Structural Insights Into RNA Bridging Between HIV-1 Vif and Antiviral Factor APOBEC3G

Great effort has been devoted to discovering the basis of A3G-Vif interaction, the key event of HIV\u27s counteraction mechanism to evade antiviral innate immune response. Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif. We present an atomic model of the A3G-Vif interface, which assembles via known amino acid determinants. This assembly is not achieved by protein-protein interaction alone, but also involves RNA. The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base preference for the interaction and a unique Vif-ribose contact. This establishes the biological significance of an RNA ligand. Further assessment of interactions between A3G, Vif, and RNA ligands show that the A3G-Vif assembly and subsequent ubiquitination can be controlled by amino acid mutations at the interface or by polynucleotide modification, suggesting that a specific chemical moiety would be a promising pharmacophore to inhibit the A3G-Vif interaction

Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 A. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5\u27-3\u27 directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics

The Structure of Physarum polycephalum Hemagglutinin I Suggests a Minimal Carbohydrate Recognition Domain of Legume Lectin Fold

Physarum polycephalum hemagglutinin I is a 104-residue protein that is secreted to extracellular space. The crystal structure of hemagglutinin I has a β-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the β-sandwich of hemagglutinin I lacks a jelly roll motif and is essentially composed of two simple up-and-down β-sheets. This up-and-down β-sheet motif is well conserved in other lectins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down β-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that hemagglutinin I lacking a jelly roll motif also binds to its target glycopeptide. Taken together, the up-and-down β-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of hemagglutinin I suggests a minimal carbohydrate recognition domain

The ssDNA Mutator APOBEC3A Is Regulated by Cooperative Dimerization

Deaminase activity mediated by the human APOBEC3 family of proteins contributes to genomic instability and cancer. APOBEC3A is by far the most active in this family and can cause rapid cell death when overexpressed, but in general how the activity of APOBEC3s is regulated on a molecular level is unclear. In this study, the biochemical and structural basis of APOBEC3A substrate binding and specificity is elucidated. We find that specific binding of single-stranded DNA is regulated by the cooperative dimerization of APOBEC3A. The crystal structure elucidates this homodimer as a symmetric domain swap of the N-terminal residues. This dimer interface provides insights into how cooperative protein-protein interactions may affect function in the APOBEC3 enzymes and provides a potential scaffold for strategies aimed at reducing their mutation load

Structural Characterization of a Trapped Folding Intermediate of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114–208 residues are broadened, indicating that conformations of the 114–208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1–113 and the 114–208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114–208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114–208 segment of PCP-0SH folds into a stable compact structure with non-native helix–helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH

'Beskrifning på et nytt Örtegenus'

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114–208 residues are broadened, indicating that conformations of the 114–208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1–113 and the 114–208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114–208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114–208 segment of PCP-0SH folds into a stable compact structure with non-native helix–helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH

Structural Characterization of a Trapped Folding Intermediate of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114–208 residues are broadened, indicating that conformations of the 114–208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1–113 and the 114–208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114–208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114–208 segment of PCP-0SH folds into a stable compact structure with non-native helix–helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH