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

Michael W Traxlmayr,Jonathan D Kiefer,Raja R Srinivas,Elisabeth Lobner,Alison W Tisdale,Naveen K Mehta,Nicole J Yang,Bruce Tidor,K Dane Wittrup

doi:10.1074/jbc.m116.741314

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.

Highlights

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
Yeast-displayed libraries that were based on this reduced charge Sso7d scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor
Examples include designed ankyrin repeat proteins [2,3,4], anticalins [5, 6], affibodies [7], OBodies [8], and cystine-knot miniproteins [9,10,11] among others. Another recently introduced class of binder scaffolds are the homologous proteins Sso7d [12, 13] and Sac7d [14, 15] from the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. Several advantages make these proteins promising as binder scaffolds. (i) They are highly stable with Tm values of 98 and 91 °C for Sso7d and Sac7d, respectively [16, 17]. (ii) They are small (7 kDa) single domain proteins. (iii) They lack cysteines and glycosylation sites

Summary

The abbreviations used are

Fn3, fibronectin type III; rcSso7d, reduced charge Sso7d; MSA, mouse serum albumin; hEGFR, human epidermal growth factor receptor; CDR, complementarity-determining region; DSC, differential scanning calorimetry; PPI, protein-protein interaction; epPCR, error-prone PCR; BLI, biolayer interferometry; EGFR, EGF receptor; SEC, size exclusion chromatography; VLR, variable lymphocyte receptor; SUMO, small ubiquitin-like modifier; 8-oxo-dGTP, 8-oxo-2Ј-deoxyguanosine 5Ј-triphosphate; dPTP, 2Ј-deoxy-Pnucleoside 5Ј-triphosphate; APC, allophycocyanin; SD-CAA, 20 g/L D-glucose, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 7.4 g/L citric acid monohydrate, 10.4 g/L sodium citrate, pH 4.5; SG-CAA, 18 g/L galactose, 2 g/L D-glucose, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 5.4 g/L Na2HPO4, 8.6 g/L NaH2PO4 monohydrate, pH 6.0. We constructed a charge-neutralized Sso7d mutant that largely maintains high thermal stability. We demonstrate that this charge neutralization reduces nonspecific interactions with mammalian cells. Binders tolerated up to four of these aromatic residues in their nine-residue binding sites and maintained high thermal stability and monomeric behavior without any observed aggregation. Together, these data demonstrate that the optimal distribution of amino acids in small, rigid binding surfaces is distinct from the composition of loopbased paratopes and that the hyperstability of the scaffold promotes an unusually high tolerance for aromatic residues

Results

Discussion

Experimental Procedures