Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein

Abstract

Cystine-knot miniproteins (knottins) are promising molecular scaffolds for protein engineering applications. Members of the knottin family have multiple loops capable of displaying conformationally constrained polypeptides for molecular recognition. While previous studies have illustrated the potential of engineering knottins with modified loop sequences, a thorough exploration into the tolerated loop lengths and sequence space of a knottin scaffold has not been performed. In this work, we used the Ecballium elaterium trypsin inhibitor II (EETI) as a model member of the knottin family and constructed libraries of EETI loop-substituted variants with diversity in both amino acid sequence and loop length. Using yeast surface display, we isolated properly folded EETI loop-substituted clones and applied sequence analysis tools to assess the tolerated diversity of both amino acid sequence and loop length. In addition, we used covariance analysis to study the relationships between individual positions in the substituted loops, based on the expectation that correlated amino acid substitutions will occur between interacting residue pairs. We then used the results of our sequence and covariance analyses to successfully predict loop sequences that facilitated proper folding of the knottin when substituted into EETI loop 3. The sequence trends we observed in properly folded EETI loop-substituted clones will be useful for guiding future protein engineering efforts with this knottin scaffold. Furthermore, our findings demonstrate that the combination of directed evolution with sequence and covariance analyses can be a powerful tool for rational protein engineering.

Combinatorial Optimization of Cystine-Knot Peptides towards High-Affinity Inhibitors of Human Matriptase-1

“…Tolerance of this loop region towards amino acid exchanges has been extensively investigated for the structurally similar knottin EETI [18], [29], [69], [70]. This loop region is thought to be involved in the early folding process of the miniprotein via formation of a type II β-turn [2], [4], [11], [18], [70]. Since this loop sequence is a folding determinant, only moderate sequence variations were included by randomizing each position to 10%.…”

Plant cystine‐knot peptides: pharmacological perspectives

“…Several studies have so far been performed in animal models, including human xenograft tumour, to evaluate the effectiveness of radio-labelled cystine-knot proteins as a diagnostic agent to be tested in glio-and medulloblastoma, melanoma, breast and pancreatic cancer and tumour neoangiogenesis. For instance, an 111 In-labelled agoutirelated protein (AgRP) with affinity for the integrin αvβ3 expressed in human glioblastoma xenograft [75] or 64 Cu-DOTA-S02 that binds integrin αvβ6 with high affinity for pancreatic [76] and lung cancer [74]. The knottin 99-Tc-SAAC-S02 was tested for SPECT imaging of integrin αvβ6 positive tumours [77] and 8 F-FP-3-4-A, an engineered peptide [78] that binds to integrin αvβ3, to obtain imaging of tumour angiogenesis.…”

Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)

“…Таким белком является барназа (РНКаза из Bacillus amyloliquefaciens), с помощью которой методом гетерологической экспрессии были получены растворимые формы кноттинов-ИСП и с правильно сформированными S-S-мостиками без рефолдинга [44,88,89]. Описано также получение библиотек, представляющих собой модифицированные аналоги ЕeТI, слитые с гемагглютинином вируса гриппа и далее с капсидным белком pIII фага Fd, и аналоги ЕеТI и МСоТI-II, слитые с поверхностным агглютинином Aga2p дрожжей Saccharomyces cerevisiae [34,90,91].…”