PDBsum entry 2r3d

Hydrolase

PDB id: 2r3d

Contents

Protein chain

258 a.a. *

Ligands

SO4 ×2

ACM

Waters ×106

* Residue conservation analysis

PDB id:

2r3d

Name:

Hydrolase

Title:

Ricin a-chain (recombinant) complex with acetamide

Structure:

Ricin a chain (ec 3.2.2.22). Chain: a. Fragment: unp residues 36-302. Engineered: yes

Source:

Ricinus communis. Castor bean. Organism_taxid: 3988. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.09Å R-factor: 0.234 R-free: 0.272

Authors:

J.H.Carra,C.A.Mchugh,S.Mulligan,L.M.Machiesky,A.S.Soares,C.B.Millard

Key ref:

J.H.Carra et al. (2007). Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site. Bmc Struct Biol, 7, 72-72. PubMed id: 17986339

Date:

29-Aug-07 Release date: 20-Nov-07

Supersedes:

1zb2

Protein chain

P02879  (RICI_RICCO) -  Ricin from Ricinus communis

Seq: 576 a.a.

Struc: 258 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.2.22 - rRNA N-glycosylase.
• Reaction: Endohydrolysis of the N-glycosidic bond at one specific adenosine on the 28S rRNA.

Bmc Struct Biol 7:72-72 (2007)

PubMed id: 17986339

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

J.H.Carra, C.A.McHugh, S.Mulligan, L.M.Machiesky, A.S.Soares, C.B.Millard.

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.