrel structure of apo- CusF is retained Metal ion is accommodated with only a few changes apo = without ligand
- 22. Highest resolution shell in parenthesis Space group : P 21 21 21 (orthorhombic) Unit cell dimensions (Å) and angles [°] a = 38.12 α = 90.00 b = 39.35 β = 90.00 c = 44.42 γ = 90.00 Resolution (Å): 29.46 – 1.00 (1.04 – 1.00)
- 23. Highest resolution shell in parenthesis I/ σ : 17.3 (3.1) Intensity / error: as resolution increases, this ratio drops Completeness (%): 97.7 (95.6) Number of measured reflections as a % of the total number of reflections at the specified resolution Redundancy: 5.52 (5.09) measured reflections / unique reflections Rmerge (%): 4.9 (38.3) measures the error of the spots, <10% is good Rwork (%): 15.9 measure of how well the refined structure predicts the observed data Rfree (%): 18.6 measures agreement between the observed and computed structures using a random subset (5%) of the data not included in the modeling and refinement process.
- 24. Refinement # protein molecules in asymmetrical unit: 1 # protein residues: 80 # Ag(I) atoms: 1 # water molecules: 93
- 25. CusF – Ag(I) With waters visible
- 26. Refinement # other entities: 2 (NO3 -), 2 (SO4 2-) Resolution range (Å ): 19.68 – 1.00 # of reflections : 34,150
- 27. CusF – Ag(I) Showing the Ag ion, and the 4 small molecules * 2 sulfate ions, 2 nitrate ions
- 28. Refinement Average B-values Protein (Å2) : 12.1 Ligand ion (Å2) : 12.8 Water (Å2) : 19.7 a B-factor of 20 equals an atomic displacement of 0.5Å, so these results are fairly good. RMS deviations from ideal values: Bond length (Å) : 0.017 Bond angles (degrees): 1.96 RMS deviations measure how well the final model conforms to expected values of bond lengths and angles. A high quality model has rmsd values lower than 0.02Å for bond lengths, and lower than 4° for angles
- 29. In the CusF-Ag(I) structure, Ag(I) is coordinated by two methionines (M47, M49) and a histidine (H36), located in beta strands 2 and 3 Residues are clustered at one end of the beta-barrel, side chains are oriented toward the interior of the barrel. A nearby triptophan (W44) caps the metal site. Image obtained using PDB ProteinWorkshop 3.9
- 30. Arrangement of ligands effectively sequesters the metal from its periplasmic environment, and thus may play a role in protecting the cell from the toxic metal ion EXAFS measurements show a similar environment for CusF-Cu(I)
- 31. Binding site region is well-ordered and has similar temperature factors as the rest of the structure electron density shows two sets of side-chain conformations for M47 and M49; only one for H36 Major conformation: 70% Minor conformation: 30%
- 33. Distances from the Ag ion to the coordinating side chain atoms H36, M47, M49 are similar to bond lengths for silver bound to small molecule complexes When compared to other protein-Ag(I) structures, there are differences other metalloproteins use cysteine residues to coordinate with the metal ions CusF uses methionines instead Methionines commonly used in the oxidizing environment of the periplasmic space of Gram- negative bacteria
- 34. Close-up of CusF-Ag(I) binding site Image obtained using RCSB PDB Ligand Explorer 3.9
- 36. CusF can also bind Cu(I), but the authors were unable to grow crystals of Cu(I)-loaded CusF Because of this, they had to resort to XAS as an alternate strategy for determining the local environment of metal centers in proteins XAS does not require diffractable crystals
- 37. XAS is a technique used to determine the local environment of metal centers in metallobiomolecules Takes advantage of the high-intensity x-ray sources at synchrotron radiation facilities Samples can be in the gas-phase, solution, or solid matter Detailed assignment of ligands, site symmetries, and metric parameters (metal-ligand distances) are available even for non-crystalline samples XAFS is the combination of EXAFS (Extended X- ray Absorption Fine Structure) and XANES (X-ray Absorption Near-Edge Structure) data
- 38. Pros Can obtain (indirect) structural information on amorphous samples Technique is element- selective; no interference from other metals Cons Information obtained is mostly qualitative Requires fairly high concentration of metal sites Requires synchrotron radiation beam time Only straightforward for elements with atomic number greater than or equal to 20 (Ca)
- 39. EXAFS is the oscillating part of the X-ray Absorption Spectrum (XAS) that extends to about 1000 eV above an absorption edge of a particular element of a sample The main advantage of EXAFS analysis over X-ray Crystallography is that structures can be studied in non-crystalline forms
- 40. Pros Can obtain (indirect) structural information on amorphous samples Technique is element- selective; no interference from other metals Metal-ligand distances can be measured very accurately (± 0.02 Å or better) Cons Requires fairly high concentration of metal sites Requires synchrotron radiation beam time Coordination numbers and atom-type determinations are relatively inaccurate Often does not give a unique determination of ligand environment; depends on simulation and curve-fitting
- 41. Now that we got that out of the way, let’s move on to some of the experimental XAFS data . . .
- 44. Well . . . that’s great, but what does it all mean?! In a nutshell, it means . . .
- 45. The absorption edge region of the spectrum shows a weak feature at 8983.0 eV with intensity equal to 0.58 of the normalized edge height The position and intensity of this peak is characteristic of Cu(I) bound to the protein in a three-coordinate environment This makes sense because we know that H36, M47, and M49 are coordinating ligands of the Ag(I) structure
- 46. Since the structure of the Ag(I) bound CusF was known, they used it as a starting point for spectral simulations of the EXAFS The initial fit did not fully reproduce the width of the FT on the high-R side of the main peak This suggests a fourth scatter The best fit was obtained with the inclusion of a nonhistidine C/O/N scatter This also makes sense because we know thatW44 is not an actual ligand, but that it may play a role in the capping of the metal site
- 47. Materials & Methods Sodium Acetate (NaC2H3O2) Ammonium Sulfate (NH4)2SO4 Silver NitrateAgNO3 CusF Protein
- 48. Want Have units Matl Qty Unit 0.1 3 M Sodium acetate 33.3 uL 2.3 3.5 M Ammonium sulfate 657.1 uL 0.002 0.02 M Silver nitrate 100 uL water 209.5 uL TOTAL 1000 uL
- 49. Sodium Acetate : 0.098 – 0.01 – 0.012 M vertically Ammonium Sulfate : 2.0 – 2.1 – 2.2 – 2.3 – 2.4 – 2.5 M horizontally Silver Nitrate 0.002 M constant
- 52. Precipitation in all wells Further trays pending results of further testing Illustrate differences betweenWT and mutants