Optimizing mechanostable anchor points of engineered lipocalin in complex with CTLA-4
Optimizing mechanostable anchor points of engineered lipocalin in complex with CTLA-4
Abstract We used single-molecule AFM force spectroscopy (AFM-SMFS) to screen residues along the backbone of a non-antibody protein binding scaffold (lipocalin/anticalin), and determine the optimal anchor point that maximizes binding strength of the interaction with its target (CTLA-4). By incorporating non-canonical amino acids into anticalin, and using click chemistry to attach an Fgβ peptide at internal sequence positions, we were able to mechanically dissociate anticalin from CTLA-4 by pulling from eight different anchoring residues using an AFM cantilever tip. 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 pulling points tested were significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1-10 nN sec-1. Anisotropic network modelling and molecular dynamics simulations using the Gō-MARTINI approach explained the mechanism underlying the geometric dependency of mechanostability. These results suggest that optimization of attachment residue position for therapeutic and diagnostic cargo can provide large improvements in binding strength, allowing affinity maturation without requiring genetic mutation of binding interface residues.
Liu Zhaowei、Moreira Rodrigo A.、Dujmovi? Ana、Yang Byeongseon、Poma Adolfo B.、Liu Haipei、Nash Michael A.
Institute of Physical Chemistry, Department of Chemistry, University of Basel||Department of Biosystems Science and EngineeringBiosystems and Soft Matter Division, Institute of Fundamental Technological Research, Polish Academy of Sciences,Institute of Physical Chemistry, Department of Chemistry, University of Basel||Department of Biosystems Science and EngineeringInstitute of Physical Chemistry, Department of Chemistry, University of Basel||Department of Biosystems Science and EngineeringBiosystems and Soft Matter Division, Institute of Fundamental Technological Research, Polish Academy of Sciences,Institute of Physical Chemistry, Department of Chemistry, University of Basel||Department of Biosystems Science and EngineeringInstitute of Physical Chemistry, Department of Chemistry, University of Basel||Department of Biosystems Science and Engineering
生物科学研究方法、生物科学研究技术生物化学分子生物学
Atomic force microscopysingle-molecule force spectroscopyprotein engineeringmechanical anisotropyCTLA-4non-canonical amino acidclick chemistry
Liu Zhaowei,Moreira Rodrigo A.,Dujmovi? Ana,Yang Byeongseon,Poma Adolfo B.,Liu Haipei,Nash Michael A..Optimizing mechanostable anchor points of engineered lipocalin in complex with CTLA-4[EB/OL].(2025-03-28)[2025-08-02].https://www.biorxiv.org/content/10.1101/2021.03.09.434559.点此复制
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