title: Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library.
abstract: The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tmof 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.
authors: From the Koch Institute for Integrative Cancer Research and michael.traxlmayr@boku.ac.at.|Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and.
authors: Michael W
authors: http://orcid.org/0000-0002-2108-582X
authors: MW
authors: Traxlmayr
authors: From the Koch Institute for Integrative Cancer Research and.
authors: Jonathan D
authors: JD
authors: Kiefer
authors: Departments of Biological Engineering and.
authors: Raja R
authors: RR
authors: Srinivas
authors: Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors: Elisabeth
authors: E
authors: Lobner
authors: From the Koch Institute for Integrative Cancer Research and.|Departments of Biological Engineering and.
authors: Alison W
authors: AW
authors: Tisdale
authors: From the Koch Institute for Integrative Cancer Research and.|Departments of Biological Engineering and.
authors: Naveen K
authors: NK
authors: Mehta
authors: From the Koch Institute for Integrative Cancer Research and.|Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and.
authors: Nicole J
authors: NJ
authors: Yang
authors: Departments of Biological Engineering and.
authors: Bruce
authors: B
authors: Tidor
authors: From the Koch Institute for Integrative Cancer Research and wittrup@mit.edu.|Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and.|Departments of Biological Engineering and.
authors: K Dane
authors: KD
authors: Wittrup
mesh_terms: D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D055550:Protein Stability; D048229:Sulfolobus solfataricus / Q000737:chemistry* / Q000235:genetics
chemical_list: D024322:Amino Acids, Aromatic; D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; C093228:Sso7d protein, Sulfolobus
keywords: aromatic amino acids; directed evolution; epidermal growth factor receptor (EGFR); molecular evolution; protein charge; protein stability; scaffold protein; yeast display