Abstract
Mass spectrometry-based hydrogen/deuterium exchange (H/DX) has been used to define the polypeptide backbone dynamics of full-length methyl CpG binding protein 2 (MeCP2) when free in solution and when bound to unmethylated and methylated DNA. Essentially the entire MeCP2 polypeptide chain underwent H/DX at rates faster than could be measured (i.e. complete exchange in ≤10 s), with the exception of the methyl DNA binding domain (MBD). Even the H/DX of the MBD was rapid compared with that of a typical globular protein. Thus, there is no single tertiary structure of MeCP2. Rather, the full-length protein rapidly samples many different conformations when free in solution. When MeCP2 binds to unmethylated DNA, H/DX is slowed several orders of magnitude throughout the MBD. Binding of MeCP2 to methylated DNA led to additional minor H/DX protection, and only locally within the N-terminal portion of the MBD. H/DX also was used to examine the structural dynamics of the isolated MBD carrying three frequent mutations associated with Rett syndrome. The effects of the mutations ranged from very little (R106W) to a substantial increase in conformational sampling (F155S). Our H/DX results have yielded fine resolution mapping of the structure of full-length MeCP2 in the absence and presence of DNA, provided a biochemical basis for understanding MeCP2 function in normal cells, and predicted potential approaches for the treatment of a subset of RTT cases caused by point mutations that destabilize the MBD.
Highlights
methyl CpG binding protein 2 (MeCP2) is a 53-kDa nuclear protein that is present in high amounts in neuronal tissues, where its stoichiometry approaches one MeCP2 per nucleosome throughout the genome [3]
At time points spanning 101 s to 104 s, the exchange reactions were quenched, MeCP2 fragmented by proteolysis, and deuterium incorporation measured by mass spectrometry
The steady-state circular dichroism (CD) experiments have documented the extensively unstructured nature of full-length MeCP2, they have raised an important question: Is the 35– 40% averaged secondary structure content calculated to be present in MeCP2 and its domains in the form of stable threedimensional structures, or does it result from rapid sampling of unfolded, random-coil state(s) during the course of the steadystate measurements? Our hydrogen/deuterium exchange (H/DX) experiments indicate that the latter possibility is correct
Summary
MeCP2 is a 53-kDa nuclear protein that is present in high amounts in neuronal tissues, where its stoichiometry approaches one MeCP2 per nucleosome throughout the genome [3]. When bound to unmethylated DNA under saturating conditions, the MBD-derived peptides were much slower to exchange, whereas there was no change observed in the very rapid H/DX at any other location in the MeCP2 protein sequence (Fig. 1 and supplemental Fig. 1).
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