Identification of the amino acids associated with the novel ribonuclease activity of cusativin via protein engineering and LC‐MS

Abstract

Ribonucleases (RNases) are widely used in detecting the location of chemical modification in RNA, an approach commonly referred to as, “RNA modification mapping” [1]. The commonly employed RNases include RNase T1 (guanosine‐specific), U2 (purine‐specific) and A (pyrimidine specific). Recently, uridine‐specific (MC1) [2], cytidine‐specific (cusativin) [3] and a non‐specific RNase U2 [4] were identified as potential tools for RNA modification mapping. Of these, RNase cusativin exhibits novel cleavage behavior where it does not cleave the bond between two consecutive cytidine nucleotides (CpC) [3]. This unique feature of cusativin has encouraged us to probe the nucleobase recognition characteristics of the enzyme binding site, which is composed of B1 and B2 sites. We employed amino acid substitutions (through protein engineering), and liquid chromatography coupled with mass spectrometry (LC‐MS) to analyze the contributions of specific amino acids in the enzyme binding site. The mutant proteins generated by specific amino acid substitutions were investigated for alteration of secondary structure, kinetic changes in enzyme activity, and substrate specificity. While single amino acid substitutions revealed minimal effects on the secondary structure, multiple substitutions resulted in significant changes as revealed by circular dichroism studies. However, the mutant proteins, irrespective of the number of substitutions, exhibited remarkable changes in their kinetic behavior with the tested RNA substrate. The LC‐MS analysis of enzymatic digestion products of RNA indicated altered nucleobase specificity depending on the location of amino acid substitutions. While mutation at one location (V99L) behaved more or less like wild type protein, mutation at different position (N97S) but within same B2 binding site pocket revealed generation of digestion products indicative of CpC bond cleavage. However, the N97S mutant also exhibited digestion products that suggest cleavage of RNA at 3′‐end of guanosine. Another mutant protein with amino acid substitutions in both B1 (R69T) and B2 (V99L and T100R) sites also exhibited significant cleavage of CpC bond. But this behavior is also accompanied with more or less equal affinity towards C and G for RNA cleavage. The implications of this preliminary experimental data on the role of specific amino acids in nucleobase recognition of RNA substrate will be discussed.Support or Funding InformationFinancial support of this work was provided by the National Institutes of Health (GM58843), and the University of Cincinnati.