biostudies-literatureBuzas DEuropean Research CouncilWellcome TrustBiotechnology and Biological Sciences Research Council342-351.e6https://www.ebi.ac.uk/biostudies/studies/S-EPMC7616808biostudies-literatureUnknown32(3)Adenovirus-derived nanoparticles (ADDomer) comprise 60 copies of adenovirus penton base protein (PBP). ADDomer is thermostable, rendering the storage, transport, and deployment of ADDomer-based therapeutics independent of a cold chain. To expand the scope of ADDomers for new applications, we engineered ADDobodies, representing PBP crown domain, genetically separated from PBP multimerization domain. We inserted heterologous sequences into hyper-variable loops, resulting in monomeric, thermostable ADDobodies expressed at high yields in Escherichia coli. The X-ray structure of an ADDobody prototype validated our design. ADDobodies can be used in ribosome display experiments to select a specific binder against a target, with an enrichment factor of ∼10⁴-fold per round. ADDobodies can be re-converted into ADDomers by genetically reconnecting the selected ADDobody with the PBP multimerization domain from a different species, giving rise to a multivalent nanoparticle, called Chimera, confirmed by a 2.2 Å electron cryo-microscopy structure. Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target.Structure (London, England : 1993)Engineering the ADDobody protein scaffold for generation of high-avidity ADDomer super-binders.PMC7616808221708BB/R000484/1202904/Z/16/Z106115/Z/14/Z206181/Z/17/Z210701/Z/18/Z221708/Z/20/Z834631Capin JYadav SKNBerger IGautam GSessions RBBufton JCToelzer CBuzas DGupta KSun HGarzoni FSchaffitzel CBorucu UfalseEngineering the ADDobody protein scaffold for generation of high-avidity ADDomer super-binders.Adenovirus-derived nanoparticles (ADDomer) comprise 60 copies of adenovirus penton base protein (PBP). ADDomer is thermostable, rendering the storage, transport, and deployment of ADDomer-based therapeutics independent of a cold chain. To expand the scope of ADDomers for new applications, we engineered ADDobodies, representing PBP crown domain, genetically separated from PBP multimerization domain. We inserted heterologous sequences into hyper-variable loops, resulting in monomeric, thermostable ADDobodies expressed at high yields in Escherichia coli. The X-ray structure of an ADDobody prototype validated our design. ADDobodies can be used in ribosome display experiments to select a specific binder against a target, with an enrichment factor of ∼10⁴-fold per round. ADDobodies can be re-converted into ADDomers by genetically reconnecting the selected ADDobody with the PBP multimerization domain from a different species, giving rise to a multivalent nanoparticle, called Chimera, confirmed by a 2.2 Å electron cryo-microscopy structure. Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target.2024-01-01T00:00:00Z2024 Mar2026-06-21T03:10:02.632Z2025-04-21T21:48:52.284ZS-EPMC76168083819895010.1016/j.str.2023.12.010