ABSTRACT
Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Šresolution. The structure together with functional analyses reveal that the B56γ (B'γ) subunit of an essential host enzyme, protein phosphatase 2 A (PP2A), is repurposed as an integral component of the intasome to mediate HTLV-1 integration. Our studies reveal a key host-virus interaction underlying the replication of an important human pathogen and highlight divergent integration strategies of retroviruses.
Subject(s)
Host-Pathogen Interactions/genetics , Human T-lymphotropic virus 1/genetics , Integrases/metabolism , Protein Phosphatase 2/genetics , Viral Proteins/metabolism , Virus Integration/genetics , Cryoelectron Microscopy , DNA, Viral/metabolism , HEK293 Cells , Human T-lymphotropic virus 1/enzymology , Humans , Integrases/ultrastructure , Models, Molecular , Point Mutation , Protein Binding/genetics , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/ultrastructure , Viral Proteins/ultrastructureABSTRACT
Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen-deuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors.
Subject(s)
Escherichia coli/enzymology , Glycolipids/metabolism , Lipid A/metabolism , Pyrophosphatases/chemistry , Pyrophosphatases/metabolism , Uridine Diphosphate/metabolism , Catalytic Domain , Crystallography, X-Ray , Escherichia coli/genetics , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Mutation , Protein Conformation , Pyrophosphatases/genetics , Substrate SpecificityABSTRACT
Most Gram-negative bacteria are surrounded by a glycolipid called lipopolysaccharide (LPS), which forms a barrier to hydrophobic toxins and, in pathogenic bacteria, is a virulence factor. During LPS biosynthesis, a membrane-associated glycosyltransferase (LpxB) forms a tetra-acylated disaccharide that is further acylated to form the membrane anchor moiety of LPS. Here we solve the structure of a soluble and catalytically competent LpxB by X-ray crystallography. The structure reveals that LpxB has a glycosyltransferase-B family fold but with a highly intertwined, C-terminally swapped dimer comprising four domains. We identify key catalytic residues with a product, UDP, bound in the active site, as well as clusters of hydrophobic residues that likely mediate productive membrane association or capture of lipidic substrates. These studies provide the basis for rational design of antibiotics targeting a crucial step in LPS biosynthesis.
