A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures

Andrea Majerle, San Hadzi, Jana Aupič, Tadej Satler, Fabio Lapenta, Žiga Strmšek, Jurij Lah, Remy Loris, Roman Jerala

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Coiled-coils (CC) are widely used in protein design since their sequence-structure relationship is quite well-understood. In the CC protein origami (CCPO) approach a polypeptide chain is composed from a defined sequence of orthogonal CC building segments which fold into different polyhedral shapes such as tetrahedron, triangular prism, and four-sided pyramid. While noninteracting residues of the CCs can be engineered, additional functionalization of CC modules would expand the versatility of protein origami scaffolds. For this purpose, we generated a panel of single-chain camelid antibodies that target different CC modules of de novo designed tetrahedral CCPO protein. To elucidate how nanobodies recognize coiled-coils we solved crystal structures of nanobodies in complex with three coiled-coil dimers: a parallel homodimer, antiparallel homodimer and a parallel heterodimer. Crystal structures revealed that nanobodies recognize CC not only by CDR loops but also by the extensive use of nanobody framework residues. Due to the modular design of CCPO proteins the characterized nanobodies can recognize the same CC modules in different polyhedral structures such as bipyramid and triangular prism, thereby expanding the ability to functionalize polyhedra with modular nanobodies. We identified a pair of allosteric nanobodies that recognize two epitopes on the antiparallel APH coiled-coil where their binding is coupled via strong positive cooperativity. This allosteric coupling is retained in the context of tetrahedra with the APH segment which opens a way to incorporate allosteric signaling into designed by CC protein origami scaffolds.
Original languageEnglish
Article numbere2021899118
Number of pages9
JournalProc Natl Acad Sci USA
Issue number17
Publication statusPublished - 27 Apr 2021


  • structural biology
  • Nanobody
  • X-ray crystallography
  • Protein design
  • Coiled-coil proteins

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