Protein scaffolds are structural frameworks used to present variable polypeptide regions that can be engineered to bind molecular targets. Cystine-knots (knottins) are promising protein scaffolds since they are thermally stable, resistant to proteolysis, and non-immunogenic. Further, knottins have multiple disulfide-bonded loops capable of displaying conformationally-constrained polypeptides for molecular recognition. In this work, we assess the structural tolerance of amino acid sequence diversity in knottin loops. We present a combinatorial approach to explore the structural tolerance of knottin loops to sequence diversity by using yeast display, directed evolution, and bioinformatics.

Our studies focus on EETI-II, a member of the knottin family that naturally binds trypsin. Libraries of EETI-II variants with randomized loop sequences (other than the trypsin binding loop) were displayed on the surface of yeast. Clones that retained the native knottin fold, determined by binding to fluorescently-labeled trypsin1, were isolated by fluorescence-activated cell sorting. Isolated clones were sequenced and then analyzed using RELIC2 bioinformatics tools. Positional pair-wise covariance analysis3,4 was performed on the variable loop sequences of isolated clones and predictive pairs of amino acids at covarying loop positions were identified. Bioinformatics analysis of EETI-II variants revealed important sequence-structure relationships of the knottin fold. Sequence diversity was best tolerated in the C-terminal loop of EETI-II, where we identified a consensus doublet at the final two loop positions and three covarying positional pairs.

From these collective analyses, we rationally designed EETI-II variants with extensive mutations (> 25% of positions mutated) that we predicted would retain the knottin fold. In addition, we constructed EETI-II variants with randomly generated variable sequences that we predicted would not adopt the native fold. Rationally designed and randomly generated EETI-II variants were displayed on the surface of yeast and tested for trypsin binding. Remarkably, all of the rationally designed EETI-II variants retained the predicted knottin fold, as indicated by wild-type levels of trypsin binding. Further, none of the randomly generated EETI-II variants bound trypsin, suggesting that these mutations were not structurally tolerated. Collectively, these results verified the importance of the observed sequence-structure relationships.

In conclusion, we found that EETI-II is generally tolerant of sequence diversity in its loop regions, suggesting it has potential to be a useful molecular scaffold. Sequence trends governing structural integrity described here can guide future protein engineering efforts with the EETI-II knottin. Importantly, our combinatorial approach can be used to probe sequence-structure relationships of proteins while accounting for compensatory mutations and the influences of the local amino acid environment.

(1) Wentzel, A.; Christmann, A.; Kratzner, R.; Kolmar, H. J Biol Chem 1999, 274, 21037-43.

(2) Mandava, S.; Makowski, L.; Devarapalli, S.; Uzubell, J.; Rodi, D. J. Proteomics 2004, 4, 1439-60.

(3) Fodor, A. A.; Aldrich, R. W. Proteins 2004, 56, 211-21.

(4) Yip, K. Y.; Patel, P.; Kim, P. M.; Engelman, D. M.; McDermott, D.; Gerstein, M. Bioinformatics 2008, 24, 290-2.

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