Neil M. McKern

Crystal Structure of a Truncated Epidermal Growth Factor Receptor Extracellular Domain Bound to Transforming Growth Factor α

We report the crystal structure, at 2.5 A resolution, of a truncated human EGFR ectodomain bound to TGFalpha. TGFalpha interacts with both L1 and L2 domains of EGFR, making many main chain contacts with L1 and interacting with L2 via key conserved residues. The results indicate how EGFR family members can bind a family of highly variable ligands. In the 2:2 TGFalpha:sEGFR501 complex, each ligand interacts with only one receptor molecule. There are two types of dimers in the asymmetric unit: a head-to-head dimer involving contacts between the L1 and L2 domains and a back-to-back dimer dominated by interactions between the CR1 domains of each receptor. Based on sequence conservation, buried surface area, and mutagenesis experiments, the back-to-back dimer is favored to be biologically relevant.

The Crystal Structure of a Truncated ErbB2 Ectodomain Reveals an Active Conformation, Poised to Interact with Other ErbB Receptors

ErbB2 does not bind ligand, yet appears to be the major signaling partner for other ErbB receptors by forming heteromeric complexes with ErbB1, ErbB3, or ErbB4. The crystal structure of residues 1-509 of ErbB2 at 2.5 A resolution reveals an activated conformation similar to that of the EGFR when complexed with ligand and very different from that seen in the unactivated forms of ErbB3 or EGFR. The structure explains the inability of ErbB2 to bind known ligands and suggests why ErbB2 fails to form homodimers. Together, the data suggest a model in which ErbB2 is already in the activated conformation and ready to interact with other ligand-activated ErbB receptors.

Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor

The type-1 insulin-like growth-factor receptor (IGF-1R) and insulin receptor (IR) are closely related members of the tyrosine-kinase receptor superfamily. IR is essential for glucose homeostasis, whereas IGF-1R is involved in both normal growth and development and malignant transformation. Homologues of these receptors are found in animals as simple as cnidarians. The epidermal growth-factor receptor (EGFR) family is closely related to the IR family and has significant sequence identity to the extracellular portion we describe here. We now present the structure of the first three domains of IGF-1R (L1–Cys-rich–L2) determined to 2.6 Å resolution. The L domains each consist of asingle-stranded right-handed β-helix. The Cys-rich region is composed of eight disulphide-bonded modules, seven of which form a rod-shaped domain with modules associated in an unusual manner. The three domains surround a central space of sufficient size to accommodate a ligand molecule. Although the fragment (residues 1–462) does not bind ligand, many of the determinants responsible for hormone binding and ligand specificity map to this central site. This structure therefore shows how the IR subfamily might interact with their ligands.

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.

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1–CR–L2) of human IR at 2.3 Å resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) β-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more α-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R.

Coat protein of Potyviruses

SummaryFour strains of potato virus Y, PVY-D, PVY-10, PVY-18, and PVY-43, obtained from different Australian sources were compared on the basis of their biological, serological and coat protein structural properties. Each of the strains could be distinguished on the basis of their reactions on selected test plant species. Two of the PVY strains, PVY-D and PVY-10, induced symptoms similar to those produced by the PVYO strain group. The reactions of PVY-18 and PVY-43, although comparable to PVYN in some hosts, did not completely match the description of the PVYN strain group. In contrast to the other three strains, PVY-18 could not be transmitted byMyzus persicae in repeated tests. No difference was observed in the serological properties of the four PVY strains in different assay systems, using polyclonal antisera.The amino acid sequences of the coat proteins of PVY-10, PVY-18, and PVY-43 were obtained and compared with the coat protein sequences of pepper mottle virus (PeMV) [Dougherty WG, Allison RF, Parks TD, Johnston RE, Feild MJ, Armstrong FB (1985) Virology 146: 282–292] and PVY-D [Shukla DD, Inglis AS, McKern NM, Gough KH (1986) Virology 152: 118–125]. The homology between the PVY strains ranged from 96.3 to 99.3% and with the PeMV sequence, 91.4 to 92.9%. Based on this high sequence homology, and the previous observation that coat protein sequences of potyvirus strains are always greater than 90% identical, PeMV could be considered a strain of PVY. However, PVY and PeMV are reported to be only distantly serologically related and on this basis PeMV is currently considered to be an independent member of the Potyvirus group.

The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an α-chain that is entirely extracellular and a β-chain that spans the cell membrane. The α-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the β-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give α-β monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the α-α dimer bond Cys524. In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the β-chain residues Cys798 and Cys807 and between the α-chain Cys647 and the β-chain Cys872. The latter bond is the sole α-β link in the molecule and implies a side-by-side alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional α-α dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys884 exists as a buried thiol in the soluble ectodomain.

Coat protein of potyviruses. 2. Amino acid sequence of the coat protein of potato virus Y.

The amino acid sequence of the coat protein of potato virus Y (PVY), the type member of the potyvirus group, has been determined by protein sequencing techniques. The protein contains 267 amino acid residues with a calculated mol wt of 29,945. A comparison of the PVY coat protein sequence with those of tobacco etch virus (TEV) and pepper mottle virus (PeMV) predicted from nucleotide sequence data (R. F. Allison, J. G. Sorenson, M. E. Kelly, F. B. Armstrong, and W. G. Dougherty, Proc. Natl. Acad. Sci. USA82, 3969-3972, 1985; W. G. Dougherty, R. F. Allison, T. D. Parks, R. E. Johnston, M. J. Feild, and F. B. Armstrong, Virology 146,282-291, 1985) shows that sequence homology between the coat proteins from PVY and PeMV is 92% and that between PVY and TEV is 62%. These data suggest that PVY and PeMV are much more closely related than previously believed from serological studies.

Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast

Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the worlds poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.

Amino acid sequence of pilin from Bacteroides nodosus (strain 198), the causative organism of ovine footrot

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