Protein species-specific characterization of conformational change induced by multisite phosphorylation.

J J Proteomics. 2016 Feb 16;134:138-43. doi: 10.1016/j.jprot.2015.12.002. Epub 2015 Dec 7.
Pan J, Zhang S, Borchers CH

Phosphorylation is a central mechanism for regulating the structure and function of proteins in the cell, but accurate characterization of a specific protein phospho-species is challenging due to the difficulty of separating it from other species, as well as the limitations of the traditional structural methods. By using selective top-down ETD, we were able to identify six specific phospho-species of Calmodulin (CaM). Phosphorylation of CaM at four sites by CK2 was found to follow a sequential order, with Ser81 as the first, Thr79 as the second, and Ser101 or Thr117 as the third. By combining top-down ETD with hydrogen/deuterium exchange, the impact of phosphorylation on CaM's structure was elucidated in a species-specific manner. A negligible structural effect was observed for mono-phosphorylation at Ser81, or di-phosphorylation at Ser81-Thr79, or tri-phosphorylation at Ser81-Thr79-Ser101 or Ser81-Thr79-Thr117. However, it was found that a significant phosphorylation-induced conformational change in CaM was caused by simultaneous phosphorylation at Ser101 and Thr117. The dramatically increased deuterium incorporation for residues between 102 and 119 strongly suggests that the structure of this region has been greatly changed.

BIOLOGICAL SIGNIFICANCE: Phosphorylation is probably the most ubiquitous and important PTM in human proteome, and has been recognized as a key mode of signal transduction. This paper describes a combination of top-down ETD with hydrogen/deuterium exchange, which was applied to phosphorylated calmodulin (CaM). By applying this method to the site-specific study of the various phosphorylated CaM species, we were able to determine the specific phosphorylation sites, the order in which these sites were filled, and the impact of phosphorylation on the protein stucture. The species-specific structural analysis strategy presented here should also be applicable to the study of the phosphorylation of other proteins and other combinatorial PTMs, using high-resolution X-ray and NMR data from the Protein Data Bank (http://www.rcsb.org) as the starting point. Although most of these structures are for proteins without the PTMs, they can be used as a foundation for deciphering the structure and function of the post-translationally modified species. We anticipate that this selective top-down HDX approach can be widely used for this purpose, particularly for proteins with multiple modificatioin sites, as demonstrated in this study using calmodulin as a model protein.