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The HEAT domains are a family of helical hairpin repeat domains, composed of four or more hairpins. HEAT is derived from the names of four family members: huntingtin, eukaryotic translation elongation factor 3 (eEF3), protein phosphatase 2 regulatory A subunit (PP2A), and mechanistic target of rapamycin (mTOR). HEAT domain-containing proteins play roles in a wide range of cellular processes, such as protein synthesis, nuclear transport and metabolism, and cell signaling. The PCI domains are a related group of helical hairpin domains, with a “winged-helix” (WH) subdomain at their C-terminus, which is responsible for multi-subunit complex formation with other PCI domains. The name is derived from the complexes, where these domains are found: the 26S Proteasome “lid” regulatory subcomplex, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). We noted that in structure similarity searches using HEAT domains, sometimes PCI domains appeared in the search results ahead of other HEAT domains, which indicated that the PCI domains could be members of the HEAT domain family, and not a related but separate group, as currently thought. Here, we report extensive structure similarity analysis of HEAT and PCI domains, both within and between the two groups of proteins. We present evidence that the PCI domains as a group have greater structural similarity with individual groups of HEAT domains than some of the HEAT domain groups have among each other. Therefore, our results indicate that the PCI domains have evolved from a HEAT domain that acquired a WH subdomain. The WH subdomain in turn mediated self-association into a multi-subunit complex, which eventually evolved into the common ancestor of the Proteasome lid/CSN/eIF3.

The HEAT domains are a family of helical hairpin repeat domains, composed of four or more hairpins. HEAT is derived from the names of four family members: h untingtin, eukaryotic translation e longation factor 3 (eEF3), protein phosphatase 2 regulatory A subunit (PP2A), and mechanistic t arget of rapamycin (mTOR). HEAT domain-containing proteins play roles in a wide range of cellular processes, such as protein synthesis, nuclear transport and metabolism, and cell signaling. The PCI domains are a related group of helical hairpin domains, with a “winged-helix” (WH) subdomain at their C-terminus, which is responsible for multi-subunit complex formation with other PCI domains. The name is derived from the complexes, where these domains are found: the 26S P roteasome “lid” regulatory subcomplex, the C OP9 signalosome (CSN), and eukaryotic translation i nitiation factor 3 (eIF3). We noted that in structure homology searches using HEAT domains, sometimes PCI domains appeared in the search results ahead of other HEAT domains, which indicated that the PCI domains could be members of the HEAT domain family, and not a related but separate group, as currently thought. Here, we report extensive structure homology analysis of HEAT and PCI domains, both within and between the two groups of proteins. We present evidence that the PCI domains as a group have greater structural homology with individual groups of HEAT domains than some of the HEAT domain groups have among each other. Therefore, our results indicate that the PCI domains have evolved from a HEAT domain that acquired a WH subdomain. The WH subdomain in turn mediated self-association into a multi-subunit complex, which eventually evolved into the common ancestor of the Proteasome lid/CSN/eIF3.

Translation initiation in eukaryotes requires multiple eukaryotic translation initiation factors (eIFs) and involves continuous remodeling of the ribosomal preinitiation complex (PIC). The GTPase eIF2 brings the initiator Met‐tRNAi to the PIC. Upon start codon selection and GTP hydrolysis promoted by its GTPase‐activating protein (GAP) eIF5, eIF2‐GDP is released in complex with eIF5. It is not known how eIF5 dissociates from its other binding partners to leave the PIC with only eIF2. Here, we report that two intrinsically disordered regions (IDRs) in eIF5, the DWEAR motif and the C‐terminal tail (CTT) dynamically contact the folded C‐terminal domain (CTD) and compete with each other. The eIF5‐CTD•CTT interaction shows modest synergy with eIF2β binding to eIF5‐CTD, whereas the eIF5‐CTD•DWEAR interaction favors eIF1A binding, instead. These findings allowed us to propose a model explaining how the rearrangement of the eIF5 intramolecular contacts can mediate remodeling of multiple interactions upon start codon selection. Using phosphomimetic mutations, we show that phosphorylation of eIF5 by Casein Kinase 2 (CK2) increases the affinity of eIF5 for eIF2β. Our results help elucidate the molecular mechanisms of stimulation of protein synthesis and cell proliferation by CK2.

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