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

Benjamin T. Porebski, Paul J. Conroy, Nyssa Drinkwater, Peter Schofield, Rodrigo Vazquez-Lombardi, Morag R. Hunter, David E. Hoke, Daniel Christ, Sheena McGowan, Ashley M. Buckle

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)541-550
Number of pages9
JournalProtein Engineering, Design and Selection
Volume29
Issue number11
DOIs
Publication statusPublished - 1 Nov 2016

Keywords

  • consensus design
  • loop grafting
  • protein engineering
  • stability-function trade-off
  • X-ray crystallography

Cite this

Porebski, Benjamin T. ; Conroy, Paul J. ; Drinkwater, Nyssa ; Schofield, Peter ; Vazquez-Lombardi, Rodrigo ; Hunter, Morag R. ; Hoke, David E. ; Christ, Daniel ; McGowan, Sheena ; Buckle, Ashley M. / Circumventing the stability-function trade-off in an engineered FN3 domain. In: Protein Engineering, Design and Selection. 2016 ; Vol. 29, No. 11. pp. 541-550.
@article{04e0ed7bff434adcb59e7935718c2815,
title = "Circumventing the stability-function trade-off in an engineered FN3 domain",
abstract = "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.",
keywords = "consensus design, loop grafting, protein engineering, stability-function trade-off, X-ray crystallography",
author = "Porebski, {Benjamin T.} and Conroy, {Paul J.} and Nyssa Drinkwater and Peter Schofield and Rodrigo Vazquez-Lombardi and Hunter, {Morag R.} and Hoke, {David E.} and Daniel Christ and Sheena McGowan and Buckle, {Ashley M.}",
year = "2016",
month = "11",
day = "1",
doi = "10.1093/protein/gzw046",
language = "English",
volume = "29",
pages = "541--550",
journal = "Protein Engineering, Design and Selection",
issn = "1741-0126",
publisher = "Oxford University Press",
number = "11",

}

Porebski, BT, Conroy, PJ, Drinkwater, N, Schofield, P, Vazquez-Lombardi, R, Hunter, MR, Hoke, DE, Christ, D, McGowan, S & Buckle, AM 2016, 'Circumventing the stability-function trade-off in an engineered FN3 domain' Protein Engineering, Design and Selection, vol. 29, no. 11, pp. 541-550. https://doi.org/10.1093/protein/gzw046

Circumventing the stability-function trade-off in an engineered FN3 domain. / Porebski, Benjamin T.; Conroy, Paul J.; Drinkwater, Nyssa; Schofield, Peter; Vazquez-Lombardi, Rodrigo; Hunter, Morag R.; Hoke, David E.; Christ, Daniel; McGowan, Sheena; Buckle, Ashley M.

In: Protein Engineering, Design and Selection, Vol. 29, No. 11, 01.11.2016, p. 541-550.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Circumventing the stability-function trade-off in an engineered FN3 domain

AU - Porebski, Benjamin T.

AU - Conroy, Paul J.

AU - Drinkwater, Nyssa

AU - Schofield, Peter

AU - Vazquez-Lombardi, Rodrigo

AU - Hunter, Morag R.

AU - Hoke, David E.

AU - Christ, Daniel

AU - McGowan, Sheena

AU - Buckle, Ashley M.

PY - 2016/11/1

Y1 - 2016/11/1

N2 - 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.

AB - 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.

KW - consensus design

KW - loop grafting

KW - protein engineering

KW - stability-function trade-off

KW - X-ray crystallography

UR - http://www.scopus.com/inward/record.url?scp=84995938487&partnerID=8YFLogxK

U2 - 10.1093/protein/gzw046

DO - 10.1093/protein/gzw046

M3 - Article

VL - 29

SP - 541

EP - 550

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

SN - 1741-0126

IS - 11

ER -