SUMMARY The HIV-1 accessory protein Vif hijacks a cellular cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 family of restriction factors. Recently, the cellular transcription cofactor CBFβ was shown to form a complex with CRL5-Vif and be essential for APOBEC3 degradation and viral infectivity. We now demonstrate that CBFβ is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and activating CRL5-Vif by direct interaction. The CRL5-Vif-CBFβ holoenzyme forms a well-defined heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFβ. Heterodimers of CBFβ and RUNX transcription factors contribute towards the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFβ is mutually exclusive of RUNX heterodimerization and impacts expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limiting coding capacity uses one factor to hijack multiple host pathways.

Cu-hydroperoxido species (CuII–OOH) have been proposed to be key intermediates in biological and synthetic oxidations. Using biotin-streptavidin (Sav) technology, artificial copper proteins have been developed to stabilize a CuII–OOH complex in solution and in crystallo. Stability is achieved because the Sav host provides a local environment around the Cu–OOH that includes a network of hydrogen bonds to the hydroperoxido ligand. Systematic deletions of individual hydrogen bonds to the Cu–OOH complex were accomplished using different Sav variants and demonstrated that stability is achieved with a single hydrogen bond to the proximal O-atom of the hydroperoxido ligand: changing this interaction to only include the distal O-atom produced a reactive variant that oxidized an external substrate.

Cupredoxins are electron transfer proteins that have active sites containing a monomeric Cu center with an unusual trigonal monopyramidal structure (Type 1 Cu). A single Cu-Scys bond is present within the trigonal plane that is responsible for its unique physical properties. We demonstrate that a cysteine-containing variant of streptavidin (Sav) can serve as a protein host to model the structure and properties of Type 1 Cu sites. A series of artificial Cu proteins are described that rely on Sav and a series of biotinylated synthetic Cu complexes. Optical and EPR measurements highlight the presence of a Cu–Scys bond and XRD analysis provide structural evidence. We further provide evidence that changes in the linker between the biotin and Cu complex within the synthetic constructs allows for small changes in the placement of Cu centers within Sav that have dramatic effects on the structural and physical properties of the resulting artificial metalloproteins. These findings highlight the utility of the biotin-Sav technology as an approach for simulating active sites of metalloproteins.

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Secondary coordination spheres of metal complexes are instrumental in controlling properties that are linked to function. To study these effects in aqueous solutions artificial Cu proteins have been developed using biotin–streptavidin (Sav) technology and their binding of external azide ions investigated. Parallel binding studies were done in crystallo on single crystals of the artificial Cu proteins. Spectroscopic changes in solution are consistent with azide binding to the Cu centers. Structural studies corroborate that a Cu–N3 unit is present in each Sav subunit and reveal the composition of hydrogen bonding (H-bonding) networks that include the coordinated azido ligand. The networks involve amino acid residues and water molecules within the secondary coordination sphere. Mutation of these residues to ones that cannot form H-bonds caused a measurble change in the equilibrium binding constants that were measured in solution. These findings further demonstrate the utility of biotin–Sav technology to prepare water-stable inorganic complexes whose structures can be controlled within both primary and secondary coordination spheres.