Mapping Mechanostable Pulling Geometries of a Therapeutic Anticalin/CTLA-4 Protein Complex

Zhaowei Liu; Rodrigo A. Moreira; Ana Dujmović; Haipei Liu; Byeongseon Yang; Adolfo B. Poma; Michael A. Nash

doi:10.1021/acs.nanolett.1c03584

Mapping Mechanostable Pulling Geometries of a Therapeutic Anticalin/CTLA-4 Protein Complex

Zhaowei Liu, Rodrigo A. Moreira, Ana Dujmović, Haipei Liu, Byeongseon Yang, Adolfo B. Poma, Michael A. Nash

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2–3 orders of magnitude over a force range of 50–200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1–10 nN s–1. Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues.

Original language	English
Pages (from-to)	179-187
Journal	Nano Letters
Volume	22
Issue number	1
DOIs	https://doi.org/10.1021/acs.nanolett.1c03584
Publication status	Published - 12 Jan 2022
Externally published	Yes

Funding

This work was supported by the University of Basel, ETH Zurich, an ERC Starting Grant (MMA-715207), the NCCR in Molecular Systems Engineering, and the Swiss National Science Foundation (Project 200021_175478). The authors thank Peter Schultz for providing the pEVOL-pAzF plasmid and Lloyd Ruddock for providing the pMJS205 plasmid. The authors thank Timothy Sharpe and the Biophysics Facility of Biozentrum, University of Basel, for the help with affinity measurements using MST. A.B.P. acknowledges financial support from the National Science Centre, Poland, under Grant No. 2017/26/D/NZ1/00466 and the grant MAB PLUS/11/2019 from the Foundation for Polish Science. Computational resources were supported by the PL-GRID infrastructure, the Jülich Supercomputing Centre on the supercomputer JURECA at Forschungszentrum Jülich, and the TUL Computing & Information Services Center infrastructure. This work was supported by the University of Basel, ETH Zurich, an ERC Starting Grant (MMA-715207), the NCCR in Molecular Systems Engineering, and the Swiss National Science Foundation (Project 200021_175478). The authors thank Peter Schultz for providing the pEVOL-pAzF plasmid and Lloyd Ruddock for providing the pMJS205 plasmid. The authors thank Timothy Sharpe and the Biophysics Facility of Biozentrum, University of Basel, for the help with affinity measurements using MST. A.B.P. acknowledges financial support from the National Science Centre, Poland, under Grant No. 2017/26/D/NZ1/00466 and the grant MAB PLUS/11/2019 from the Foundation for Polish Science. Computational resources were supported by the PL-GRID infrastructure, the Ju?lich Supercomputing Centre on the supercomputer JURECA at Forschungszentrum Ju?lich, and the TUL Computing & Information Services Center infrastructure.

Funders	Funder number
Biophysics Facility of Biozentrum
NCCR in Molecular Systems Engineering
TUL Computing & Information Services Center infrastructure
Timothy Sharpe
Universität Basel
Horizon 2020 Framework Programme	715207
Horizon 2020 Framework Programme
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung	200021_175478
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
Fundacja na rzecz Nauki Polskiej
Eidgenössische Technische Hochschule Zürich	MMA-715207
Eidgenössische Technische Hochschule Zürich
Narodowe Centrum Nauki	MAB PLUS/11/2019, 2017/26/D/NZ1/00466
Narodowe Centrum Nauki

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title = "Mapping Mechanostable Pulling Geometries of a Therapeutic Anticalin/CTLA-4 Protein Complex",

abstract = "We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2–3 orders of magnitude over a force range of 50–200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1–10 nN s–1. Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues.",

author = "Zhaowei Liu and Moreira, {Rodrigo A.} and Ana Dujmovi{\'c} and Haipei Liu and Byeongseon Yang and Poma, {Adolfo B.} and Nash, {Michael A.}",

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AU - Liu, Zhaowei

AU - Moreira, Rodrigo A.

AU - Dujmović, Ana

AU - Liu, Haipei

AU - Yang, Byeongseon

AU - Poma, Adolfo B.

AU - Nash, Michael A.

PY - 2022/1/12

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N2 - We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2–3 orders of magnitude over a force range of 50–200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1–10 nN s–1. Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues.

AB - We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2–3 orders of magnitude over a force range of 50–200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1–10 nN s–1. Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues.

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Mapping Mechanostable Pulling Geometries of a Therapeutic Anticalin/CTLA-4 Protein Complex

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