Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a β-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magicangle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. 31 P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand-rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. 1 H-31 P 2D correlation spectra and 2 H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation.solid-state NMR spectroscopy | small-angle X-ray scattering | conformational polymorphism | membrane curvature | peptide-membrane interactions V iral fusion proteins mediate entry of enveloped viruses into cells by merging the viral lipid envelope and the cell membrane. The membrane-interacting subunit of these glycoproteins contains two hydrophobic domains: a fusion peptide (FP) that is usually located at the N terminus and a transmembrane domain (TMD) at the C terminus (1). Together, these domains sandwich a water-soluble ectodomain with a helical segment that trimerizes into a coiled coil. During virus-cell fusion, the trimeric protein, which initially adopts a compact structure, unfolds to an extended intermediate that exposes the FP to the target cell membrane while keeping the TMD in the virus envelope. This extended conformation then folds onto itself to form a trimer of hairpins, in so doing pulling the cell membrane and the virus envelope into close proximity (2, 3). Subsequently, the FP and TMD are hypothesized to deform the two membranes and dehydrate them (4), eventually causing a fusion pore and a fully merged membrane. In the postfusion state, most viral fusion proteins exhibit a six-helixbundle structure in the ectodomain due to the trimer of hairpins (5).This model of virus-cell fusion largely derives from crystal structures of fusion proteins in the pre-and postfusion st...

Viral fusion proteins catalyze the merger of the virus envelope and the target cell membrane through multiple steps of protein conformational changes. The fusion peptide domain of these proteins is important for membrane fusion, but how it causes membrane curvature and dehydration is still poorly understood. We now use solid-state NMR spectroscopy to investigate the conformation, topology, and lipid and water interactions of the fusion peptide of the PIV5 virus F protein in three lipid membranes, POPC/POPG, DOPC/DOPG, and DOPE. These membranes allow us to investigate the effects of lipid chain disorder, membrane surface charge, and intrinsic negative curvature on the fusion peptide structure. Chemical shifts and spin diffusion data indicate that the PIV5 fusion peptide is inserted into all three membranes but adopts distinct conformations: it is fully α-helical in the POPC/POPG membrane, adopts a mixed strand/helix conformation in the DOPC/DOPG membrane, and is primarily a β-strand in the DOPE membrane. 31P NMR spectra show that the peptide retains the lamellar structure and hydration of the two anionic membranes. However, it dehydrates the DOPE membrane, destabilizes its inverted hexagonal phase, and creates an isotropic phase that is most likely a cubic phase. The ability of the β-strand conformation of the fusion peptide to generate negative Gaussian curvature and to dehydrate the membrane may be important for the formation of hemifusion intermediates in the membrane fusion pathway.