Structure and activity of an active site substitution of ricin A chain.
The A chain of ricin (RTA) is an N-glycosidase which inactivates ribosomes by removing a single adenine base from a conserved region of rRNA. X-ray structures and site-directed mutagenesis revealed that Arg 180 interacts with the target adenine hydrogen bonding with N3. It may fully or partially protonate that atom as part of the hydrolysis mechanism. Arg 180 was previously converted to His (R180H) and shown to greatly reduce activity. Here R180H is shown to reduce overall activity 500-fold against Artemia salina ribosomes. A 2.2 A crystal structure reveals the mutation causes a rearrangement of the active site cleft, with Tyr 80 moving to block access to the adenine recognition site. His 180 forms a strong aromatic interaction with Trp 211, Tyr 80, and Tyr 123. A complex is formed with 250 mM AMP. The nucleotide binds in the active site region, but in an apparently nonproductive orientation. His 180 cannot bond to N3 and is screened from the substrate analog by the intervening Tyr 80. It may be that natural polynucleotide substrates, using additional interactions, can displace Tyr 80 and effect a productive binding.
Day
P J
PJ
Department of Chemistry and Biochemistry, University of Texas, Austin 78712, USA.
Ernst
S R
SR
Frankel
A E
AE
Monzingo
A F
AF
Pascal
J M
JM
Molina-Svinth
M C
MC
Robertus
J D
JD
D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001156:Artemia / Q000187:drug effects / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism; D012276:Ricin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012995:Solubility; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D012276:Ricin; D006026:Glycoside Hydrolases; D014357:Trypsin