Functional implications of hexameric assembly of RraA proteins from Vibrio vulnificus.

Saemee Song,Hyung-Kyoon Choi,Ji-Hyun Yeom,Nam-Chul Ha,Minho Lee,Jinyang Jang,Yeri Lim,Jaejin Lee,Jun-Yeong Jeon,Seokho Hong,Nohra Park,Kangseok Lee,Eric Cascales

doi:10.1371/journal.pone.0190064

Saemee Song, Hyung-Kyoon Choi + Show 11 more

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https://doi.org/10.1371/journal.pone.0190064

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Abstract

RNase E has a pivotal role in the degradation and processing of RNAs in Escherichia coli, and protein inhibitors RraA and RraB control its enzymatic activity. The halophilic pathogenic bacterium Vibrio vulnificus also expresses orthologs of RNase E and RraA—RNase EV, RraAV1, and RraAV2 (herein renamed as VvRNase E, VvRraA1, and VvRraA2). A previous study showed that VvRraA1 actively inhibits the ribonucleolytic activity of VvRNase E by interacting with the C-terminal region of VvRNase E. However, the molecular mechanism underlying the effect of VvRraA1 on the ribonucleolytic activity of VvRNase E has not yet been elucidated. In this study, we report that the oligomer formation of VvRraA proteins affects binding efficiency to VvRNase E as well as inhibitory activity on VvRNase E action. The hexameric structure of VvRraA1 was converted to lower oligomeric forms when the Cys 9 residue was substituted with an Asp residue (VvRraA1-C9D), showing decreased inhibitory activity of VvRraA1 on VvRNase E in vivo. These results indicated that the intermolecular disulfide linkage contributed critically to the hexamerization of VvRraA1 for its proper function. On the contrary, the VvRraA2 that existed in a trimeric state did not bind to or inhibit VvRNase E. An in vitro cleavage assay further showed the reduced inhibitory effect of VvRraA-C9D on VvRNase E activity compared to wild-type VvRraA1. These findings provide insight into how VvRraA proteins can regulate VvRNase E action on its substrate RNA in V. vulnificus. In addition, based on structural and functional comparison of RraA homologs, we suggest that hexameric assembly of RraA homologs may well be required for their action on RNase E-like proteins.

Highlights

RNase E is an essential endoribonuclease, which was initially discovered as an enzyme that is involved in the processing of ribosomal RNA in Escherichia coli [1]
To test whether structural changes in VvRraA1 protein affect its known inhibitory effect on ribonucleolytic activity of VvRNase E (VvRne) [6], and whether VvRraA2 and VvRraA2-C9D can modulate the activity of VvRne, we investigated in vivo activity of these VvRraA proteins
Co-expression of VvRraA2 or VvRraA2-C9D did not affect growth rates of DK001 overexpressing VvRne. These results indicate that (i) VvRraA1 can effectively modulate the activity of over-produced VvRNase E, rendering VvRne-overproducing cells to normally grow, and this VvRraA1 activity is largely abolished by the C9D mutation. (ii) Coexpression of VvRraA2 and VvRraA2-C9D does not inhibit the enzymatic activity of VvRne in VvRne-overproducing cells

Summary

Introduction

RNase E is an essential endoribonuclease, which was initially discovered as an enzyme that is involved in the processing of ribosomal RNA in Escherichia coli [1]. It is well known for its role in mRNA decay, the processing of tRNA and rRNA, and the regulation of ColE1-type plasmid replication [1,2,3]. The endonucleolytic activity of RNase E is controlled by protein inhibitors RraA and RraB (regulator of ribonuclease activity A or B) They bind to separate sites in the CTH and repress the activity of RNase E. Two proteins exert distinct effects on the composition of the degradosome complex [2, 9]

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