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Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library*

Posted by on in 2016
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  • Traxlmayr MW1,2Kiefer JD3Srinivas RR4Lobner E5Tisdale AW3,4Mehta NK3,4Yang NJ3,2Tidor B4Wittrup KD6,2,4. 2016. J Biol Chem 291 (43): 22496-22508. Epub 2016 Aug 30.
    1From the Koch Institute for Integrative Cancer Research and
    2Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and.
    3From the Koch Institute for Integrative Cancer Research and.
    4Departments of Biological Engineering and
    5Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
    6From the Koch Institute for Integrative Cancer Research and


The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 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.

*The Chicken anti-CMYC used in this publication is a product of Gallus Immunotech Inc.




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