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Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site.

Carra JH, McHugh CA, Mulligan S, Machiesky LM, Soares AS, Millard CB - BMC Struct. Biol. (2007)

Bottom Line: We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80.Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function.Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Army Medical Research Institute of Infectious Diseases, 1425 Porter St,, Fort Detrick, MD 21702, USA. john.carra@amedd.army.mil

ABSTRACT

Background: Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions.

Results: We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a DeltaH of -13 +/- 2 kJ/mol and a DeltaS of -0.04 +/- 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site.

Conclusion: We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

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Electron density maps of the RTA-NMU complex instereo. (A) Initial 2mFo-DFc map before repositioning of Tyr80. Contoured at 1σ (blue), 2σ (purple), 3σ (pink), 4σ (magenta), and 5σ (red). (B) Density recalculated after removal of the Tyr80 ring. (C) After repositioning Tyr80 to fit the observed density and one round of refinement. (D) Density before placement of N-methylurea and water molecules at the active site. (E) Final structure after placement of ligand and water.
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Figure 2: Electron density maps of the RTA-NMU complex instereo. (A) Initial 2mFo-DFc map before repositioning of Tyr80. Contoured at 1σ (blue), 2σ (purple), 3σ (pink), 4σ (magenta), and 5σ (red). (B) Density recalculated after removal of the Tyr80 ring. (C) After repositioning Tyr80 to fit the observed density and one round of refinement. (D) Density before placement of N-methylurea and water molecules at the active site. (E) Final structure after placement of ligand and water.

Mentions: To test for ligand binding, we soaked RTA crystals in solutions of mother liquor containing 2 M NMU, 1 M urea, or 2 M acetamide. These concentrations were chosen to maximize ligand occupancy while avoiding protein denaturation. The highest quality result was obtained with NMU. This structure was refined to 1.8 Å resolution with an Rcryst of 22.5% and an Rfree of 24.3% (Table 1). The electron density for tyrosine 80 obtained by molecular replacement phasing with the RTA structural model 1IFT indicated that this residue had moved in the presence of NMU (Fig. 2). Upon refining tyrosine 80 in an alternate conformation at full occupancy, positive-difference electron density consistent with an NMU molecule at the active site became clear in 2mFo-DFc maps. We refined an NMU molecule in this density as shown in Figure 2, along with a proximal water molecule. The asymmetry and hydrogen-bonding potential of NMU supported placement of the ligand with a unique orientation.


Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site.

Carra JH, McHugh CA, Mulligan S, Machiesky LM, Soares AS, Millard CB - BMC Struct. Biol. (2007)

Electron density maps of the RTA-NMU complex instereo. (A) Initial 2mFo-DFc map before repositioning of Tyr80. Contoured at 1σ (blue), 2σ (purple), 3σ (pink), 4σ (magenta), and 5σ (red). (B) Density recalculated after removal of the Tyr80 ring. (C) After repositioning Tyr80 to fit the observed density and one round of refinement. (D) Density before placement of N-methylurea and water molecules at the active site. (E) Final structure after placement of ligand and water.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194779&req=5

Figure 2: Electron density maps of the RTA-NMU complex instereo. (A) Initial 2mFo-DFc map before repositioning of Tyr80. Contoured at 1σ (blue), 2σ (purple), 3σ (pink), 4σ (magenta), and 5σ (red). (B) Density recalculated after removal of the Tyr80 ring. (C) After repositioning Tyr80 to fit the observed density and one round of refinement. (D) Density before placement of N-methylurea and water molecules at the active site. (E) Final structure after placement of ligand and water.
Mentions: To test for ligand binding, we soaked RTA crystals in solutions of mother liquor containing 2 M NMU, 1 M urea, or 2 M acetamide. These concentrations were chosen to maximize ligand occupancy while avoiding protein denaturation. The highest quality result was obtained with NMU. This structure was refined to 1.8 Å resolution with an Rcryst of 22.5% and an Rfree of 24.3% (Table 1). The electron density for tyrosine 80 obtained by molecular replacement phasing with the RTA structural model 1IFT indicated that this residue had moved in the presence of NMU (Fig. 2). Upon refining tyrosine 80 in an alternate conformation at full occupancy, positive-difference electron density consistent with an NMU molecule at the active site became clear in 2mFo-DFc maps. We refined an NMU molecule in this density as shown in Figure 2, along with a proximal water molecule. The asymmetry and hydrogen-bonding potential of NMU supported placement of the ligand with a unique orientation.

Bottom Line: We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80.Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function.Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Army Medical Research Institute of Infectious Diseases, 1425 Porter St,, Fort Detrick, MD 21702, USA. john.carra@amedd.army.mil

ABSTRACT

Background: Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions.

Results: We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a DeltaH of -13 +/- 2 kJ/mol and a DeltaS of -0.04 +/- 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site.

Conclusion: We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

Show MeSH
Related in: MedlinePlus