Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.     

Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.     

Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca(2+) and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr(348), Thr(353), Ser(366) and Ser(445), all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser(78), a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser(78), Thr(348) and Ser(366) to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr(348) was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr(348) appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607-2616]. Ser(366) phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases.     

Solution Structure of the Carboxy-Terminal Tandem Repeat Domain of Eukaryotic Elongation Factor 2 Kinase and Its Role in Substrate Recognition

Eukaryotic elongation factor 2 kinase (eEF-2K), an atypical calmodulin-activated protein kinase, regulates translational elongation by phosphorylating its substrate, eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome. The activation and activity of eEF-2K are critical for survival under energy-deprived conditions and is implicated in a variety of essential physiological processes. Previous biochemical experiments have indicated that the binding site for the substrate eEF-2 is located in the C-terminal domain of eEF-2K, a region predicted to harbor several α-helical repeats. Here, using NMR methodology, we have determined the solution structure of a C-terminal fragment of eEF-2K, eEF-2K_562–725 that encodes two α-helical repeats. The structure of eEF-2K_562–725 shows signatures characteristic of TPR domains and of their SEL1-like sub-family. Furthermore, using the analyses of NMR spectral perturbations and ITC measurements, we have localized the eEF-2 binding site on eEF-2K_562–725. We find that eEF-2K_562–725 engages eEF-2 with an affinity comparable to that of the full-length enzyme. Furthermore, eEF-2K_562–725 is able to inhibit the phosphorylation of eEF-2 by full-length eEF-2K in trans. Our present studies establish that eEF-2K_562–725 encodes the major elements necessary to enable the eEF-2K/eEF-2 interactions.     

Roles of three domains of Tetrahymena eEF1A in bundling F-actin

The conventional role of eukaryotic elongation factor 1A (eEF1A) is to transport aminoacyl tRNA to the A site of ribosomes during the peptide elongation phase of protein synthesis. eEF1A also is involved in regulating the dynamics of microtubules and actin filaments in cytoplasm. In Tetrahymena, eEF1A forms homodimers and bundles F-actin. Ca(2+)/calmodulin (CaM) causes reversion of the eEF1A dimer to the monomer, which loosens F-actin bundling, and then Ca(2+)/CaM/eEF1A monomer complexes dissociate from F-actin. eEF1A consists of three domains in all eukaryotic species, but the individual roles of the Tetrahymena eEF1A domains in bundling F-actin are unknown. In this study, we investigated the interaction of each domain with F-actin, recombinant Tetrahymena CaM, and eEF1A itself in vitro, using three glutathione-S-transferase-domain fusion proteins (GST-dm1, -2, and -3). We found that only GST-dm3 bound to F-actin and influences dimer formation, but that all three domains bound to Tetrahymena CaM in a Ca(2+)-dependent manner. The critical Ca(2+) concentration for binding among three domains of eEF1A and CaM were < or =100 nM for domain 1, 100 nM to 1 microM for domain 3, and >1 microM for domain 2, whereas stimulation of and subsequent Ca(2+) influx through Ca(2+) channels raise the cellular Ca(2+) concentration from the basal level of approximately 100 nM to approximately 10 microM, suggesting that domain 3 has a pivotal role in Ca(2+)/CaM regulation of eEF1A.     

Regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase

Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of alphaCaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca(2+)/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile(441) is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu(440), exhibited enhanced Ca(2+)/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile(441) remained Ca(2+)/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile(441) for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp(444)) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.     

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

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^2 + 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

Eukaryotic elongation factor 2 kinase (eEF‐2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF‐2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM‐regulated kinases. We had previously identified a minimal construct of eEF‐2K (TR) that is activated similarly to the wild‐type enzyme by CaM in vitro and retains its ability to phosphorylate eEF‐2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1‐methyls of TR and Ile δ1‐ and Met ε‐methyls of CaM, as probes of their mutual interaction and the influence of Ca²⁺ thereon. We find that in the absence of Ca²⁺, CaM exclusively utilizes its C‐terminal lobe (CaM_C) to engage the N‐terminal CaM‐binding domain (CBD) of TR in a high‐affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca²⁺‐loaded N‐terminal lobe of CaM (CaM_N) at increased Ca²⁺ levels serves to enhance the affinity further. These latter interactions under Ca²⁺ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca²⁺‐free state. Our data are consistent with a scenario in which Ca²⁺ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM‐bound species without significantly modifying the conformation of TR within the final, active complex.     

Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis

Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.     

Cyclic AMP inhibits translation of cyclin D3 in T lymphocytes at the level of elongation by inducing eEF2-phosphorylation

The purpose of the present study was to understand the mechanism by which activated protein kinase A (PKA) leads to down-regulation of cyclin D3 in lymphocytes. By using Jurkat cells as a model system, we have been able to demonstrate that cyclin D3 is reduced at the level of translation by inhibition of elongation. One of the important factors involved in translational elongation is the eukaryotic elongation factor 2 (eEF2). eEF2 promotes translation in its unphosphorylated form, and we observed a rapid phosphorylation of the eEF2-protein upon forskolin treatment. When using specific inhibitors of the eEF2-kinase prior to forskolin treatment, we were able to inhibit the increased phosphorylation of eEF2. Furthermore, inhibition of eEF2-kinase prevented the forskolin-mediated down-regulation of cyclin D3. Taken together, it appears that activation of PKA in Jurkat cells reduces the expression of cyclin D3 at the level of translational elongation by increasing the phosphorylation of eEF2 and thereby inhibiting its activity.     

Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398

Protein synthesis consumes a high proportion of the metabolic energy of mammalian cells, and most of this is used by peptide chain elongation. An important regulator of energy supply and demand in eukaryotic cells is the AMP-activated protein kinase (AMPK). The rate of peptide chain elongation can be modulated through the phosphorylation of eukaryotic elongation factor (eEF) 2, which inhibits its activity and is catalyzed by a specific calcium/calmodulin-dependent protein kinase termed eEF2 kinase. Here we show that AMPK directly phosphorylates eEF2 kinase, and we identify the major site of phosphorylation as Ser-398 in a regulatory domain of eEF2 kinase. AMPK also phosphorylates two other sites (Ser-78 and Ser-366) in eEF2 kinase in vitro. We develop appropriate phosphospecific antisera and show that phosphorylation of Ser-398 in eEF2 kinase is enhanced in intact cells under a range of conditions that activate AMPK and increase the phosphorylation of eEF2. Ser-78 and Ser-366 do not appear to be phosphorylated by AMPK within cells. Although cardiomyocytes appear to contain a distinct isoform of eEF2 kinase, it also contains a site corresponding to Ser-398 that is phosphorylated by AMPK in vitro. Stimuli that activate AMPK and increase eEF2 phosphorylation within cells increase the activity of eEF2 kinase. Thus, AMPK and eEF2 kinase may provide a key link between cellular energy status and the inhibition of protein synthesis, a major consumer of metabolic energy.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40).

Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.     

Myocardial ischemia and increased heart work modulate the phosphorylation state of eukaryotic elongation factor-2

Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in cellular energy homeostasis. Therefore, we tested the hypothesis that, as in liver, it could mediate the inhibition of protein synthesis by oxygen deprivation in heart by modulating the phosphorylation of eukaryotic elongation factor-2 (eEF2), which becomes inactive in its phosphorylated form. In anoxic cardiomyocytes, AMPK activation was associated with an inhibition of protein synthesis and an increase in phosphorylation of eEF2. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), did not mimic the effect of oxygen deprivation to inhibit protein synthesis in cardiomyocytes or lead to eEF2 phosphorylation in perfused hearts, suggesting that AMPK activation did not inhibit mTOR/p70 ribosomal protein S6 kinase (p70S6K) signaling. Human recombinant eEF2 kinase (eEF2K) was phosphorylated by AMPK in a time- and AMP-dependent fashion, and phosphorylation led to eEF2K activation, similar to that observed in extracts from ischemic hearts. In contrast, increasing the workload resulted in a dephosphorylation of eEF2, which was rapamycin-insensitive, thus excluding a role for mTOR in this effect. eEF2K activity was unchanged by increasing the workload, suggesting that the decrease in eEF2 phosphorylation could result from the activation of an eEF2 phosphatase.     

Analysis of the domain structure of elongation factor-2 kinase by mutagenesis.

A number of elongation factor-2 kinase (eEF-2K) mutants were constructed to investigate features of this kinase that may be important in its activity. Typical protein kinases possess a highly conserved lysine residue in subdomain II which follows the GXGXXG motif of subdomain I. Mutation of two lysine residues, K340 and K346, which follow the GXGXXG motif in eEF-2K had no effect on activity, showing that such a lysine residue is not important in eEF-2K activity. Mutation of a conserved pair of cysteine residues C-terminal to the GXGXXG sequence, however, completely inactivated eEF-2K. The eEF-2K CaM binding domain was localised to residues 77-99 which reside N-terminal to the catalytic domain. Tryptophan 84 is an important residue within this domain as mutation of this residue completely abolishes CaM binding and eEF-2K activity. Removal of approximately 130 residues from the C-terminus of eEF-2K completely abolished autokinase activity; however, removal of only 19 residues inhibited eEF-2 kinase activity but not autokinase activity, suggesting that a short region at the C-terminal end may be important in interacting with eEF-2. Likewise, removal of between 75 and 100 residues from the N-terminal end completely abolished eEF-2K activity.     

Solution structure of the 162 residue C-terminal domain of human elongation factor 1Bgamma

The multisubunit elongation factor 1 (eEF1) is required for the elongation step of eukaryotic protein synthesis. The eEF1 complex consists of four subunits: eEF1A, a G-protein that shuttles aminoacylated tRNAs to the ribosome; eEF1Balpha and eEF1Bbeta, two guanine nucleotide exchange factors, and eEF1Bgamma. Although its exact function remains unknown, this latter subunit is present in all eukaryotes. Recombinant human eEF1Bgamma has been purified and shown to consist of two independent domains. We have utilized high resolution NMR to determine the three-dimensional structure of the 19 kDa C-terminal fragment (domain 2). The structure consists of a five-stranded anti-parallel beta-sheet surrounded by alpha-helices and resembles a contact lens. Highly conserved residues are mainly located on the concave face, suggesting thereby that this side of the molecule might be involved in some biologically relevant interface(s). Although the isolated domain 2 appears to be mostly monomeric in solution, biochemical and structural data indicate a potential homodimer. The proposed dimer model can be further positioned within the quaternary arrangement of the whole eEF1 assembly.     

Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.     

Mutations in the Chromodomain-like Insertion of Translation Elongation Factor 3 Compromise Protein Synthesis through Reduced ATPase Activity

Translation elongation is mediated by ribosomes and multiple soluble factors, many of which are conserved across bacteria and eukaryotes. During elongation, eukaryotic elongation factor 1A (eEF1A; EF-Tu in bacteria) delivers aminoacylated-tRNA to the A-site of the ribosome, whereas eEF2 (EF-G in bacteria) translocates the ribosome along the mRNA. Fungal translation elongation is striking in its absolute requirement for a third factor, the ATPase eEF3. eEF3 binds close to the E-site of the ribosome and has been proposed to facilitate the removal of deacylated tRNA from the E-site. eEF3 has two ATP binding cassette (ABC) domains, the second of which carries a unique chromodomain-like insertion hypothesized to play a significant role in its binding to the ribosome. This model was tested in the current study using a mutational analysis of the Sac7d region of the chromodomain-like insertion. Specific mutations in this domain result in reduced growth rate as well as slower translation elongation. In vitro analysis demonstrates that these mutations do not affect the ability of eEF3 to interact with the ribosome. Kinetic analysis revealed a larger turnover number for ribosomes in comparison to eEF3, indicating that the partial reactions involving the ribosome are significantly faster than that of eEF3. Mutations in the chromodomain-like insertion severely compromise the ribosome stimulated ATPase of eEF3, strongly suggesting that it exerts an allosteric effect on the hydrolytic activity of eEF3. The chromodomain-like insertion is, therefore, vital to eEF3 function and may be targeted for developing novel antifungal drugs.