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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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van't Hoff plots. Temperature dependence of binding of urea (triangles) or adenine (circles) to RTA.
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Figure 5: van't Hoff plots. Temperature dependence of binding of urea (triangles) or adenine (circles) to RTA.

Mentions: To find the enthalpy and entropy changes associated with binding of urea or adenine to RTA, titration experiments were carried out over a temperature range from 5 to 30°C (Fig. 5). The temperature range was limited to avoid effects from irreversible unfolding of the protein. Van't Hoff analysis gave a ΔH° for urea binding of -13 ± 2 kJ/mol. The amount of scatter in the data was too large and the temperature range too narrow to determine ΔCp by fitting with a temperature-dependent enthalpy change. However, the slight deviation from linearity of this plot (R = 0.85) indicated that the ΔCp of binding must be relatively small. An unfavorable entropy change of -0.04 ± 0.01 kJ/(K*mol) opposed the enthalpy change, resulting in a ΔG° of -2 ± 0.6 kJ/mol. For the larger and tighter-binding ligand adenine, ΔH° was -53 kJ/mol (R = 0.97) with a ΔS° of -0.12 ± 0.01 kJ/(K*mol). NMU, acetamide, and formamide gave complex non-linear results in van't Hoff analysis that did not allow determination of ΔH and ΔS, perhaps due to their lower affinities for the protein.


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)

van't Hoff plots. Temperature dependence of binding of urea (triangles) or adenine (circles) to RTA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: van't Hoff plots. Temperature dependence of binding of urea (triangles) or adenine (circles) to RTA.
Mentions: To find the enthalpy and entropy changes associated with binding of urea or adenine to RTA, titration experiments were carried out over a temperature range from 5 to 30°C (Fig. 5). The temperature range was limited to avoid effects from irreversible unfolding of the protein. Van't Hoff analysis gave a ΔH° for urea binding of -13 ± 2 kJ/mol. The amount of scatter in the data was too large and the temperature range too narrow to determine ΔCp by fitting with a temperature-dependent enthalpy change. However, the slight deviation from linearity of this plot (R = 0.85) indicated that the ΔCp of binding must be relatively small. An unfavorable entropy change of -0.04 ± 0.01 kJ/(K*mol) opposed the enthalpy change, resulting in a ΔG° of -2 ± 0.6 kJ/mol. For the larger and tighter-binding ligand adenine, ΔH° was -53 kJ/mol (R = 0.97) with a ΔS° of -0.12 ± 0.01 kJ/(K*mol). NMU, acetamide, and formamide gave complex non-linear results in van't Hoff analysis that did not allow determination of ΔH and ΔS, perhaps due to their lower affinities for the protein.

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