Domain Analysis

Figure 1. Phyre predicted 3D structure of eEF2K rendered using Pymol. The different eEF2K domains are labelled in different colours.

1. Alpha kinase domain

The alpha kinase domain is the catalytic domain of eEF2K, which spans from the residue 116-326 (Uniprot). The catalytic domain of eEF2K shares high sequence homology to that of Dicytostelium MHCK A (1). Hence, its crystallised structure was used as a model to elucidate the structure of eEF2K catalytic domain.

EEF2K, despite lacking sequence homology to conventional protein kinases, was found to have a domain similar in structure to that of conventional protein kinase domains. All protein kinases share the GXGXXG motif which is part of the ATP-binding site (1). This glycine-richloop interacts with the phosphate groups of the bound ATP. The annotated ATP-binding site of eEF2K according to Uniprot, contains a -GDGNLGV- motif located between the residues 296-302, which coincides with the GXGXXG motif (2). Furthermore, there is a pair cysteine residues near the GXGXXG motif which are conserved in the C-terminal region in alpha kinases (C314 and C318 in the human isozyme) (3). It has been suggested that these are involved in zinc ion coordination as the binding of the metal ion would stabilise the tertiary structure of the catalytic core of eEF2K. This was proven in numerous studies as the mutation of these cysteine residues led to the abolishment of eEF2K activity (3, 4).

Figure 2. 3D structure of the alpha kinase domain in eEF2K isolated from the Phyre model.

2. Calmodulin-binding domain

The activity of eEF2K is dependent on calcium/calmodulin as calmodulin-binding is suggested to stabilise the kinase domain of eEF2K for its catalytic activity (3). However, little is known about its structure and location.

Computer prediction estimated that the calmodulin-binding domain of eEF2K will lie C-terminus to the catalytic domain, between the residues 593 -609 due to the presence of amphipathic alpha helices (1). However, experiments using the truncated protein showed that calmodulin is bound to the region between amino acids 51-96, which is N-terminus to the catalytic domain. Pavur et al further postulated that the exact binding site could be between the residues 81-94 which expresses the sequence FKEAWKHAIEKAKH. This is because these residues form a basic amphipathic helix which is characteristic for calmodulin-binding domains. W85 was shown to be important for calmodulin-binding because when it was substituted for alanine, calmodulin-binding was inhibited. Besides that, this sequence also resembles the type A, 1-8- 14 motif, present in many calmodulin-dependent enzymes (5).

Figure 3. 3D structure of the Calmodulin-binding domain in eEF2K isolated from the Phyre model.

3. SEL 1-like repeats

Based on structural studies, eEF2K lacking the C-terminal region between residues 521-725 was unable to phosphorylate eEF2 despite being activated after autophosphorylation at threonine 348 (5). This region was found to contain the SEL1-like repeats, which are a subfamily of tetratricopeptide repeat sequences (TPR). These alpha-helix pair repeats serve

as a scaffold for protein-protein interactions (6).

There are four predicted SEL1-like motifs within the C-terminal region of eEF2K. This agrees to the predicted 3D model as 4 alpha-helix pairs are seen. Due to data discrepancy in the possible numbers and positions of the repeats stated in different databases, we have used Pfam and TPRpred to detect potential SEL1-like repeats within eEF2K. Pfam detected a SEL1- like repeat between the residues 526-563 whereas TPRpred detected 3 possible SEL-like repeats at residues 575-610, 615-650 and 667-702. The TPRpred results had a p-value of 2.4E-08 and a 66.13% predictability score.

Furthermore, the 15 residues at the extreme C-terminal is highly conserved in mammalian isoforms of eEF2K and it was suggested that these could interact with eEF2 to facilitate its phosphorylation by the catalytic domain. Deletion of these residues abrogated eEF2 phosphorylation but MH-1 peptide substrate could still be phosphorylated (3). Hence, these conserved 15 residues could be crucial in eEF2 recognition.

Figure 4. 3D structure of SEL 1-like repeats in eEF2K isolated from the Phyre model.

As seen in Figure 5 below, after the eEF2 has been bound by the C-terminal residues, the SEL1-like repeats would bring eEF2 into close proximity with the catalytic domain so that the target protein can be phosphorylated.

Figure 5. The diagrammatic representation of the interaction between the catalytic domain and SEL1 domain of eEF2K in eEF2 phosphorylation. Image adapted from Pigott et al, 2012. (3)

4. Linker region

The linker region is estimated to span between the residues 336-520 (5). This unstructured region serves to link the N-terminal domains with the C-terminal domain.

Figure 6. 3D structure of Linker region in eEF2K isolated from the Phyre model.

3D Structure

References

1. Ryazanov AG, Ward MD, Mendola CE, Pavur KS, Dorovkov MV, Wiedmann M, et al. Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc Natl Acad Sci U S A. 1997;94(10):4884-9. Available from:

http://www.ncbi.nlm.nih.gov/pubmed/9144159

2. UniProtKB – O00418 (EF2K_Human) [Internet]. Available from:http://www.uniprot.org/uniprot/O00418

3. Pigott CR, Mikolajek H, Moore CE, Finn SJ, Phippen CW, Werner JM, et al. Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains. Biochem J. 2012;442(1):105-18. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22115317

4. Diggle TA, Seehra CK, Hase S, Redpath NT. Analysis of the domain structure of elongation factor-2 kinase by mutagenesis. FEBS Lett. 1999;457(2):189-92. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10471776

5. Pavur KS, Petrov AN, Ryazanov AG. Mapping the functional domains of elongation factor-2kinase. Biochemistry. 2000;39(40):12216-24. Available from:http://www.ncbi.nlm.nih.gov/pubmed/11015200

6. Kenney JW, Moore CE, Wang X, Proud CG. Eukaryotic elongation factor 2 kinase, an unusual enzyme with multiple roles. Adv Biol Regul. 2014;55:15-27. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24853390