This thesis describes the structural organization of recombinant eEF1Bα, eEF1Bβ, eEF1Bγ subunits, stoichiometry and architecture of their complex, eEF1B, and functional activity of eEF1Bα and eEF1Bβ as the guanine nucleotide exchange factors of eEF1A.
Protein biosynthesis in eukaryotic cell is spatially and structurally organized that ensures high efficiency of this process. One of the distinguishing features of the eukaryotic cell is the presence of the stable macromolecular complexes of aminoacyl-tRNA synthetases and translation elongation factors. Until now, the structural organization of the eEF1B translation elongation factor complex, as well as its individual subunits, remains unknown. Therefore, the aim of this thesis is to establish the structural organization of the human eEF1B complex and characterize the structural features and functional properties of its individual subunits.
We determined that eEF1Bα is a monomeric protein with a moderately elongated shape in solution. It consists of two rigidly structured domains (N-terminal and GEF) connected by a long structurally dynamic region. eEF1Bβ is a stable trimer of a highly elongated shape in solution. Trimerization of eEF1Bβ is mediated by its leucine-zipper motif, which forms a compact supercoiled trimeric bundle. Three GEF domains are connected to this bundle via unstructured regions and CAR domains on one side of this bundle; three N-terminal domains with a dynamic α-helical organization are located on the other side. eEF1Bγ is also a moderately elongated protein and its aggregation state depends on the protein concentration. At a concentration below 1.8 μM, eEF1Bγ forms monomer-dimer equilibrium. Increasing protein concentration results in the formation of stable dimers and tetramers.
We explained a mechanism of the stimulatory effect of eEF1Bγ on the rate of guanine nucleotide exchange reaction mediated by eEF1Bα. We demonstrated that the N-terminal domain of eEF1Bα inhibits its nucleotide exchange activity by interfering with eEF1A binding to the C-terminal domain of eEF1Bα. The formation of the eEF1Bαγ complex confines the N-terminal domain of eEF1Bα in eEF1Bγ that consequently eliminates this inhibitory effect. In contrast to eEF1Bα, eEF1Bγ did not affect functional activity of eEF1Bβ.
We found that the eEF1Bαγ and eEF1Bβγ complexes are formed at an equimolar subunits ratio. Using the method of hydrogen deuterium exchange coupled to mass spectrometry, we outlined the regions involved in the protein-protein interaction for each subunit. Amino acid residues 6-58 of the eEF1Bα N-terminal domain acquire rigidly structured conformation when interacting with eEF1Bγ. In turn, two short regions of the eEF1Bγ N-terminal domain (residues 144-161 and 170-190) are responsible for the interaction with eEF1Bα. The N-terminal domains of eEF1Bβ and eEF1Bγ are responsible for the eEF1Bβγ complex formation as well, particularly, amino acid residues 11-29 of eEF1Bβ and the entire N-terminal domain of eEF1Bγ, with the exception of peptides interacting with eEF1Bα, display high protection in the complex.
Using the molecular docking method, we built an atomistic model of the eEF1B complex. The N-terminal domain of eEF1Bγ interacts with the N-terminal domains of eEF1Bα and eEF1Bβ simultaneously. The eEF1Bβ subunit is trimerized by the leucine-zipper motif interaction, thus, forming the eEF1B(αβγ)3 complex. Since eEF1Bα and eEF1Bβ proteins have structurally similar GEF-domains, their total number in the complex is equal to six. Therefore, the eEF1B(αβγ)3 complex is able to bind up to six molecules of eEF1A2. Such, so far, unique structural assembly of the guanine-nucleotide exchange factors within a stable complex may be considered as a “GEF-hub” that provides efficient conversion of eEF1A from the GDP-bound state to the active GTP-bound conformation in higher eukaryotes.
Key words: protein biosynthesis, eukaryotic translation elongation factors, protein-protein interactions, stable protein complexes.
