Evidence for the importance of electrostatics in the function of two distinct families of ribosome inactivating toxins

Abstract

a-Sarcin and ricin represent two structurally and mechanistically distinct families of site-specific enzymes that block translation by irreversibly modifying the sarcin/ricin loop (SRL) of 23S-28S rRNA. a-Sarcin family enzymes are designated as ribotoxins and act as endonucleases. Ricin family enzymes are designated as ribosome inactivating proteins (RIP) and act as N-glycosidases. Recently, we demonstrated that basic surface residues of the ribotoxin restrictocin promote rapid and specific ribosome targeting by this endonuclease. Here, we report that three RIP: ricin A, saporin, and gypsophilin depurinate the ribosome with strong salt sensitivity and achieve unusually fast k cat /K m ;10 9 -10 10 M À1 s À1 , implying that RIP share with ribotoxins a common mechanism of electrostatically facilitated ribosome targeting. Bioinformatics analysis of RIP revealed that surface charge properties correlate with the presence of the transport chain in the RIP molecule, suggesting a second role for the surface charge in RIP transport. These findings put forward surface electrostatics as an important determinant of RIP activity.

Arginine Residues on the Opposite Side of the Active Site Stimulate the Catalysis of Ribosome Depurination by Ricin A Chain by Interacting with the P-protein Stalk

“…R193A/R235A and R189A/R234A could not bind intact ribosomes by Biacore analysis. Because the ribosome is about 120 times bigger than RTA (molecular mass of 30 kDa for RTA and 3600 kDa for the yeast ribosome), to reach the high depurination rate, RTA must have a very high local concentration near the ribosome (52,53). We show here that the seven arginines at the RTA/RTB interface contribute to the positive surface charge, which allows RTA to interact with the ribosome via electrostatic interactions.…”

“…The electrostatic character of the ribosomal surface has been shown to be responsible for the unusually high catalytic activity of ribotoxins and RIPs (52,53). We previously showed that electrostatic interactions concentrate RTA on the ribosome surface and facilitate its interaction with the stalk (18).…”

Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

“…The faster dissociation rate of RTA with ribosomes than with the isolated stalk might be determined by the ribosomal particle, where dynamics of the stalk may deliver RTA to the SRL by undergoing conformational changes, as proposed for the interaction between the C termini of the stalk proteins and the translation factors (10,42). The electrostatic character of the ribosomal surface was shown to be critical for its interaction with a ribotoxin (43) and for the enzymatic activity of RIPs (44). Similarly, we showed that electrostatic interactions dominated the interaction between RTA and ribosomes, enabling rapid target localization, and accounted for much higher depurination rate of intact ribosomes than of isolated rRNA (40).…”

Investigating the 3' RNA Phosphodiesterase and 3'-5' Exoribonuclease Activities of APE1.

“…Thus, APE1 is a potential candidate enzyme that cleaves damaged abasic-site-containing ribosomal RNA (rRNA) and transfer RNA (tRNA) molecules which pose hindrance to normal protein synthesis (Korennykh et al 2007). APE1…”

“…The ability to cleave AP-site-containing RNA implies that APE1 can remove damaged templates from the endogenous pool. This activity provides a possible mechanism for the cell to eliminate damaged RNA prior to association with the ribosome and execution of non-productive translation, i.e., damage to rRNA and tRNA (Berquist et al 2008;Korennykh et al 2007;Cochella et al 2005). Future in vivo work in 70 CHAPTER 5 -GENERAL DISCUSSION understanding the role of these variants may generate valuable insights into understanding their associations with cancers.…”