Victor A. Streltsov

Structure of the insulin receptor ectodomain reveals a folded-over conformation

The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation. Here we present the crystal structure at 3.8 Å resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.

Mechanism-Based Covalent Neuraminidase Inhibitors with Broad Spectrum Influenza Antiviral Activity

Adding to the Antiviral Arsenal The envelope of influenza virus contains two immunodominant glycoproteins: hemagglutinin and neuraminidase (NA). Existing antivirals like zanamivir (Relenza) and oseltamivir (Tamiflu) target NA; however, the development of drug resistance is a problem. Kim et al. (p. 71, published online 21 February) now report a different class of NA inhibitors. NA catalyzes the removal of sialic acids from the surface of host cells to initiate entry. Discovery of a NA–sialic acid intermediate led to the production of sialic acid analogs that bound covalently to NA and inhibited its enzymatic activity. These compounds showed activity against a wide variety of influenza strains, inhibited viral replication in cell culture, and were able to protect mice against influenza infection. Protection of mice was equivalent to protection seen from zanamivir. Moreover, the compounds showed activity against drug-resistant strains in vitro. These compounds represent a potentially useful addition to the arsenal of antivirals used to treat influenza infection. Looking deeply into the mechanism of enzyme inhibition provides a clue for the development of new drugs to fight flu. Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here on a new class of specific, mechanism-based anti-influenza drugs that function through the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and we confirm this mode of action with structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad-spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates.

Electron density and optical anisotropy in rhombohedral carbonates. III: Synchrotron X-ray studies of CaCO3, MgCO3 and MnCO3

Diffraction-deformation electron-density (Δp) images for small, naturally faced single crystals of synthetic calcite (CaCO 3 ), magnesite (MgCO 3 ) and mineral rhodochrosite (MnCO 3 ) were measured with focused λ = 0.7 and 0.9 A synchrotron (SR) X-radiation. Mo Kα (λ = 0.71073 A) structure factors were also measured for MnCO 3 . Lattice mode frequencies predicted from eigenvalues of T and L tensors for CO 3 rigid-group motion in these structures are close to spectroscopic values. High approximate Δp symmetry around the cations increases towards 6/mmm in the sequence CaCO 3 , MgCO 3 to MnCO 3 . The Δp topography near the CO 3 groups shows the influence of the cations, and correlates strongly with the refractive indices, as required for a cause and effect relationship between electron density and optical anisotropy. Aspherical electron density around the Mn atom can be attributed to the effect of a non-ideal octahedral crystal field on the 3d electron distribution. The relationship of the Δp topography near the Mn atom with that near the CO 3 group in MnCO 3 is consistent with magnetic interactions. Space group R3c, hexagonal, Z = 6, T= 295K : CaCO 3 , M r = 100.09, a = 4.988(2), c = 17.068(2) A, V = 367.8(3) A 3 , D x = 2.711 Mg m -3 , μ 0.7 = 1.93 mm -1 , F(000) = 300, R = 0.015, wR = 0.012, S = 3.0 for 437 unique reflections ; MgCO 3 , M r = 84.31, a = 4.632(1), c = 15.007 (2) A, V = 278.8 (2) A 3 , D x = 3.013 Mg m -3 , μ 0.9 = 0.99 mm -1 , F(000) = 252, R = 0.015, wR = 0.021, S = 4.34 for 270 unique reflections ; MnCO 3 , M r = 114.95, a = 4.772(3), c = 15.637 (3) A, V = 308.4 (4) A 3 , D x = 3.713 Mg m -3 , p 0.7 = 5.62 m -1 , F(000) = 330, R = 0.015, wR = 0.039, S = 3.38 for 386 unique reflections of the SR data set and a = 4.773 (1), c = 15.642(1) A, V = 308.6 (1) A 3 , D x = 3.711 Mg m -3 , μ(Mo Kα) = 5.86 mm -1 , R = 0.017, wR = 0.024, S = 2.79 for 368 unique Mo Kα reflections.

X-ray Study of the Electron Density in Calcite, CaCO3

The electron density in synthetic calcite, CaCO 3 , has been determined using diffraction data for a naturally faced single crystal measured with X-ray Mo Kα (λ=0.71073 A) radiation. Extinction corrections that minimize differences between equivalent reflection intensities are closely approximated by the values which optimize the extinction parameter as part of the least-squares structure refinement. Deformation electron densities evaluated with the two techniques are closely similar. There are 0.26 e A -3 high-density maxima in the C-O bonds and 0.28 e A -3 maxima at the 0-atom lone pairs

Crystal Structure of the Amyloid-β p3 Fragment Provides a Model for Oligomer Formation in Alzheimer's Disease

Alzheimers disease is a progressive neurodegenerative disorder associated with the presence of amyloid-β (Aβ) peptide fibrillar plaques in the brain. However, current evidence suggests that soluble nonfibrillar Aβ oligomers may be the major drivers of Aβ-mediated synaptic dysfunction. Structural information on these Aβ species has been very limited because of their noncrystalline and unstable nature. Here, we describe a crystal structure of amylogenic residues 18–41 of the Aβ peptide (equivalent to the p3 α/γ-secretase fragment of amyloid precursor protein) presented within the CDR3 loop region of a shark Ig new antigen receptor (IgNAR) single variable domain antibody. The predominant oligomeric species is a tightly associated Aβ dimer, with paired dimers forming a tetramer in the crystal caged within four IgNAR domains, preventing uncontrolled amyloid formation. Our structure correlates with independently observed features of small nonfibrillar Aβ oligomers and reveals conserved elements consistent with residues and motifs predicted as critical in Aβ folding and oligomerization, thus potentially providing a model system for nonfibrillar oligomer formation in Alzheimers disease.

The Structure of the Amyloid-β Peptide High-Affinity Copper II Binding Site in Alzheimer Disease

Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-beta (Abeta) protein bound primarily to copper ions. The evidence for an oxidative stress role of Abeta-Cu redox chemistry is still incomplete. Details of the copper binding site in Abeta may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of Abeta peptides complexed with Cu(2+) in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl) concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length Abeta-Cu(2+) peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the Abeta-Cu(2+) complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu(2+) binding site is consistent with the hypothesis that the redox activity of the metal ion bound to Abeta can lead to the formation of dityrosine-linked dimers found in AD.

Synchrotron X-ray study of the electron density in α-Al2O3

Structure factors for synthetic haematite, α-Fe 2 O 3 , have been measured for two small crystals using focused λ=0.7 A synchrotron radiation. The structure factors from the two data sets are consistent. Approximate symmetry in the concordant densities, related more closely to the Fe-Fe geometry than to the nearest-neighbour Fe-O interactions, is similar to that in the corundum α-Al 2 O 3 structure. Deformation density maxima are located at the midpoint of the Fe-Fe vector along the c axis, on a common face for O-octahedra, perpendicular to c. Maxima also occur at the midpoint of the Fe-Fe vector bisecting the edges of the O-octahedra. These results are in accordance with theoretical predictions for metal-metal bonding

Structural and functional basis of resistance to neuraminidase inhibitors of influenza B viruses.

We have identified a virus, B/Perth/211/2001, with a spontaneous mutation, D197E in the neuraminidase (NA), which confers cross-resistance to all NA inhibitors. We analyzed enzyme properties of the D197 and E197 NAs and compared these to a D197N NA, known to arise after oseltamivir treatment. Zanamivir and peramivir bound slowly to the wild type NA, but binding of oseltamivir was more rapid. The D197E/N mutations resulted in faster binding of all three inhibitors. Analysis of the crystal structures of D197 and E197 NAs with and without inhibitors showed that the D197E mutation compromised the interaction of neighboring R150 with the N-acetyl group, common to the substrate sialic acid and all NA inhibitors. Although rotation of the E275 in the NA active site occurs upon binding peramivir in both the D197 and E197 NAs, this does not occur upon binding oseltamivir in the E197 NA. Lack of the E275 rotation would also account for the loss of slow binding and the partial resistance of influenza B wild type NAs to oseltamivir.

Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype.

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen‐driven affinity‐matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an “upright” conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3‐specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low‐sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen‐binding surface.

Structural insights into ligand-induced activation of the insulin receptor

The current model for insulin binding to the insulin receptor proposes that there are two binding sites, referred to as sites 1 and 2, on each monomer in the receptor homodimer and two binding surfaces on insulin, one involving residues predominantly from the dimerization face of insulin (the classical binding surface) and the other residues from the hexamerization face. High‐affinity binding involves one insulin molecule using its two surfaces to make bridging contacts with site 1 from one receptor monomer and site 2 from the other. Whilst the receptor dimer has two identical site 1‐site 2 pairs, insulin molecules cannot bridge both pairs simultaneously. Our structures of the insulin receptor (IR) ectodomain dimer and the L1‐CR‐L2 fragments of IR and insulin‐like growth factor receptor (IGF‐1R) explain many of the features of ligand‐receptor binding and allow the two binding sites on the receptor to be described. The IR dimer has an unexpected folded‐over conformation which places the C‐terminal surface of the first fibronectin‐III domain in close juxtaposition to the known L1 domain ligand‐binding surface suggesting that the C‐terminal surface of FnIII‐1 is the second binding site involved in high‐affinity binding. This is very different from previous models based on three‐dimensional reconstruction from scanning transmission electron micrographs. Our single‐molecule images indicate that IGF‐1R has a morphology similar to that of IR. In addition, the structures of the first three domains (L1‐CR‐L2) of the IR and IGF‐1R show that there are major differences in the two regions governing ligand specificity. The implications of these findings for ligand‐induced receptor activation will be discussed.