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The Crystal Structure of Hexamer RraA from Pseudomonas Aeruginosa Reveals Six Conserved Protein–Protein Interaction Sites

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Abstract

RNase E functions as the rate-limiting enzyme in the global mRNA metabolism as well as in the maturation of functional RNAs. The endoribonuclease, binding to the PNPase trimer, the RhlB monomer, and the enolase dimer, assembles into an RNA degradosome necessary for effective RNA metabolism. The RNase E processing is found to be negatively regulated by the protein modulator RraA which appears to work by interacting with the non-catalytic region of the endoribonuclease and significantly reduce the interaction between RNase E and PNPase, RhlB and enolase of the RNA degradosome. Here we report the crystal structure of RraA from P. aeruginosa to a resolution of 2.0 Å. The overall architecture of RraA is very similar to other known RraAs, which are highly structurally conserved. Gel filtration and dynamic light scattering experiments suggest that the protein regulator is arranged as a hexamer, consistent with the crystal packing of “a dimer of trimer” arrangement. Structure and sequence conservation analysis suggests that the hexamer RraA contains six putative charged protein–protein interaction sites which may serve as binding sites for RNase E.

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Abbreviations

RraA:

regulator of ribonuclease activity A

PaRraA:

regulator of ribonuclease activity A from P. aeruginosa

PNPase:

Polynucleotide Phosphorylase

RhlB:

RNase helicase B

IPTG:

isopropyl β-D-thiogalactopyranoside

PEG:

Polyethylene glycol

MPD:

2-methyl-1,3- propanediol

PDB:

Protein Data Bank

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Acknowledgments

The authors would like to acknowledge X-ray facility for Key Laboratory of Structural Biology of Chinese Academy of Science, and to thank Prof. Liwen Niu, Prof. Maikun Teng, and Dr. Zhiqiang Zhu, School of Life Science, the University of Science and Technology of China. Financial support for this project to Deqiang Wang was provided by research grants from the Chinese National Natural Science Foundation (grant Nos. 30600101, 30770481 and 30970563) and Natural Science Foundation of Chongqing (grants Nos.2006BB5275 and 2009BB5413). We are also grateful to Dr. David Worthylake and Louis LeCour in Louisiana State University Health Science Center on paper editing.

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Authors and Affiliations

  1. Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, 400016, People’s Republic of China

    Jian Tang, Miao Luo, Siqiang Niu, Hua Zhou, Xuefei Cai, Wenlu Zhang, Yuan Hu, Ailong Huang & Deqiang Wang

  2. Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Department of Laboratory Medicine in Chongqing Medical University, Chongqing, 400016, People’s Republic of China

    Jian Tang, Miao Luo, Siqiang Niu, Hua Zhou, Yibing Yin & Deqiang Wang

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  1. Jian Tang
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  2. Miao Luo
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  3. Siqiang Niu
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  9. Ailong Huang
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  10. Deqiang Wang
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Correspondence to Deqiang Wang.

Additional information

Jian Tang, Miao Luo contributed equally to this work.

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Tang, J., Luo, M., Niu, S. et al. The Crystal Structure of Hexamer RraA from Pseudomonas Aeruginosa Reveals Six Conserved Protein–Protein Interaction Sites. Protein J 29, 583–590 (2010). https://doi.org/10.1007/s10930-010-9293-x

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