Core-Binding Factor β Increases the Affinity between Human Cullin 5 and HIV-1 Vif within an E3 Ligase Complex
ABSTRACT HIV-1 Vif masquerades as a receptor for a cellular E3 ligase harboring Elongin B, Elongin C, and Cullin 5 (EloB/C/Cul5) proteins that facilitate degradation of the antiretroviral factor APOBEC3G (A3G). This Vif-mediated activity requires human core-binding factor β (CBFβ) in contrast to cellular substrate receptors. We observed calorimetrically that Cul5 binds tighter to full-length Vif((1-192))/EloB/C/CBFβ (K(d) = 5 ± 2 nM) than to Vif((95-192))/EloB/C (K(d) = 327 ± 40 nM), which cannot bind CBFβ. A comparison of heat capacity changes supports a model in which CBFβ prestabilizes Vif((1-192)) relative to Vif((95-192)), consistent with a stronger interaction of Cul5 with Vif's C-terminal Zn(2+)-binding motif. An additional interface between Cul5 and an N-terminal region of Vif appears to be plausible, which has therapeutic design implications.
- SourceAvailable from: Alessio Ciulli[Show abstract] [Hide abstract]
ABSTRACT: Proteins of the ankyrin repeat and SOCS-box (ASB) family act as substrate recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyse the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. ASB multimeric complexes are therefore involved in numerous cell processes and pathways, however, their interactions, assembly and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC) and Cullin 5. Here we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization and ion mobility-mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modelling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to other CRL systems, and provide a platform for further molecular investigation of this important protein family. Data reported here will also be of use in the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.Biochemistry 07/2013; 52(31). DOI:10.1021/bi400758h · 3.19 Impact Factor
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ABSTRACT: Several members of the APOBEC3 family of cellular restriction factors provide intrinsic immunity to the host against viral infection. Specifically, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H haplotypes II, V, and VII provide protection against HIV-1Δvif through hypermutation of the viral genome, inhibition of reverse transcription, and inhibition of viral DNA integration into the host genome. HIV-1 counteracts APOBEC3 proteins by encoding the viral protein Vif, which contains distinct domains that specifically interact with these APOBEC3 proteins to ensure their proteasomal degradation, allowing virus replication to proceed. Here, we review our current understanding of APOBEC3 structure, editing and non-editing mechanisms of APOBEC3-mediated restriction, Vif-APOBEC3 interactions that trigger APOBEC3 degradation, and the contribution of APOBEC3 proteins to restriction and control of HIV-1 replication in infected patients.Journal of Molecular Biology 11/2013; DOI:10.1016/j.jmb.2013.10.033 · 3.96 Impact Factor
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ABSTRACT: CBFβ was recently found to be a key regulator of HIV-1 Vif's ability to overcome host anti-viral APOBEC3 proteins. However, the detailed molecular requirements for the Vif-CBFβ interaction are still not clear. Here, we mapped the minimum Vif domain required for CBFβ binding. In terms of CBFβ binding, the Vif N-terminus was very sensitive to deletions. We determined that Vif fragment 5-126 was sufficient to form a stable complex with CBFβ in vitro. We also observed that ionic interactions were not the main contributor to the interaction between Vif and CBFβ. Instead, hydrophobic interactions were important for maintaining the Vif-CBFβ complex, since it could be disrupted by non-ionic detergent. Site-directed mutagenesis of conserved hydrophobic amino acids revealed novel residues in Vif that were important for CBFβ binding and APOBEC3 inactivation. At least part of the well-characterized HCCH domain (residues 108-139) was required to form a stable Vif-CBFβ complex. Thus, the HCCH motif may have dual role in binding both Cul5 and CBFβ. Considering the importance of Vif in HIV-1 infection, this unique Vif-CBFβ interaction represents an attractive pharmacological intervention target against HIV-1.Journal of Virology 12/2013; 88(5). DOI:10.1128/JVI.03604-13 · 4.65 Impact Factor