The 1918 influenza pandemic resulted in about 20 million deaths. This enormous impact, coupled with renewed interest in emerging infections, makes characterization of the virus involved a priority. Receptor binding, the initial event in virus infection, is a major determinant of virus transmissibility that, for influenza viruses, is mediated by the hemagglutinin (HA) membrane glycoprotein. We have determined the crystal structures of the HA from the 1918 virus and two closely related HAs in complex with receptor analogs. They explain how the 1918 HA, while retaining receptor binding site amino acids characteristic of an avian precursor HA, is able to bind human receptors and how, as a consequence, the virus was able to spread in the human population.

Escherichia coli GlpG is an integral membrane protein that belongs to the widespread rhomboid protease family. Rhomboid proteases, like site-2 protease (S2P) and gamma-secretase, are unique in that they cleave the transmembrane domain of other membrane proteins. Here we describe the 2.1 A resolution crystal structure of the GlpG core domain. This structure contains six transmembrane segments. Residues previously shown to be involved in catalysis, including a Ser-His dyad, and several water molecules are found at the protein interior at a depth below the membrane surface. This putative active site is accessible by substrate through a large 'V-shaped' opening that faces laterally towards the lipid, but is blocked by a half-submerged loop structure. These observations indicate that, in intramembrane proteolysis, the scission of peptide bonds takes place within the hydrophobic environment of the membrane bilayer. The crystal structure also suggests a gating mechanism for GlpG that controls substrate access to its hydrophilic active site.

The three-dimensional structures of avian H5 and swine H9 influenza hemagglutinins (HAs) from viruses closely related to those that caused outbreaks of human disease in Hong Kong in 1997 and 1999 were determined bound to avian and human cell receptor analogs. Emerging influenza pandemics have been accompanied by the evolution of receptor-binding specificity from the preference of avian viruses for sialic acid receptors in ␣2,3 linkage to the preference of human viruses for ␣2,6 linkages. The four new structures show that HA binding sites specific for human receptors appear to be wider than those preferring avian receptors and how avian and human receptors are distinguished by atomic contacts at the glycosidic linkage. ␣2,3-Linked sialosides bind the avian HA in a trans conformation to form an ␣2,3 linkage-specific motif, made by the glycosidic oxygen and 4-OH of the penultimate galactose, that is complementary to the hydrogen-bonding capacity of Gln-226, an avian-specific residue. ␣2,6-Linked sialosides bind in a cis conformation, exposing the glycosidic oxygen to solution and nonpolar atoms of the receptor to Leu-226, a human-specific residue. The new structures are compared with previously reported crystal structures of HA͞sialoside complexes of the H3 subtype that caused the 1968 Hong Kong Influenza virus pandemic and analyzed in relation to HA sequences of all 15 subtypes and to receptor affinity data to make clearer how receptor-binding sites of HAs from avian viruses evolve as the virus adapts to humans.

Amyloid beta-peptide, which forms neuronal and vascular amyloid deposits in Alzheimer's disease, is derived from an integral membrane protein precursor. The biological function of the precursor is currently unclear. Here we describe the X-ray structure of E2, the largest of the three conserved domains of the precursor. The structure of E2 consists of two coiled-coil substructures connected through a continuous helix and bears an unexpected resemblance to the spectrin family of protein structures. E2 can reversibly dimerize in the solution, and the dimerization occurs along the longest dimension of the molecule in an antiparallel orientation, which enables the N-terminal substructure of one monomer to pack against the C-terminal substructure of a second monomer. Heparan sulfate proteoglycans, the putative ligand for the precursor present in extracellular matrix, bind to E2 at a conserved and positively charged site near the dimer interface.

There are 15 subtypes of in¯uenza A virus (H1±H15), all of which are found in avian species. Three caused pandemics in the last century: H1 in 1918H1 in (and 1977H1 in ), H2 in 1957H1 in and H3 in 1968H1 in . In 1997H1 in , an H5 avian virus and in 1999 an H9 virus caused outbreaks of respiratory disease in Hong Kong. We have determined the three-dimensional structures of the haemagglutinins (HAs) from H5 avian and H9 swine viruses closely related to the viruses isolated from humans in Hong Kong. We have compared them with known structures of the H3 HA from the virus that caused the 1968 H3 pandemic and of the HA±esterase±fusion (HEF) glycoprotein from an in¯uenza C virus. Structure and sequence comparisons suggest that HA subtypes may have originated by diversi®cation of properties that affected the metastability of HAs required for their membrane fusion activities in viral infection. Keywords: avian in¯uenza/in¯uenza A virus/sialic acid/ swine in¯uenza/virus evolution IntroductionThe haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins of in¯uenza A viruses, which function respectively as the receptor-binding and membrane fusion glycoprotein in cell entry (HA) and as the receptordestroying enzyme in virus release (NA), are divided into subtypes on the basis of differences in their antigenicities and amino acid sequences. Fifteen subtypes of HA (H1±H15) that share between 40 and 60% sequence identity and nine of NA (N1±N9, 40±60% identity) have been distinguished (World Health Organization, 1980;Rohm et al., 1996). Viruses containing all 15 HAs have been isolated from avian species and more limited numbers from equine, H3 and H7; seals, H3, H4 and H7; whales, H1 and H13; and swine, H1, H3 and H9. Viruses containing three different HA subtypes, H1, H2 and H3, caused pandemics in the last century : in 1918, H1; in 1957, H2; in 1968, H3; and H1 again in 1977. Recent outbreaks of in¯uenza in humans in Hong Kong and Southern China have resulted from infections with H5 and H9 viruses (Claas et al., 1998;Lin et al., 2000).In¯uenza viruses that infect humans or the genes for their HAs initially derive from avian viruses either directly by cross-species infection or by gene reassortment during mixed infections. Direct infection is proposed to have occurred in 1918(Reid et al., 1999), in 1997(Claas et al., 1998(Lin et al., 2000 and, on the last two occasions, viruses from local birds were found to have infected humans. Reassortant viruses appear to have caused the pandemics of 1957 and 1968; the 1957 H2 virus differed by three genes, those for HA, NA and the RNA polymerase subunit PB1, from the H1 virus that infected humans between 1918 and 1957; the 1968 H3 virus differed by two genes, those for HA and PB1, from the H2 virus that infected humans between 1957 and 1968(Kawaoka et al., 1989. In both cases, the genes for the H2 and H3 HAs are proposed to have been contributed by avian viruses, during infection of an unknown host that was infected simultaneously by the prevalent human virus.In the int...

How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >1011 -fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a "shutter" that cytoplasmic factors might open or close to regulate iron release in vivo.Ferritins are vesicle-like assemblies of 24 polypeptide (4-helix bundle) subunits that concentrate iron in cells by directing the formation of a ferric mineral in the hollow protein interior (8 nm diameter) (1-3). Effective cellular iron concentrations Ͼ1011 times the solubility of the ferric ion are achieved by ferritins, which are found in microorganisms, plants, and animals. The complexity and the sophistication of the genetic regulation of the ferritins, involving both DNA and mRNA (4 -7), emphasize the central role of iron and ferritin in life. Rates of Fe(II) oxidation, translocation of Fe(II) and Fe(III) (1.0 -2.0 nm), and mineralization are all controlled by the protein (1, 2). Fe(II) release from ferritin following reduction of the mineral is slow and poorly understood (8, 9) but is important for the biosynthesis of iron-proteins, such as those required in respiration, photosynthesis, nitrogen fixation, and cell division, (1, 2) and as dietary iron (10). How and where the iron exits from ferritin in vivo is not known.We now show that localized unfolding in the assembled protein, at sites of cooperative subunit interactions, can increase the rate of exit of iron from ferritin. When conserved leucine 134 was replaced by proline (L134P), the protein assembled, oxidized Fe(II), and mineralized Fe(III), but the time for complete dissolution of mineral (480 iron) in vitro was greatly decreased (5 min compared with 150 min for the parent protein). X-ray diffraction studies of crystals of H-L134P ferritin showed a flexible region localized near the termini of two subunit helices (C, D), which form the interfaces of subunit trimers and a channel. The results indicate that iron can exit from ferritin at the trimer subunit junction. A possible mechanism for regulated iron release in vivo could be localized disorder in the assembled protein, enhanced by cytoplasmic changes with effects analogous to the effect of H-L134P. EXPERIMENTAL PROCEDURES Expression and Purification of Recombinant Ferritin Proteins-The coding sequence for H ferritin, H-L134P, K82Q, and H-L134P, R86Qwere obtained by the mutagenesis of PJD5F12L134P sequence (12) with a Chameleon TM double-stranded, site-directed mutagenesis kit (Stratagene). The oligonucleotides, 5F12P134L (5Ј-CACCTGTTCCTC-CAGGTATTCAGTCTCC-3Ј), 5F12K82Q (5Ј-CGCTCTGGTTTCTGGA-CATCCTGCAG-3Ј), and 5F12R86Q (5Ј-CCCCATTCATCCTGCTCTG-GTTTCTTGACATCC-3Ј), were used as the...

Comparing the structures of H3, H5 and H9 subtype haemagglutinins, we deduced a structural basis for including all 15 influenza subtypes in four clades. H3, H5 and H9 represent three of these clades; we now report the structure of an H7 HA as a representative of the fourth clade. We confirm the structure of the turn at the N-terminus of the conserved central alpha-helix of HA2, and the combination of ionisable residues near the "fusion peptide" as clade-specific features. We compare the structures of three H1 HAs with H5 HA in the same clade, to refine our previous classification and we confirm the division of the clades into two groups of two. We also show the roles of carbohydrate side chains in the esterase-fusion domain boundaries in the formation of clade-specific structural markers.

Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.