Biopanning of Phylomer¹ Phage Display libraries against hCD40L yielded a cluster of CD40L-specific overlapping peptides corresponding to the conserved catalytic domain from the tetrameric Gα2β2 class of Glycyl tRNA synthetases. Structural analysis of these overlapping peptides described a scaffold consisting of a central β-sheet, comprising 4 anti-parallel β-strands, flanked by N- and C-terminal α-helices. Further analysis revealed that these key features are conformationally conserved across both tetrameric Gα2β2 and dimeric Gα2 Glycyl tRNA synthetases, yet importantly, are only weakly conserved in sequence. Given the identical function of the  domain and it’s structural conservation, we postulated that members of the dimeric Gα2 class would display similar CD40L-specific binding as the tetrameric Gα2β2 class, despite the sequence dissimilarity. To test this hypothesis, structurally equivalent peptides from representative bacterial, archaeal and eukaryotic genomes comprising the dimeric Gα2 class were tested for CD40L-binding in a process we termed ortholog scanning. The results showed that both archaeal and eukaryotic structurally equivalent peptides bound to CD40L with good specificity and inhibited the CD40:CD40L interaction with comparable IC50’s to the primary Gα2β2 class peptides. Similar results were also observed for the representative bacterial Gα2 class peptides. These findings have important implications to the affinity enhancement strategies to develop the scaffold as a therapeutic agent, and in improving its “drug-like” properties.