Instytut Podstawowych Problemów Techniki
Polskiej Akademii Nauk

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B. Yang


Ostatnie publikacje
1.  Liu Z., Moreira R.A., Dujmović A., Liu H., Yang B., Poma A.B., Nash M.A., Mapping mechanostable pulling geometries of a therapeutic anticalin/CTLA-4 protein complex, Nano Letters, ISSN: 1530-6984, DOI: 10.1021/acs.nanolett.1c03584, Vol.22, pp.179-187, 2022

Streszczenie:
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.

Słowa kluczowe:
atomic force microscopy, protein engineering, single-molecule force spectroscopy, mechanical anisotropy, click chemistry, Go̅-Martini model, PCA

Afiliacje autorów:
Liu Z. - inna afiliacja
Moreira R.A. - IPPT PAN
Dujmović A. - inna afiliacja
Liu H. - Imperial College London (GB)
Yang B. - inna afiliacja
Poma A.B. - IPPT PAN
Nash M.A. - inna afiliacja
200p.

Abstrakty konferencyjne
1.  Liu Z., Moreira R., Dujmović A., Liu H., Yang B., Poma Bernaola A., Nash M., Mapping mechanostable pulling geometries of protein-ligand complexes, 65th Annual Meeting of the Biophysical Society, 2021-02-22/02-26, virtual meeting (US), DOI: 10.1016/j.bpj.2020.11.2233, pp.362a, 2021

Streszczenie:
Anticalin is a non-immunoglobulin protein scaffold with potential as an alternative to monoclonal antibodies for nanoparticle-based drug delivery to cells displaying cytotoxic T-lymphocyte antigen 4 (CTLA-4). In this context, one limiting factor is the resistance of the anticalin:CTLA-4 complex to mechanical forces exerted by fluid shear stress. Here, we used single-molecule AFM force spectroscopy to screen residues along the anticalin backbone and determine the optimal pulling point that achieves maximum mechanical stability of the anticalin:CTLA-4 complex. We used non-canonical amino acid incorporation by amber suppression in the anticalin combined with click chemistry to attach an Fgβ peptide at internal residues of the anticalin. We then used the Fgβ peptide as a handle to mechanically dissociate anticalin from CTLA-4 by applying tension at 8 different anchor residues, and measure the unbinding energy landscape for each pulling geometry. We found that pulling from amino acid position 60 on the anticalin resulted in ∼100% higher mechanical stability of the complex as compared with either the N- or C-terminus. Molecular dynamics (MD) simulations using the coarse-grained Martini force field showed strong agreement with experiments and help explain the mechanisms underlying the geometric dependency of mechanical stability in this therapeutic molecular complex. These results demonstrate that the mechanical stability of receptor-ligand complexes can be optimized by controlling the loading geometry without making any changes to the binding interface.

Afiliacje autorów:
Liu Z. - inna afiliacja
Moreira R. - IPPT PAN
Dujmović A. - inna afiliacja
Liu H. - Imperial College London (GB)
Yang B. - inna afiliacja
Poma Bernaola A. - IPPT PAN
Nash M. - inna afiliacja

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