Structural Dynamics of the Activation of Elongation Factor 2 Kinase by Ca2+-Calmodulin

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM)-activated α-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry and small-angle X-ray scattering measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen/deuterium exchange mass spectrometry and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.

The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase

“…Using native mass spectrometry, we had previously demonstrated that CaM and TR form a high‐affinity heterodimeric complex of molecular weight of ~77.3 kDa in the presence of Ca 2+ . Given the size of the CaM•TR complex and its low stability even at modest concentrations (~50 μM) over extended periods of time, we took advantage of the excellent sensitivity provided by methyl groups for NMR in large, proton‐sparse systems .…”

“…The 20 Ile residues on TR (Figure S2A,S2B) are distributed over all its functional domains including the N‐terminal CBD (Ile89, that constitutes the fifth position of 1–5–8–14 binding mode seen in the structure of the Ca 2+ ‐CaM•eEF‐2K CBD complex), on the N‐lobe of the kinase domain (Figure S2A; KD N ; Ile107, Ile131, Ile173, Ile214, Ile215, and Ile232), the C‐lobe of the kinase domain (Figure S2A; KD C ; Ile209, Ile237, Ile251, Ile271, Ile275, Ile287, and Ile317), on the loop connecting the N‐ and C‐terminal regions of TR (a remnant of the R‐loop; Ile349 that lies next to Thr348, the site of primary activating autophosphorylation) and on the CTD (Figure S2B; Ile518, Ile522, Ile567, Ile581, and Ile614). Of these 20 resonances, 15 were unambiguously assigned using a mutation‐based approach as described previously (see Figure S3 for examples that illustrate this approach).…”

“…This structure illustrates what has been suggested to embody the primary interaction mode between eEF‐2K and CaM in its Ca 2+ ‐loaded state. Furthermore, we had developed a minimal eEF‐2K construct, we termed TR (for truncated), that is activated by CaM to a similar extent in vitro as wild‐type enzyme and retains the ability to phosphorylate eEF‐2 in cells with high efficiency . TR (shown schematically in Figure ) contains the intact CBD at its N‐terminus, the intact α‐kinase domain, and the entire C‐terminal domain (CTD), which encodes multiple helical repeats of wild‐type eEF‐2K.…”

“…TR lacks the extreme N‐terminus (1–70), a significant portion of the central regulatory loop (R‐loop; 359–489) and several sites of up‐ and down‐regulating phosphorylation contained therein, but includes both the primary (Thr348) and secondary (Ser500) sites of autophosphorylation . Our recent studies using hydrogen exchange mass spectrometry (HXMS), chemical cross‐linking mass spectrometry (XLMS), small‐angle X‐ray scattering (SAXS), and computational modeling have helped ascertain the relative organization of CaM and the structural domains of TR within the Ca 2+ ‐CaM•TR complex . However, additional information is required to elucidate the conformational changes that drive TR to its active state by CaM, the corresponding conformational changes in CaM itself, and the role of Ca 2+ therein.…”

The Role of Calmodulin in Tumor Cell Migration, Invasiveness, and Metastasis

“…Finally, it is worth mentioning that PDGF-BB-mediated activation of the eukaryotic elongation factor 2 kinase (eEF2K), also named CaMK-III, which is negatively regulated by CaM [157] (reviewed in [158]), enhances the proliferation and migration of VSMCs. This works via the ERK/MAPK, p38MAPK, Akt pathways, and Hsp27 as a downstream substrate of p38MAPK, shown by results using the eEF2K inhibitor A-484954 [159].…”