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

Onderzoeksoutput: Articlepeer review

11 Citaten (Scopus)
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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.
Originele taal-2English
Aantal pagina's9
TijdschriftProc Natl Acad Sci USA
Nummer van het tijdschrift17
StatusPublished - 27 apr 2021

Bibliografische nota

Funding Information:
ACKNOWLEDGMENTS. This research was supported by the Slovenian Research Agency (Program No. P4-0176 to R.J. and Project No. J1-1706 to J.L.), the European Research Council (Advanced Grant MaCChines 787115 to R.J.), the ERA-NET project MediSurf (Project3193 coordinated by Alexander Kros), and Vrije Universiteit Brussel OZR Grant SPR13 to R.L. We acknowledge the use of the PX1 and PX2 beamlines at SOLEIL Synchrotron and the P12 beamline at Deutsches Elektronen-Synchrotron and Advanced Light Source. We thank Bojana Stevovićand students Nuša Krivec, Uroš Zavrtanik, and Domen Oblak for their technical support in cloning or in ITC measurements.

Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.

Copyright 2021 Elsevier B.V., All rights reserved.


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