Functional analyses of Dph1•Dph2 in synthesis of diphthamide, a clinically relevant modification on eEF2

Diphthamide is a unique post-translational modification of a conserved histidine on the eukaryotic translation elongation factor 2 (eEF2). During translation elongation, eEF2 induces translocation of codon-anticodon duplexes across the ribosomal decoding center. Diphthamide is involved in fine-tuning of this process by stabilizing the codon-anticodon duplex and maintaining reading frame accuracy. Initially, diphthamide was identified as the target for diphtheria toxin, enabling ADP-ribosylation of eEF2 and resulting in cell death. Hypomodification of eEF2 is associated with a range of cellular defects and reflected by relevance in human disease. Mutations in genes coding for diphthamide synthesis protein Dph1 are associated with various cancer diseases. Dph1 and Dph2 mutations are also associated with an autosomal recessive neurodevelopmental disorder named diphthamide deficiency syndrome (DDS). Affected individuals display mutual traits including intellectual disability, craniofacial malformations and short stature. In S. cerevisiae, diphthamide is synthesized in four catalytic steps requiring eight proteins (Dph1-Dph8). The first step revolves around a radical reaction catalysed by the heterodimer Dph1•Dph2, processing S-adenosylmethionine (SAM) into the diphthamide precursor ACP (3-amino-3-carboxypropyl). This biological redox reaction requires [Fe4-S4]-clusters as inorganic cofactors. The subunits of Dph1•Dph2 are proposedly asymmetric in function, with Dph1 being the catalyst Dph2 a regulator. Their [Fe4-S4] binding motifs include four cysteines, respectively and two of these form tandem-cysteines motifs (TCMs) with an ill-defined role. Based on mutagenesis in yeast cells, chromosomally coded substitution mutants of Dph1 and Dph2 were phenotypically and biochemically analysed. Results show that tandem-cysteines in Dph2 (but not in Dph1) are cooperatively important in function, suggesting four cysteines in Dph2 qualify as [Fe4-S4]-cluster ligands. All essential cysteines in Dph1•Dph2 were revealed as critical integrity factors for both subunits unanimously. Unlike Dph2, Dph1 (the catalytic subunit) contains an amino acid sequence qualifying for SAM binding motif (SAM pocket). Dph1 residues which were structurally predicted to surround the SAM methionine moiety are critical for functionality, suggesting an involvement in SAM orientation for efficient ACP-formation. Based on obtained insights into critical Dph1•Dph2 regions, rare human DPH1 and DPH2 variants (occurring heterozygous) were analysed in human cells and their yeast counterparts as well. Ten DPH1 and two DPH2 variants have been identified as prone to trigger DDS. Some functionally compromised variants affected DPH1 residues close to the active center, which may indicate a role in DPH1•DPH2 activation by DPH3. Lastly, in a collaborative effort, a novel DPH5-related diphthamide deficiency syndrome was identified including studies on Dph5 counterparts in yeast cells. Taken together, this thesis is supposed to provide insights in understanding Dph1•Dph2 and facilitate DDS diagnosis.