Person:

Li, Zongli

We have previously described the interactions of aquaporin-0 (AQP0) with dimyristoyl phosphatidylcholine (DMPC) lipids. We have now determined the 2.5 Å structure of AQP0 in two-dimensional (2D) crystals formed with Escherichia coli polar lipids (EPLs), which differ from DMPC both in headgroups and acyl chains. Comparison of the two structures shows that AQP0 does not adapt to the different length of the acyl chains in EPLs and that the distance between the phosphodiester groups in the two leaflets of the DMPC and EPL bilayers is almost identical. The EPL headgroups interact differently with AQP0 than do those of DMPC, but the acyl chains in the EPL and DMPC bilayers occupy similar positions. The interactions of annular lipids with membrane proteins seem to be driven by the propensity of the acyl chains to fill gaps in the protein surface. Interactions of the lipid headgroups may be responsible for the specific interactions found in tightly bound lipids but seem to have a negligible effect on interactions of generic annular lipids with membrane proteins.

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.

Cellular immunity against viral infection and tumor cells depends on antigen presentation by the major histocompatibility complex class 1 molecules (MHC I). Intracellular antigenic peptides are transported into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) and then loaded onto the nascent MHC I, which are exported to the cell surface and present peptides to the immune system1. Cytotoxic T lymphocytes recognize non-self peptides and program the infected or malignant cells for apoptosis. Defects in TAP account for immunodeficiency and tumor development. To escape immune surveillance, some viruses have evolved strategies to either down-regulate TAP expression or directly inhibit TAP activity. To date neither the architecture of TAP nor the mechanism of viral inhibition has been elucidated at the structural level. In this study we describe the cryo-electron microscopy (cryo-EM) structure of human TAP in complex with its inhibitor ICP47, a small protein produced by the herpes simplex virus I. We show that the twelve transmembrane helices and two cytosolic nucleotide-binding domains (NBDs) of the transporter adopt an inward-facing conformation with the two NBDs separated. The viral inhibitor ICP47 forms a long helical hairpin, which plugs the translocation pathway of TAP from the cytoplasmic side. Association of ICP47 precludes substrate binding and also prevents NBD closure necessary for ATP hydrolysis. This work illustrates a striking example of immune evasion by persistent viruses. By blocking viral antigens from entering the ER, herpes simplex virus is hidden from cytotoxic T lymphocytes, which may contribute to establishing a lifelong infection in the host.

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na+-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4–S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π–π bonds and cation–π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.