Peter A. Hoyne

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.

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.

Antibodies specifically targeting a locally misfolded region of tumor associated EGFR

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR287–302 complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFRC271A/C283A mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFRC271A/C283A. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.

Molecular Characterization of a Secreted Enzyme with Phospholipase B Activity from Moraxella bovis

A candidate for a vaccine against infectious bovine keratoconjunctivitis (IBK) has been cloned and characterized from Moraxella bovis. The plb gene encodes a protein of 616 amino acids (molecular mass of ~65.8 kDa) that expresses phospholipase B activity. Amino acid sequence analysis revealed that PLB is a new member of the GDSL (Gly-Asp-Ser-Leu) family of lipolytic enzymes.

High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper

Insulin receptors (IRs) that are truncated at the end of the ectodomain form dimers that bind insulin with different characteristics to wild type receptors. These soluble IRs have lowered affinity for insulin compared with full‐length IR, and exhibit linear Scatchard plots in contrast to the curvilinear plots obtained with full‐length IR, IR truncated at the C‐terminus of the transmembrane region and IR ectodomains fused to the self‐associating constant domains from Fc or λ immunoglobulins. In this report, we have fused the IR ectodomain to the 33 residue leucine zipper from the transcriptional activator GCN4 of Saccharomyces cerevisiae. This fusion protein binds insulin with high affinity in a manner comparable to native receptor. The respective dissociation constants were K d1 8.2×10−11 M and K d2 1.6×10−8 M for hIRedZip and K d1 5.7×10−11 M and K d2 6.3×10−9 M for membrane‐anchored, native receptor.

Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin‐producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.

Properties of an insulin receptor with an IGF-1 receptor loop exchange in the cysteine-rich region.

The insulin receptor (IR) and the insulin‐like growth factor‐I receptor (IGF‐1R) show differential binding of insulin and IGFs. The specificity determinants for IGF‐1 binding are known to be located in the cysteine‐rich (Cys‐rich) region between residues 223 and 274 of human IGF‐1R, which includes a loop that protrudes into the putative ligand binding site. In this report we have replaced residues 260–277 of human IR with residues 253–266 of the human IGF‐1R to produce an IR‐based, cysteine loop exchange chimaera, termed hIR‐Cys loop exchange (CLX), in which all 14 amino acid residues in the exchanged loop differ from wild‐type insulin receptor. This loop exchange had a detrimental effect on the efficiency of pro‐receptor processing and on the binding of the mouse monoclonal antibody 83‐7. However, this antibody, which binds hIR but not hIGF‐1R, was still capable of immunoprecipitating the mature chimaeric receptor, indicating that the conformational epitope recognised by this antibody is not primarily determined by the loop region exchanged. The loop exchange did not significantly affect the ability of insulin to displace bound radiolabelled insulin, but increased the capacity of IGF‐1 to competitively displace labelled insulin by at least 10 fold.

Sequence of pilin from Bacteroides nodosus 351 (serogroup H) and implications for serogroup classification

The nucleotide sequence of the pilin gene from Bacteroides nodosus strain 351, currently classified as serogroup H, subgroup 2 (H2) has been determined. The gene encodes a single polypeptide (prepilin) of 160 amino acids and Mr 17,150. However, pilin isolated from B. nodosus 351 migrates as two distinct bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, due to an internal peptide bond cleavage. Amino acid sequence studies of pilin from B. nodosus 351 have established that the cleavage occurs between 72Ala and 73Ser of the mature protein sequence. Comparisons of gene and amino acid sequences of pilin from B. nodosus 351 with the corresponding sequences from strains of serogroups D and H1 indicate that these sequences share a close relationship. However, the level of sequence identity between B. nodosus 351 pilin and pilin from strain 265 of serogroup H1 is lower than anticipated for strains within a serogroup and suggests that B. nodosus 265 and B. nodosus 351 should not be classified within the same serogroup.