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Using combinatorial phage\u2010displayed libraries, we applied a saturation scan approach to dissect the entire T20 sequence for binding to a prefusogenic five\u2010helix bundle (5HB) mimetic of HIV\u20101 gp41. Our data set compares all possible amino acid substitutions at all positions, and affords a complete view of the complex molecular interactions governing the binding of T20 to 5HB. The scan of T20 revealed that 12 of its 36 positions were conserved for 5HB binding, which cluster into three epitopes: hydrophobic epitopes at the ends and a central dyad of hydrophilic residues. The scan also revealed that the T20 sequence was highly adaptable to mutations at most positions, demonstrating a striking structural plasticity that allows multiple amino acid substitutions at contact points to adapt to conformational changes, and also at noncontact points to fine\u2010tune the interface. Based on the scan result and structural knowledge of the gp41 fusion intermediate, a library was designed with tailored diversity at particular positions of T20 and was used to derive a variant (T20v1) that was found to be a highly effective inhibitor of infection by multiple HIV\u20101 variants, including a common T20\u2010escape mutant. These findings show that the plasticity of the T20 functional sequence space can be exploited to develop variants that overcome resistance of HIV\u20101 variants to T20 itself, and demonstrate the utility of saturation scanning for rapid epitope mapping and protein engineering.<\/jats:p>","DOI":"10.1002\/pro.3669","type":"journal-article","created":{"date-parts":[[2019,6,22]],"date-time":"2019-06-22T12:59:13Z","timestamp":1561208353000},"page":"1501-1512","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Optimization of peptidic HIV\u20101 fusion inhibitor T20 by phage display"],"prefix":"10.1002","volume":"28","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6304-9173","authenticated-orcid":false,"given":"Gang","family":"Chen","sequence":"first","affiliation":[{"name":"Banting and Best Department of Medical Research, The Terrence Donnelly Center for Cellular and Biomolecular Research University of Toronto  Toronto 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