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Structural and interaction analysis of the Rrp5 C-terminal region.

ABSTRACT: Rrp5 is an essential factor during the ribosome biogenesis process. The protein contains a series of 12 S1 RNA-binding domains followed by a TetratricoPeptide Repeat (TPR) domain. In the past, several studies aiming at defining the function of the TPR domain have used nonequivalent Rrp5 constructs, as these protein fragments include not only the TPR module, but also three or four S1 domains. We solved the structure of the Rrp5 TPR module and demonstrated in vitro that the TPR region alone does not bind RNA, while the three S1 domains preceding the TPR module can associate with homopolymeric RNA. Finally, we tested the association of our Rrp5 constructs with several proposed interactors, in support of cryo-EM-based models. Coordinates:Atomic coordinates and structure factors have been deposited to the Protein Data Bank under the accession number 5NLG.

SUBMITTER: Perebaskine N

PROVIDER: S-EPMC6168700 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Structural and interaction analysis of the Rrp5 C-terminal region.

Pérébaskine Natacha N Thore Stéphane S Fribourg Sébastien S

FEBS open bio 20180830 10

Rrp5 is an essential factor during the ribosome biogenesis process. The protein contains a series of 12 S1 RNA-binding domains followed by a TetratricoPeptide Repeat (TPR) domain. In the past, several studies aiming at defining the function of the TPR domain have used nonequivalent Rrp5 constructs, as these protein fragments include not only the TPR module, but also three or four S1 domains. We solved the structure of the Rrp5 TPR module and demonstrated <i>in vitro</i> that the TPR region alone ...[more]

PMID: 30338212

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Project description:BACKGROUND: The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. RESULTS: The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC) in different temperatures, and Kd was observed to be in the low muM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. CONCLUSION: Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure. The observed affinity can be physiologically relevant, given the high abundance of both binding partners in the nervous system.

Dataset Information

Structural and interaction analysis of the Rrp5 C-terminal region.

Publications

Structural and interaction analysis of the Rrp5 C-terminal region.

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