A two-step binding model proposed for the electrostatic interactions of ricin a chain with ribosomes.
Ricin is a ribosome inactivating protein that catalytically removes a universally conserved adenine from the alpha-sarcin/ricin loop (SRL) of the 28S rRNA. We recently showed that ricin A chain (RTA) interacts with the P1 and P2 proteins of the ribosomal stalk to depurinate the SRL in yeast. Here we examined the interaction of RTA with wild-type and mutant yeast ribosomes deleted in the stalk proteins by surface plasmon resonance. The interaction between RTA and wild-type ribosomes did not follow a single-step binding model but was best characterized by two distinct types of interactions. The AB1 interaction had very fast association and dissociation rates, was saturable, and required an intact stalk, while the AB2 interaction had slower association and dissociation rates, was not saturable, and did not require the stalk. RTA interacted with the mutant ribosomes by a single type of interaction, which was similar to the AB2 interaction with the wild-type ribosomes. Both interactions were dominated by electrostatic interactions, and the AB1 interaction was stronger than the AB2 interaction. On the basis of these results, we propose a two-step interaction model. The slow and ribosomal stalk nonspecific AB2 interactions concentrate the RTA molecules on the surface of the ribosome. The AB2 interactions facilitate the diffusion of RTA toward the stalk and promote the faster, more specific AB1 interactions with the ribosomal stalk. The electrostatic AB1 and AB2 interactions work together allowing RTA to depurinate the SRL at a much higher rate on the intact ribosomes than on the naked 28S rRNA.
Li
Xiao-Ping
XP
Biotechnology Center for the Agriculture and the Environment, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey 08901-8520, USA.
Chiou
Jia-Chi
JC
Remacha
Miguel
M
Ballesta
Juan P G
JP
Tumer
Nilgun E
NE
D008024:Ligands; D008956:Models, Chemical; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D012276:Ricin / Q000737:chemistry / Q000378:metabolism*; D055672:Static Electricity; D020349:Surface Plasmon Resonance
D008024:Ligands; D011994:Recombinant Proteins; D012276:Ricin