Structural Dynamics of the GW182 Silencing Domain Including its RNA Recognition motif (RRM) Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry

Maja K Cieplak-Rotowska,Michal Dadlez,Nahum Sonenberg,Marc R Fabian,Marcin Rubin,Krzysztof Tarnowski,Anna Niedzwiecka

doi:10.1007/s13361-017-1830-9

Abstract

The human GW182 protein plays an essential role in micro(mi)RNA-dependent gene silencing. miRNA silencing is mediated, in part, by a GW182 C-terminal region called the silencing domain, which interacts with the poly(A) binding protein and the CCR4-NOT deadenylase complex to repress protein synthesis. Structural studies of this GW182 fragment are challenging due to its predicted intrinsically disordered character, except for its RRM domain. However, detailed insights into the properties of proteins containing disordered regions can be provided by hydrogen–deuterium exchange mass spectrometry (HDX/MS). In this work, we applied HDX/MS to define the structural state of the GW182 silencing domain. HDX/MS analysis revealed that this domain is clearly divided into a natively unstructured part, including the CCR4-NOT interacting motif 1, and a distinct RRM domain. The GW182 RRM has a very dynamic structure, since water molecules can penetrate the whole domain in 2 h. The finding of this high structural dynamics sheds new light on the RRM structure. Though this domain is one of the most frequently occurring canonical protein domains in eukaryotes, these results are – to our knowledge – the first HDX/MS characteristics of an RRM. The HDX/MS studies show also that the α2 helix of the RRM can display EX1 behavior after a freezing-thawing cycle. This means that the RRM structure is sensitive to environmental conditions and can change its conformation, which suggests that the state of the RRM containing proteins should be checked by HDX/MS in regard of the conformational uniformity.Graphical

Highlights

I n recent years, there have appeared a significant number of works proving that structural dynamics governs the function of biological molecules to no less extent than the protein sequence and its 3D structure [1,2,3]
Homology modeling of the human GW182 RNA recognition motif (RRM) structure (Figure 1c) shows that this domain is similar to the D. melanogaster GW182 RRM except for a slight distortion of the shortest β3′-strand
Our work reveals the hydrogen–deuterium exchange mass spectrometric description of an RNA Recognition Motif, which is one of the most frequently occurring canonical protein domains

Summary

Introduction

I n recent years, there have appeared a significant number of works proving that structural dynamics governs the function of biological molecules to no less extent than the protein sequence and its 3D structure [1,2,3]. No evidence of such direct interactions was found, even though the presence of the RRM domain in the GW182 sequence was shown to be required for efficient gene silencing [12, 32] This observation corresponds better to another putative role that could be fulfilled by RRM domains, i.e., stabilization of a protein structure that enables assembling of a required functional bipartite motif composed of unstructured regions flanking the folded RRM, as, e.g., in case of a bimodular non-classical nuclear localization signal of ADAR1 enzyme [35]. These proteins use hydrophobic or aromatic residues to mediate interactions more frequently than ordered proteins do [45, 47] Such protein structures are neither suitable for X-ray crystallography nor for NMR due to their dynamic character or limited solubility. These results are – to our knowledge – the first hydrogen–deuterium exchange mass spectrometry ‘fingerprint’ of an RRM domain

Materials and Methods

Results and Discussion

Conclusions