Structural and Functional Differences between Homologous Bacterial Ribonucleases.

Vera Ulyanova,Alsu Nadyrova,Aleksandra Kuznetsova,Dzhigangir Faizullin,Darya Novopashina,Nikita Kuznetsov,Olga Ilinskaya,Albina Ahmetgalieva,Yulia Surchenko,Yuriy Zuev,Elena Dudkina

doi:10.3390/ijms23031867

Abstract

Small cationic guanyl-preferring ribonucleases (RNases) produced by the Bacillus species share a similar protein tertiary structure with a high degree of amino acid sequence conservation. However, they form dimers that differ in conformation and stability. Here, we have addressed the issues (1) whether the homologous RNases also have distinctions in catalytic activity towards different RNA substrates and interactions with the inhibitor protein barstar, and (2) whether these differences correlate with structural features of the proteins. Circular dichroism and dynamic light scattering assays revealed distinctions in the structures of homologous RNases. The activity levels of the RNases towards natural RNA substrates, as measured spectrometrically by acid-soluble hydrolysis products, were similar and decreased in the row high-polymeric RNA >>> transport RNA > double-stranded RNA. However, stopped flow kinetic studies on model RNA substrates containing the guanosine residue in a hairpin stem or a loop showed that the cleavage rates of these enzymes were different. Moreover, homologous RNases were inhibited by the barstar with diverse efficiency. Therefore, minor changes in structure elements of homologous proteins have a potential to significantly effect molecule stability and functional activities, such as catalysis or ligand binding.

Highlights

RNA molecules represent promising therapeutic targets for a multitude of important human diseases including infections and cancer
Binase and balnase differ by a single amino acid substitution: threonine at position 106 in the binase is replaced by alanine in balnase, while binase and balifase has 73% identity of primary structures [21]
We have found that in the barnase dimer, both active centers are blocked by barstar, in the binase dimer, only one of two active centers is bound by the barstar, and in the balifase dimer, both active centers are free of an inhibitor (Figure 7)

Summary

Introduction

RNA molecules represent promising therapeutic targets for a multitude of important human diseases including infections and cancer. Ribonucleases (RNases) play an important role in cell physiology by the regulation of RNA metabolism [3]. Catalyzing the degradation of RNA polymers, RNases indirectly participate in many cellular processes such as gene expression, cell growth and differentiation, and immune response [4–6]. The ability of RNases to inhibit the growth of tumor cells and the reproduction of viruses in infected cells was shown [7,8]. The application of RNases as antitumor and antiviral agents implies their penetration into mammalian cells which contain a cytosolic RNase inhibitor (RI). RI regulates the activity of mammalian RNases and protects cellular RNA from their cytotoxic action [9]. Microbial RNases are insensitive to blocking action of RI [10]; thereby they are considered as perspective therapeutics

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