Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain

Porebski, Benjamin T.; Nickson, Adrian A.; Hoke, David E.; Hunter, Morag R.; Zhu, Liguang; McGowan, Sheena; Webb, Geoffrey I.; Buckle, Ashley M.

doi:10.1093/protein/gzv002

Cited by 40 publications

(50 citation statements)

References 64 publications

(108 reference statements)

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“…3E, Supplementary Figs S2A and S4). In close proximity to G30 is R32 and R71, which belong to a stability enhancing electrostatic mesh on the surface of FN3con (Porebski et al ., 2015) that were retained in the grafting process. As these are long and charged residues, we therefore hypothesize that R32 and R71 in FN3con-α-lys (G33 and Y74 in FNfn10-α-lys) may impose steric clashes with lysozyme, as suggested by modeling of the complex, thereby further restricting tight complex formation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…4A and B). We have previously shown FN3con to be extremely rigid due to an optimized hydrophobic core (Porebski et al ., 2015). For this reason, we did not expect to be able to easily engineer strand D to mimic the conformation seen in the FNfn10-α-lys structure (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…4C). To mimic the remaining FNfn10-α-lys paratope, we made three more mutations (G30Y, R32G and R71Y), which predominantly removed charged residues from FN3con's stability enhancing electrostatic mesh (Porebski et al ., 2015). We did so with the hypothesis that this will restore packing and remove the predicted steric clashes, thereby allowing for tighter complex formation.…”

Section: Resultsmentioning

confidence: 99%

“…In order to further explore the stability-function trade-off in FN3 domains, we questioned whether the potent binding activity of FNfn10-α-lys could be achieved without a loss in stability. We previously reported the consensus design of a FN3 domain, FN3con , which exhibits an extremely high degree of thermodynamic and kinetic stability ( T m > 100°C, reversible folding and aggregation resistant) (Porebski et al ., 2015). In this study, we describe loop grafting from FNfn10-α-lys onto the stable FN3con scaffold (creating FN3con-α-lys).…”

Section: Introductionmentioning

confidence: 99%

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Circumventing the stability-function trade-off in an engineered FN3 domain

Porebski

Conroy

Drinkwater

et al. 2016

Protein Engineering, Design and Selection

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The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-α-lys in complex with lysozyme revealed unanticipated interactions at the protein–protein interface involving framework residues of FNfn10-α-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-α-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-α-lys). FN3con therefore provides an exceptional window of stability to tolerate deleterious mutations, resulting in a substantial advantage for functional design. This study emphasizes the utility of consensus design for the generation of highly stable scaffolds for downstream protein engineering studies.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Circumventing the stability-function trade-off in an engineered FN3 domain

Porebski

Conroy

Drinkwater

et al. 2016

Protein Engineering, Design and Selection

Self Cite

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“…Although Fn3 repeats have considerable sequence variability, they adopt a similar three‐dimensional fold. We aligned the sequence of Fn3(1) with the closest hit in the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do), an engineered Fn3 domain (RCSB code 4U3H; 32% sequence identity) (Porebski et al., ). The sequence of Fn3(1) was then aligned with this structure to gain information on the locations of Arg533 and Gly556 (Fig.…”

Section: Sequence Variations In Bocmentioning

confidence: 99%

BOC is a modifier gene in holoprosencephaly

et al. 2017

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Holoprosencephaly (HPE), a common developmental defect of the forebrain and midface, has a complex etiology. Heterozygous, loss-of-function mutations in the Sonic hedgehog (SHH) pathway are associated with HPE. However, mutation carriers display highly variable clinical presentation, leading to an “autosomal dominant with modifier” model, in which the penetrance and expressivity of a predisposing mutation is graded by genetic or environmental modifiers. Such modifiers have not been identified. Boc encodes a SHH co-receptor and is a silent HPE modifier gene in mice. Here, we report identification of missense BOC variants in HPE patients. Consistent with these alleles functioning as HPE modifiers, individual variant BOC proteins had either loss- or gain-of-function properties in cell-based SHH signaling assays. Therefore, in addition to heterozygous loss-of-function mutations in specific SHH pathway genes and an ill-defined environmental component, our findings identify a third variable in HPE: low frequency modifier genes, BOC being the first identified.

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