Structural dynamics of the complex of calmodulin with a minimal functional construct of eukaryotic elongation factor 2 kinase and the role of Thr348 autophosphorylation

Piserchio, Andrea; Long, Kimberly; Lee, Kwang-Woon; Kumar, Eric A.; Abzalimov, Rinat R.; Dalby, Kevin N.; Ghose, Ranajeet

doi:10.1002/pro.4087

Cited by 10 publications

(7 citation statements)

References 43 publications

(126 reference statements)

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“…This assembly is consistent with changes in protection seen in previous hydrogen exchange mass spectrometry (HXMS) analyses (fig. S4) ( 26 , 35 ). All four CaM C helices engage the CaM-targeting motif (CTM) through their hydrophobic faces in a Ca 2+ -bound “open” conformation of the lobe, and all, except α H , interact with KD N using their predominantly hydrophilic faces.…”

Section: Resultsmentioning

confidence: 99%

“…The protocols used for the expression and purification of eEF-2K TR and CaM, the selective phosphorylation of the former at T348 (to generate p eEF-2K TR ), and the subsequent purification of the 1:1 heterodimeric CaM• p eEF-2K TR complex were performed as described previously ( 26 , 35 ). The final samples used in the crystallization trials consisted of ~11.0 mg/ml of the CaM• p eEF-2K TR complex in a buffer containing 20 mM tris (pH 7.5), 100 mM NaCl, 3.0 mM CaCl 2 , 1.0 mM tris(2-carboxyethyl)phosphine (TCEP; GoldBio), 1.5 mM MgCl 2 , and 1.0 mM AMP-PNP (MilliporeSigma).…”

Section: Methodsmentioning

confidence: 99%

“…13 C, 1 H-Ile-1, Met-, 2 H, 15 N-labeled CaM (IM-labeled CaM) (41) and unlabeled peEF-2K TR (35) were prepared as previously described, and the corresponding 1:1 heterodimeric complex was purified as described above. All NMR samples (unless specifically noted) were prepared in a buffer (NMR buffer) containing 20 mM Hepes (pH 7.5), 100 mM NaCl, 1.0 mM TCEP, and 5% 2 H 2 O.…”

Section: Solution Nmr Spectroscopymentioning

confidence: 99%

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Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

et al. 2022

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Translation is a tightly regulated process that ensures optimal protein quality and enables adaptation to energy/nutrient availability. The α-kinase eukaryotic elongation factor 2 kinase (eEF-2K), a key regulator of translation, specifically phosphorylates the guanosine triphosphatase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and autophosphorylation at the primary stimulatory site, T348. Biochemical studies predict a calmodulin-mediated activation mechanism for eEF-2K distinct from other calmodulin-dependent kinases. Here, we resolve the atomic details of this mechanism through a 2.3-Å crystal structure of the heterodimeric complex of calmodulin and the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights an intimate association of the kinase with the calmodulin C-lobe, creating an “activation spine” that connects its amino-terminal calmodulin-targeting motif to its active site through a conserved regulatory element.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Solution Nmr Spectroscopymentioning

confidence: 99%

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Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

et al. 2022

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“…eEF-2K TR and CaM were expressed and purified, the former was selectively phosphorylated at T348 (to generate p eEF-2K TR ), and the 1:1 heterodimeric CaM• p eEF-2K TR complex was generated using previously described protocols ( 32 , 61 ). Wild-type full-length eEF-2K ( 62 ) was expressed using a codon-optimized construct using essentially the same protocol used for the expression and purification of eEF-2K TR .…”

Section: Methodsmentioning

confidence: 99%

ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin

Piserchio

Long

Browning

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α–kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP.

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“…Until recently, no structure of the entire eEF2K protein existed, yet despite this, several groups have been instrumental in characterizing the activity and properties of this unique enzyme [ 9 , 10 , 11 , 12 , 24 , 25 , 26 ]. This review will discuss protein modeling advances used to guide eEF2K inhibitor design, the effectiveness of a variety of eEF2K inhibitors, and the use of eEF2K inhibitors in disease models.…”

Section: Introductionmentioning

confidence: 99%

eEF2K Inhibitor Design: The Progression of Exemplary Structure-Based Drug Design

Klupt

Jia

2023

Molecules

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The α-kinase, eEF2K, phosphorylates the threonine 56 residue of eEF2 to inhibit global peptide elongation (protein translation). As a master regulator of protein synthesis, in combination with its unique atypical kinase active site, investigations into the targeting of eEF2K represents a case of intense structure-based drug design that includes the use of modern computational techniques. The role of eEF2K is incredibly diverse and has been scrutinized in several different diseases including cancer and neurological disorders—with numerous studies inhibiting eEF2K as a potential treatment option, as described in this paper. Using available crystal structures of related α-kinases, particularly MHCKA, we report how homology modeling has been used to improve inhibitor design and efficacy. This review presents an overview of eEF2K related drug discovery efforts predating from the 1990’s, to more recent in vivo studies in rat models. We also provide the reader with a basic introduction to several approaches and software programs used to undertake such drug discovery campaigns. With the recent exciting publication of an eEF2K crystal structure, we present our view regarding the future of eEF2K drug discovery.

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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

Cited by 10 publications

References 43 publications

Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin

eEF2K Inhibitor Design: The Progression of Exemplary Structure-Based Drug Design

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