X-ray structure of recombinant ricin A-chain at 1.8 A resolution.
Ricin is a potent plant toxin which acts by removing a specific adenine residue from the ribosome. The X-ray crystal structure of a new, tetragonal crystal form of the recombinant ricin A-chain diffracting to 1.8 A resolution has been determined via molecular replacement methods and refined to a crystallographic R-factor of 18.6%. The higher resolution electron density allowed improvements to be made upon previously published models, resulting in an increase in the assigned secondary structure of the protein. The enzyme adopts the same global conformation in this crystal form with differences in detail due only partly to crystal packing. The active site superimposes closely with those of previously published models but the locations of the active-site water molecules differ in this structure. To address the current mechanistic model, an additional two structures are presented: recombinant ricin A-chain complexed with the substrate analogue formycin monophosphate as well as with adenosine monophosphate, which is cleaved by the crystalline enzyme. The formycin monophosphate displaces a putative catalytic water molecule. This supports the notion that the analogue does not bind in a transition state conformation and that contacts from other elements of the 28 S RNA natural substrate are required to achieve full reactivity. The structure of the adenosine monophosphate complex suggests a mechanism for the release of the adenine product via of the side-chain Tyr80. The structures suggest that Glu177 is better positioned for the activation of the catalytic water molecule than Arg180.
Weston
S A
SA
Protein Structure Laboratory, Zeneca Pharmaceuticals, Macclesfield, Cheshire, United Kingdom.
Tucker
A D
AD
Thatcher
D R
DR
Derbyshire
D J
DJ
Pauptit
R A
RA
D000249:Adenosine Monophosphate / Q000378:metabolism; D018360:Crystallography, X-Ray; D005573:Formycins / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012265:Ribonucleotides / Q000378:metabolism; D012276:Ricin / Q000737:chemistry*
D005573:Formycins; D011994:Recombinant Proteins; D012265:Ribonucleotides; C021683:formycin 5'-phosphate; D000249:Adenosine Monophosphate; D012276:Ricin