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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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Ligand effects on active site conformation. (A) RTA without bound ligand (1IFT [7]). Figure made in stereo with SPDBV [46] and POV-Ray. (B) RTA with bound adenine. The position of Tyr80 in structure 1IFS is shown in cyan, and in 2P8N in yellow. (C) Closer view of the effect of binding adenine on the active site. Tyr80 in structure 1IFT (without adenine) is in green, 1IFS in cyan, and 2P8N in yellow. The 2mFo-DFc electron density of Tyr80 in 2P8N is contoured at 1.5σ. (D) RTA-NMU complex. A single water molecule is shown in light blue. (E) RTA-urea complex.
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Figure 3: Ligand effects on active site conformation. (A) RTA without bound ligand (1IFT [7]). Figure made in stereo with SPDBV [46] and POV-Ray. (B) RTA with bound adenine. The position of Tyr80 in structure 1IFS is shown in cyan, and in 2P8N in yellow. (C) Closer view of the effect of binding adenine on the active site. Tyr80 in structure 1IFT (without adenine) is in green, 1IFS in cyan, and 2P8N in yellow. The 2mFo-DFc electron density of Tyr80 in 2P8N is contoured at 1.5σ. (D) RTA-NMU complex. A single water molecule is shown in light blue. (E) RTA-urea complex.

Mentions: As in the RTA-adenine complex 1IFS [7], arginine 180 rotated out of plane with the stationary indole ring of tryptophan 211 in the presence of NMU (Fig. 3), and to a greater angle. In the complex with NMU, a single ligand molecule was observed occupying the same position as the adenine base, lying nearly in the same plane. No other molecule of NMU was found. A water molecule (number 384) was positioned to donate an H-bond to the carbonyl group of NMU, while simultaneously forming bidentate H-bonds with the side-chain of arginine 180. This water was associated with a strong electron density peak and a B-factor of 20.7 Å2, suggesting that it was relatively tightly bound. NMU also was positioned to form H-bonds with the peptide backbone at residues valine 81 and glycine 121, comparable with those proposed for the adenine complex. The refined structure of RTA in the N-methylurea complex was overall very similar to that of unbound RTA (1IFT) with a root-mean-square-deviation of 0.84 Å for all protein atoms.


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)

Ligand effects on active site conformation. (A) RTA without bound ligand (1IFT [7]). Figure made in stereo with SPDBV [46] and POV-Ray. (B) RTA with bound adenine. The position of Tyr80 in structure 1IFS is shown in cyan, and in 2P8N in yellow. (C) Closer view of the effect of binding adenine on the active site. Tyr80 in structure 1IFT (without adenine) is in green, 1IFS in cyan, and 2P8N in yellow. The 2mFo-DFc electron density of Tyr80 in 2P8N is contoured at 1.5σ. (D) RTA-NMU complex. A single water molecule is shown in light blue. (E) RTA-urea complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Ligand effects on active site conformation. (A) RTA without bound ligand (1IFT [7]). Figure made in stereo with SPDBV [46] and POV-Ray. (B) RTA with bound adenine. The position of Tyr80 in structure 1IFS is shown in cyan, and in 2P8N in yellow. (C) Closer view of the effect of binding adenine on the active site. Tyr80 in structure 1IFT (without adenine) is in green, 1IFS in cyan, and 2P8N in yellow. The 2mFo-DFc electron density of Tyr80 in 2P8N is contoured at 1.5σ. (D) RTA-NMU complex. A single water molecule is shown in light blue. (E) RTA-urea complex.
Mentions: As in the RTA-adenine complex 1IFS [7], arginine 180 rotated out of plane with the stationary indole ring of tryptophan 211 in the presence of NMU (Fig. 3), and to a greater angle. In the complex with NMU, a single ligand molecule was observed occupying the same position as the adenine base, lying nearly in the same plane. No other molecule of NMU was found. A water molecule (number 384) was positioned to donate an H-bond to the carbonyl group of NMU, while simultaneously forming bidentate H-bonds with the side-chain of arginine 180. This water was associated with a strong electron density peak and a B-factor of 20.7 Å2, suggesting that it was relatively tightly bound. NMU also was positioned to form H-bonds with the peptide backbone at residues valine 81 and glycine 121, comparable with those proposed for the adenine complex. The refined structure of RTA in the N-methylurea complex was overall very similar to that of unbound RTA (1IFT) with a root-mean-square-deviation of 0.84 Å for all protein atoms.

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