Partner: M. Nash


Recent publications
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
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.

Keywords:

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

Affiliations:
Liu Z.-other affiliation
Moreira R.A.-IPPT PAN
Dujmović A.-other affiliation
Liu H.-Imperial College London (GB)
Yang B.-other affiliation
Poma A.B.-IPPT PAN
Nash M.A.-other affiliation

Conference abstracts
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
Abstract:

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.

Affiliations:
Liu Z.-other affiliation
Moreira R.-IPPT PAN
Dujmović A.-other affiliation
Liu H.-Imperial College London (GB)
Yang B.-other affiliation
Poma Bernaola A.-IPPT PAN
Nash M.-other affiliation
2.Poma Bernaola A., Moreira R., Liu Z., Nash M., Optimisation of the mechanical stability of anticalin:CTLA-4 protein complex via GoMARTINI simulations, Hünfeld 2021: Workshop on Computer Simulation and Theory of Macromolecules, 2021-04-23/04-24, Virtual Meeting (DE), pp.110, 2021
Abstract:

A variety of non-immunoglobulin protein scaffolds with potential as alternatives to monoclonal antibodies for nanoparticle-based drug delivery are of high interest for targeting T-cells displaying cytotoxic T-lymphocyte antigen 4 (CTLA-4), a limiting factor is the resistance of the anticalin:CTLA-4 complex to mechanical forces exerted by local shear stress. Here, we used a multi scale approach based on Go-MARTINI approach and single-molecule AFM force spectroscopy (AFM-SMFS) to screen residues along the anticalin backbone and determine the optimal anchor point that maximizes binding strength of the anticalin:CTLA-4 complex. We parametrize the Go-MARTINI approach based on the AFM_SMFS data and the molecular dynamics (MD) simulations using parametrized approach help to explain the mechanisms underlying the geometric dependency of mechanostability in the complex. This process can be related to an unzipping-shear mechanism which is commonly seen in nucleic acids strands. These results suggest that optimization of attachment residue position for therapeutic and diagnostic cargo can provide large improvements without requiring genetic mutation of binding interface residues.

Keywords:

Biomechanics, CTLA4-anticalin complex, SMFS, Gō-Martini, mechanostabiity, MD simulation, PCA, protein engineering

Affiliations:
Poma Bernaola A.-IPPT PAN
Moreira R.-IPPT PAN
Liu Z.-other affiliation
Nash M.-other affiliation