ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.
Subject(s)
HIV/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , vif Gene Products, Human Immunodeficiency Virus/chemistry , vif Gene Products, Human Immunodeficiency Virus/metabolism , APOBEC-3G Deaminase , Amino Acid Sequence , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , HIV/genetics , Humans , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Thermodynamics , Ubiquitin-Protein Ligases/genetics , Ubiquitination , vif Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
All canonical transfer RNAs (tRNAs) have a uridine at position 8, involved in maintaining tRNA tertiary structure. However, the hyperthermophilic archaeon Methanopyrus kandleri harbors 30 (out of 34) tRNA genes with cytidine at position 8. Here, we demonstrate C-to-U editing at this location in the tRNA's tertiary core, and present the crystal structure of a tRNA-specific cytidine deaminase, CDAT8, which has the cytidine deaminase domain linked to a tRNA-binding THUMP domain. CDAT8 is specific for C deamination at position 8, requires only the acceptor stem hairpin for activity, and belongs to a unique family within the "cytidine deaminase-like" superfamily. The presence of this C-to-U editing enzyme guarantees the proper folding and functionality of all M. kandleri tRNAs.
Subject(s)
Cytidine Deaminase/chemistry , Cytidine Deaminase/metabolism , Euryarchaeota/metabolism , RNA Editing , RNA, Archaeal/metabolism , RNA, Transfer/metabolism , Amino Acid Motifs , Catalytic Domain , Crystallography, X-Ray , Deamination , Euryarchaeota/enzymology , Euryarchaeota/genetics , Genes, Archaeal , Models, Chemical , Models, Molecular , Nucleic Acid Conformation , Protein Multimerization , Protein Structure, Tertiary , RNA, Archaeal/chemistry , RNA, Archaeal/genetics , RNA, Transfer/chemistry , RNA, Transfer/geneticsABSTRACT
Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.
Subject(s)
Gene Products, vif/chemistry , Gene Products, vif/metabolism , HIV/genetics , Alanine/metabolism , Amino Acid Sequence , Amino Acid Substitution , Cell Line , Consensus Sequence , Conserved Sequence , Crystallization , Cullin Proteins/chemistry , Cullin Proteins/metabolism , Escherichia coli/genetics , Gene Products, vif/genetics , Humans , Hydrophobic and Hydrophilic Interactions , Kidney/cytology , Models, Biological , Models, Molecular , Molecular Sequence Data , Mutation , Plasmids , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Transfection , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , X-Ray DiffractionABSTRACT
The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.
