Faculty Bibliography

We report the generation of a chimeric monoclonal antibody (ch806) with specificity for an epitope on the epidermal growth factor receptor (EGFR) that is different from that targeted by all other anti-EGFR therapies. Ch806 antibody is reactive to both de2-7 and overexpressed wild-type (wt) EGFR but not native EGFR expressed in normal tissues at physiological levels. Ch806 was stably expressed in CHO (DHFR -/-) cells and purified for subsequent characterisation and validated for use in preliminary immunotherapy investigations. Ch806 retained the antigen binding specificity and affinity of the murine parental antibody. Furthermore, ch806 displayed enhanced antibody-dependent cellular cytotoxicity against target cells expressing the 806 antigen in the presence of human effector cells. Ch806 was successfully radiolabelled with both iodine-125 and indium-111 without loss of antigen binding affinity or specificity. The radioimmunoconjugates were stable in the presence of human serum at 37 degrees C for up to 9 days and displayed a terminal half-life (T(1/2beta)) of approximately 78 h in nude mice. Biodistribution studies undertaken in BALB/c nude mice bearing de2-7 EGFR-expressing or amplified EGFR-expressing xenografts revealed that (125)I-labelled ch806 failed to display any significant tumour retention. However, specific and prolonged tumour localisation of (111)In-labelled ch806 was demonstrated with uptake of 31%ID g(-1) and a tumour to blood ratio of 5 : 1 observed at 7 days postinjection. In vivo therapy studies with ch806 demonstrated significant antitumour effects on established de2-7 EGFR xenografts in BALB/c nude mice compared to control, and both murine 806 and the anti-EGFR 528 antibodies. These results support a potential therapeutic role of ch806 in the treatment of suitable EGFR-expressing tumours, and warrants further investigation of the potential of ch806 as a therapeutic agent

The major facilitator superfamily represents the largest group of secondary active membrane transporters in prokaryotic and eukaryotic cells. They transport a vast variety of substrates, presumably via similar mechanisms, yet the details of these mechanisms remain unclear. Here we report the 3.3 A resolution structure of a member of this superfamily--GlpT, the glycerol-3-phosphate transporter from the E. coli inner membrane, in the absence of a substrate. The antiporter mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Its N- and C-terminal domains exhibit a pseudo 2-fold symmetry along an axis perpendicular to the membrane. Eight of the twelve transmembrane alpha-helices are arranged around a centrally located substrate translocation pore that is closed off at the periplasmic surface. Present at the beginning of the pore are two arginine residues that presumably comprise the substrate-binding site which is accessible only from the cytosol, suggesting an inward-facing conformation for the transporter. The central loop connecting the N- and C-terminal domains is partially disordered and exhibits reduced susceptibility to trypsin in the presence of substrate, indicating conformational changes. We propose that GlpT operates via a single binding-site, alternating-access mechanism

Glycerol-3-phosphate (G3P) plays a major role in glycolysis and phospholipid biosynthesis in the cell. Escherichia coli uses a secondary membrane transporter protein, GlpT, to uptake G3P into the cytoplasm. The crystal structure of the protein was recently determined to 3.3 A resolution. The protein consists of an N- and a C-terminal domain, each formed by a compact bundle of six transmembrane alpha-helices. The substrate-translocation pore is found at the domain interface and faces the cytoplasm. At the closed end of the pore is the substrate binding site, which is formed by two arginine residues. In combination with biochemical data, the crystal structure suggests a single binding site, alternating access mechanism for substrate translocation, namely, the substrate bound at the N- and C-terminal domain interface is transported across the membrane via a rocker-switch type of movement of the domains. Furthermore, GlpT may serve as a structural and mechanistic paradigm for other secondary active membrane transporters

The major facilitator superfamily represents the largest group of secondary active membrane transporters in the cell. The 3.3A resolution structure of a member of this protein superfamily, the glycerol-3-phosphate transporter from the Escherichia coli inner membrane, reveals two domains connected by a long central loop. These N- and C-terminal domains, each containing a six-helix bundle, are related by pseudo-twofold symmetry. A substrate translocation pore is located between the two domains and is open to the cytoplasm. Two arginines at the closed end of the pore comprise the substrate-binding site. Biochemical experiments show that, upon substrate binding, the protein adopts a more compact conformation. The crystal structure suggests that the transporter operates through a single binding site, alternating access mechanism via a rocker-switch type of movement of the N- and C-terminal domains. The structure and mechanism of the glycerol-3-phosphate transporter form a paradigm for other members of the major facilitator superfamily

Glycogen storage disease type Ib is caused by mutations in the glucose 6-phosphate transporter (G6PT) in the endoplasmic reticulum membrane in liver and kidney. Twenty-eight missense and two deletion mutations that cause the disease were previously shown to reduce or abolish the transporter's activity. However, the mechanisms by which these mutations impair transport remain unknown. On the basis of the recently determined crystal structure of its Escherichia coli homologue, the glycerol 3-phosphate transporter, we built a three-dimensional structural model of human G6PT by homology modeling. G6PT is proposed to consist of 12 transmembrane alpha-helices that are divided into N- and C-terminal domains, with the substrate-translocation pore located between the two domains and the substrate-binding site formed by R28 and K240 at the domain interface. The disease-causing mutations were found to occur at four types of positions: (I) in the substrate-translocation pore, (II) at the N-/C-terminal domain interface, (III) in the interior of the N- and C-terminal domains, and (IV) on the protein surface. Whereas class I mutations affect substrate binding directly, class II mutations, mostly involving changes in side chain size, charge, or both, hinder the conformational change required for substrate translocation. On the other hand, class III and class IV mutations, often introducing a charged residue into a helix bundle or at the protein-lipid interface, probably destabilize the protein. These results also suggest that G6PT operates by a similar antiport mechanism as its E. coli homologue, namely, the substrate binds at the N- and C-terminal domain interface and is then transported across the membrane via a rocker-switch type of movement of the two domains

Voltage-dependent ion channels serve as field-effect transistors by opening a gate in response to membrane voltage changes. The gate's response to voltage is mediated by voltage sensors, which are arginine-containing structures that must move with respect to the membrane electric field. We have analysed by electron microscopy a voltage-dependent K(+) channel from Aeropyrum pernix (KvAP). Fab fragments were attached to 'voltage sensor paddles' and identified in the electron microscopy map at 10.5 A resolution. The extracellular surface location of the Fab fragments in the map is consistent with the membrane-depolarized, open conformation of the channel in electrophysiological experiments. Comparison of the map with a crystal structure demonstrates that the voltage sensor paddles are 'up' (that is, near the channel's extracellular surface) and situated at the protein-lipid interface. This finding supports the hypothesis that in response to changes in voltage the sensors move at the protein-lipid interface rather than in a gating pore surrounded by protein

The major facilitator superfamily represents the largest group of secondary membrane transporters in the cell. Here we report the 3.3 angstrom resolution structure of a member of this superfamily, GlpT, which transports glycerol-3-phosphate into the cytoplasm and inorganic phosphate into the periplasm. The amino- and carboxyl-terminal halves of the protein exhibit a pseudo two-fold symmetry. Closed off to the periplasm, a centrally located substrate-translocation pore contains two arginines at its closed end, which comprise the substrate-binding site. Upon substrate binding, the protein adopts a more compact conformation. We propose that GlpT operates by a single-binding site, alternating-access mechanism through a rocker-switch type of movement