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Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: β -grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the β -grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames.
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Evidence for the emergence of β-trefoils by ‘Peptide Budding’ from an IgG-like β-sandwich
As sequence and structure comparison algorithms gain sensitivity, the intrinsic interconnectedness of the protein universe has become increasingly apparent. Despite this general trend, β-trefoils have emerged as an uncommon counterexample: They are an isolated protein lineage for which few, if any, sequence or structure associations to other lineages have been identified. If β-trefoils are, in fact, remote islands in sequence-structure space, it implies that the oligomerizing peptide that founded the β-trefoil lineage itself arose de novo . To better understand β-trefoil evolution, and to probe the limits of fragment sharing across the protein universe, we identified both ‘β-trefoil bridging themes’ (evolutionarily-related sequence segments) and ‘β-trefoil-like motifs’ (structure motifs with a hallmark feature of the β-trefoil architecture) in multiple, ostensibly unrelated, protein lineages. The success of the present approach stems, in part, from considering β-trefoil sequence segments or structure motifs rather than the β-trefoil architecture as a whole, as has been done previously. The newly uncovered inter-lineage connections presented here suggest a novel hypothesis about the origins of the β-trefoil fold itself–namely, that it is a derived fold formed by ‘budding’ from an Immunoglobulin-like β-sandwich protein. These results demonstrate how the evolution of a folded domain from a peptide need not be a signature of antiquity and underpin an emerging truth: few protein lineages escape nature’s sewing table.
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- Award ID(s):
- 1724300
- PAR ID:
- 10328826
- Editor(s):
- Elofsson, Arne
- Date Published:
- Journal Name:
- PLOS Computational Biology
- Volume:
- 18
- Issue:
- 2
- ISSN:
- 1553-7358
- Page Range / eLocation ID:
- e1009833
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pcbi.1009833
