Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library
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Strong enrichment of aromatic.pdf
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Author(s)
Lobner, Elisabeth
Traxlmayr, Michael
Kiefer, Jonathan
Srinivas, Raja Ram
Tisdale, Alison W.
Mehta, Naveen
Yang, Nicole Jie Yeon
Tidor, Bruce
Wittrup, Karl Dane
Date Issued
August 2016
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry and Molecular Biology (ASBMB)
Citation
Traxlmayr, Michael W.; Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce and Wittrup, K. Dane. “Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-Neutralized Hyperthermostable Sso7d Scaffold Library.” Journal of Biological Chemistry 291, no. 43 (August 2016): 22496–22508 © 2016 American Society for Biochemistry and Molecular Biology (ASBMB)
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Final published version
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 (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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Chemical Engineering
Koch Institute for Integrative Cancer Research at MIT
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DOI of Published Version
https://doi.org/10.1074/jbc.M116.741314
