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Ubiquitin-derived artificial binding proteins targeting oncofetal fibronectin reveal scaffold plasticity by β-strand slippage

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Katzschmann, Anja, Haupts, Ulrich, Reimann, Anja, Settele, Florian, Gloser-Bräunig, Manja, Fiedler, Erik, Parthier, Christoph·2024·Communications Biology
DOI 10.1038/s42003-024-06569-9PMC PMC11283464

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Abstract

Affilin proteins, artificial binding proteins based on the ubiquitin scaffold, have been generated by directed protein evolution to yield de-novo variants that bind the extra-domain B (EDB) of oncofetal fibronectin, an established marker of tumor neovasculature. The crystal structures of two EDB-specific Affilin variants reveal a striking structural plasticity of the ubiquitin scaffold, characterised by β-strand slippage, leading to different negative register shifts of the β5 strands. This process recruits amino acid residues from β5 towards the N-terminus to an adjacent loop region and subsequent residues into β5, respectively, remodeling the binding interface and leading to target specificity and affinity. Protein backbone alterations resulting from β-strand register shifts, as seen in the ubiquitin fold, can pose additional challenges to protein engineering as structural evidence of these events is still limited and they are difficult to predict. However, they can surface under the selection pressure of directed evolution and suggest that backbone plasticity allowing β-strand slippages can increase structural diversity, enhancing the evolutionary potential of a protein scaffold.

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Fig. 1 Analysis of binding properties and thermal stability of evolved Affilin variants. a SPR analysis of increasing Affilin concentrations binding to the immobilized target 67B89. Affilin concentrations used (sensorgram colours from light to dark blue): 0 nM, 1.2 nM, 3.7 nM, 11.1 nM and 33.3 nM. Resulting data are given in Table 1. b Analysis of Affilin thermal stabilities by DSF in comparison with the non-evolved parental diubiquitin variant (diUb-WAA, Tm = 71 °C). Vertical lines indicate the midpoint transition temperatures (Tm). c EDB-specific binding analysed by ELISA, left panel: binding of Affilin variants to 67B89 (filled circles) and absence of binding to 6789 (open circles). Right panel: comparison of Af2 binding to different oncofetal Fn fragments (67B89, 7B8, B89), no affinity to 67B and 6789. Mean data points and error bars shown with black contours, individual measurements superimposed as coloured crosses. d Binding of Af2s to human foetal lung fibroblasts cells (Wi-38, high EBD expression, detected by immunofluorescence in green, cell nuclei stained in blue) and no binding of Af2s to neonatal human dermal fibroblasts (NHDF, no EDB expression). No binding is observed for (non-evolved) diubiquitin variant diUb-WAA. Scale bar: 100 µm.
Fig. 1 Analysis of binding properties and thermal stability of evolved Affilin variants. a SPR analysis of increasing Affilin concentrations binding to the immobilized target 67B89. Affilin concentrations used (sensorgram colours from light to dark blue): 0 nM, 1.2 nM, 3.7 nM, 11.1 nM and 33.3 nM. Resulting data are given in Table 1. b Analysis of Affilin thermal stabilities by DSF in comparison with the non-evolved parental diubiquitin variant (diUb-WAA, Tm = 71 °C). Vertical lines indicate the midpoint transition temperatures (Tm). c EDB-specific binding analysed by ELISA, left panel: binding of Affilin variants to 67B89 (filled circles) and absence of binding to 6789 (open circles). Right panel: comparison of Af2 binding to different oncofetal Fn fragments (67B89, 7B8, B89), no affinity to 67B and 6789. Mean data points and error bars shown with black contours, individual measurements superimposed as coloured crosses. d Binding of Af2s to human foetal lung fibroblasts cells (Wi-38, high EBD expression, detected by immunofluorescence in green, cell nuclei stained in blue) and no binding of Af2s to neonatal human dermal fibroblasts (NHDF, no EDB expression). No binding is observed for (non-evolved) diubiquitin variant diUb-WAA. Scale bar: 100 µm.CC BY · PubMed Central
Fig. 2 Crystal structure of Affilin Af2 in complex with the target 7B8. a The crystal structure of Affilin Af2 bound to oncofetal Fn fragment 7B8 revealing three target-binding regions (I-III, dotted red elipsoids) involving the Fn domains EDB and Fn8. The β5-strands of Af2 Ubi-N (darker blue) and Ubi-C (darker red) exhibit distinct negative register shifts, remodelling the α2β5 loops involved in binding (coloured in cyan and magenta, respectively). Detailed view of key interactions between (b) Ub-N and EDB (interface I), (c) Ub-C and EDB (interface II) and (d) Ub-C and Fn8 (interface III). Residues involved in binding shown as sticks, other evolved amino acids as lines, hydrogen bonds depicted as yellow dashed lines, water molecules as red spheres. Residue colouring of C-atoms in cyan/light blue (Ub-N), magenta/light red (Ub-C), orange/blue (EDB). Ub-C residue numbers labelled with asterisk correspond to the residue numbering of the Ub-N domain.
Fig. 2 Crystal structure of Affilin Af2 in complex with the target 7B8. a The crystal structure of Affilin Af2 bound to oncofetal Fn fragment 7B8 revealing three target-binding regions (I-III, dotted red elipsoids) involving the Fn domains EDB and Fn8. The β5-strands of Af2 Ubi-N (darker blue) and Ubi-C (darker red) exhibit distinct negative register shifts, remodelling the α2β5 loops involved in binding (coloured in cyan and magenta, respectively). Detailed view of key interactions between (b) Ub-N and EDB (interface I), (c) Ub-C and EDB (interface II) and (d) Ub-C and Fn8 (interface III). Residues involved in binding shown as sticks, other evolved amino acids as lines, hydrogen bonds depicted as yellow dashed lines, water molecules as red spheres. Residue colouring of C-atoms in cyan/light blue (Ub-N), magenta/light red (Ub-C), orange/blue (EDB). Ub-C residue numbers labelled with asterisk correspond to the residue numbering of the Ub-N domain.CC BY · PubMed Central
Fig. 3 The different β5 register shifts observed in the 7B8-bound structure of Af2. a Overall structure of 7B8-bound Af2 depicting the distribution of the evolved residues of Ub-N and Ub-C (side chains shown as lines, C-atoms of Ub-N cyan, C-atoms of Ub-C magenta). Linker residues coloured in green. The β5 strands of Af2 Ubi-N (darker blue) and Ubi-C (darker red) exhibit distinct negative register shifts, extending the α2β5 loops involved in target binding. Structural superposition of Ub-wt (PDB id 1UBQ, light grey, black labels) and (b) the Ub-N domain of Af2 (light blue, blue labels, evolved residues shown in cyan) revealing the -2 register-shift of β5 (darker blue) and (c) the Ub-C domain of Af2 (light red, red labels with asterisk, corresponding to residue numbering of Ub-N domain, evolved residues shown in magenta) exhibiting a -4 register-shift of β5 (darker red). Af2 residues relocated by the β5 register shifts and corresponding residues of Ub-wt shown as sticks.
Fig. 3 The different β5 register shifts observed in the 7B8-bound structure of Af2. a Overall structure of 7B8-bound Af2 depicting the distribution of the evolved residues of Ub-N and Ub-C (side chains shown as lines, C-atoms of Ub-N cyan, C-atoms of Ub-C magenta). Linker residues coloured in green. The β5 strands of Af2 Ubi-N (darker blue) and Ubi-C (darker red) exhibit distinct negative register shifts, extending the α2β5 loops involved in target binding. Structural superposition of Ub-wt (PDB id 1UBQ, light grey, black labels) and (b) the Ub-N domain of Af2 (light blue, blue labels, evolved residues shown in cyan) revealing the -2 register-shift of β5 (darker blue) and (c) the Ub-C domain of Af2 (light red, red labels with asterisk, corresponding to residue numbering of Ub-N domain, evolved residues shown in magenta) exhibiting a -4 register-shift of β5 (darker red). Af2 residues relocated by the β5 register shifts and corresponding residues of Ub-wt shown as sticks.CC BY · PubMed Central
Fig. 4 Crystal structure of unbound Affilin Af1. a Overall structure of Af1 depicting the distribution of the evolved residues of Ub-N and Ub-C (side chains shown as lines, C-atoms of Ub-N cyan, C-atoms of Ub-C magenta). Linker residues coloured in green. The β5 strands of Af1 Ub-N (bright blue) and Ub-C (lighter red) exhibit -2 negative register shifts. W142 (corresponding to Y141 in Af2) shown as sticks. b Structural superposition of Af1 and the Af2:7B8 complex, based on the alignment of the Ub-N domains, showing the different relative orientations of Ub-N und Ub-C. Regions of evolved residues coloured in cyan (Ub-N) and magenta (Ub-C). Y141 (Af2) and W142 (Af1) shown as sticks. c The two β5 register shifts of Af1 are apparent from the structural superposition of Ub-wt (PDB id 1UBQ, light grey, black labels) with Af1 Ub-N (blue, blue labels) and Ub-C (dark red, red labels). Residues of β5 shown as sticks, evolved residues outside β5 shown as lines. Structural comparison of (d) the Ub-N domains of Af1 (dark blue, black labels) and Af2 (light blue, blue labels), both exhibiting -2 register shifts and (e) the Ub-C domains of af Af1 (dark red, black labels) and Af2 (light red, red labels) with distinct β5 register shifts (−2/−4). Ub-C residue numbers labelled with asterisk correspond to the residue numbering of the Ub-N domain. Residues H68/H68* and R72/R72*, shown as sticks, mark the different structural rearrangements around β5 caused by the register shifts.
Fig. 4 Crystal structure of unbound Affilin Af1. a Overall structure of Af1 depicting the distribution of the evolved residues of Ub-N and Ub-C (side chains shown as lines, C-atoms of Ub-N cyan, C-atoms of Ub-C magenta). Linker residues coloured in green. The β5 strands of Af1 Ub-N (bright blue) and Ub-C (lighter red) exhibit -2 negative register shifts. W142 (corresponding to Y141 in Af2) shown as sticks. b Structural superposition of Af1 and the Af2:7B8 complex, based on the alignment of the Ub-N domains, showing the different relative orientations of Ub-N und Ub-C. Regions of evolved residues coloured in cyan (Ub-N) and magenta (Ub-C). Y141 (Af2) and W142 (Af1) shown as sticks. c The two β5 register shifts of Af1 are apparent from the structural superposition of Ub-wt (PDB id 1UBQ, light grey, black labels) with Af1 Ub-N (blue, blue labels) and Ub-C (dark red, red labels). Residues of β5 shown as sticks, evolved residues outside β5 shown as lines. Structural comparison of (d) the Ub-N domains of Af1 (dark blue, black labels) and Af2 (light blue, blue labels), both exhibiting -2 register shifts and (e) the Ub-C domains of af Af1 (dark red, black labels) and Af2 (light red, red labels) with distinct β5 register shifts (−2/−4). Ub-C residue numbers labelled with asterisk correspond to the residue numbering of the Ub-N domain. Residues H68/H68* and R72/R72*, shown as sticks, mark the different structural rearrangements around β5 caused by the register shifts.CC BY · PubMed Central
Fig. 5 Binding and functional analysis of an EDB-specific Affilin-IL-2 fusion. a Binding of Af2-IL-2 to the target 67B89 (filled circles) and lack of binding to the off-target 6789 (open circles) analysed by ELISA (binding parameters given in Table 1). Mean data points and error bars shown in black, individual measurements superimposed as coloured crosses. b Binding of Af2-IL-2 to human foetal lung fibroblasts cells (Wi-38, high EBD expression, detected by immunofluorescence in green, cell nuclei stained in blue) and no binding of Af2-IL-2 to neonatal human dermal fibroblasts (NHDF, no EDB expression), scale bar: 100 µm. c IL-2-dependent growth of murine cytotoxic T-cells (CTLL-2) after application of Af2-IL-2 (filled black circles, black error bars), recombinant human IL-2 (open grey circles, grey error bars) serving as reference.
Fig. 5 Binding and functional analysis of an EDB-specific Affilin-IL-2 fusion. a Binding of Af2-IL-2 to the target 67B89 (filled circles) and lack of binding to the off-target 6789 (open circles) analysed by ELISA (binding parameters given in Table 1). Mean data points and error bars shown in black, individual measurements superimposed as coloured crosses. b Binding of Af2-IL-2 to human foetal lung fibroblasts cells (Wi-38, high EBD expression, detected by immunofluorescence in green, cell nuclei stained in blue) and no binding of Af2-IL-2 to neonatal human dermal fibroblasts (NHDF, no EDB expression), scale bar: 100 µm. c IL-2-dependent growth of murine cytotoxic T-cells (CTLL-2) after application of Af2-IL-2 (filled black circles, black error bars), recombinant human IL-2 (open grey circles, grey error bars) serving as reference.CC BY · PubMed Central

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Metadata

Authors
Katzschmann, A., Haupts, U., Reimann, A., Settele, F., Gloser-Bräunig, M., Fiedler, E., & Parthier, C.
Affiliations
Martin Luther University Halle-Wittenberg (DE)
Corresponding author
  • Fiedler, Erik
  • Parthier, Christoph
ORCID
Venue
Communications Biology
Publisher
Springer Science and Business Media LLC
Published
2024-07-27
Volume
7
Issue
1
Pages
907-907
ISSN
2399-3642 (ISSN-L)
DOI
10.1038/s42003-024-06569-9
PMC
PMC11283464
OpenAlex
W4401046350
Cited by
2
References
2
Citations by year
2026: 2
FWCI
0.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Communications Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Digitalen Hochschulbibliothek Sachsen-Anhalt (Universitäts- und Landesbibliothek Sachsen-Anhalt) (cc-by)
Funders
Helmholtz-Zentrum Berlin für Materialien und Energie
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
oncofetal fibronectin, directed protein evolution, tumor neovasculature, crystal structure
MeSH terms
Protein Conformation, beta-Strand, Fibronectins, Humans, Models, Molecular, Protein Binding, Protein Engineering, Crystallography, X-Ray, Ubiquitin

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Katzschmann, A., Haupts, U., Reimann, A., Settele, F., Gloser-Bräunig, M., Fiedler, E., & Parthier, C. (2024). Ubiquitin-derived artificial binding proteins targeting oncofetal fibronectin reveal scaffold plasticity by β-strand slippage. Communications Biology. https://doi.org/10.1038/s42003-024-06569-9