Structural Dynamics of the GW182 Silencing Domain Including its RNA Recognition motif (RRM) Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry
- University of Warsaw, Division of Biophysics, Institute of Experimental Physics, Faculty of Physics (Poland)
- Polish Academy of Sciences, Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics (Poland)
- Jewish General Hospital, Lady Davis Institute for Medical Research (Canada)
- McGill University, Department of Biochemistry (Canada)
- Polish Academy of Sciences, Laboratory of Biological Physics, Institute of Physics (Poland)
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. .
- OSTI ID:
- 22777059
- Journal Information:
- Journal of the American Society for Mass Spectrometry, Vol. 29, Issue 1; Other Information: Copyright (c) 2018 American Society for Mass Spectrometry; Article Copyright (c) 2017 The Author(s); http://www.springer-ny.com; Country of input: International Atomic Energy Agency (IAEA); ISSN 1044-0305
- Country of Publication:
- United States
- Language:
- English
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