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Structural dynamics of the complex of calmodulin with a minimal functional construct of eukaryotic elongation factor 2 kinase and the role of Thr348 autophosphorylation
Author(s) -
Piserchio Andrea,
Long Kimberly,
Lee Kwangwoon,
Kumar Eric A.,
Abzalimov Rinat,
Dalby Kevin N.,
Ghose Ranajeet
Publication year - 2021
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.4087
Subject(s) - autophosphorylation , calmodulin , elongation factor , phosphorylation , microbiology and biotechnology , chemistry , biophysics , kinase , biochemistry , protein kinase a , biology , ribosome , rna , enzyme , gene
The calmodulin (CaM) activated α‐kinase, eukaryotic elongation factor 2 kinase (eEF‐2K), plays a central role in regulating translational elongation by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its ability to associate with the ribosome and suppressing global protein synthesis. Using TR (for truncated), a minimal functional construct of eEF‐2K, and utilizing hydrogen/deuterium exchange mass spectrometry (HXMS), solution‐state nuclear magnetic resonance (NMR) and biochemical approaches, we investigate the conformational changes accompanying complex formation between Ca 2+ ‐CaM and TR and the effects of autophosphorylation of the latter at Thr348, its primary regulatory site. Our results suggest that a CaM C‐lobe surface, complementary to the one involved in recognizing the calmodulin‐binding domain (CBD) of TR, provides a secondary TR‐interaction platform. CaM helix F, which is part of this secondary surface, responds to both Thr348 phosphorylation and pH changes, indicating its integration into an allosteric network that encompasses both components of the Ca 2+ ‐CaM•TR complex. Solution NMR data suggest that CaM H107K , which carries a helix F mutation, is compromised in its ability to drive the conformational changes in TR necessary to enable efficient Thr348 phosphorylation. Biochemical studies confirm the diminished capacity of CaM H107K to induce TR autophosphorylation compared to wild‐type CaM.