The antibody zalutumumab inhibits epidermal growth factor receptor signaling by limiting intra- and intermolecular flexibility.

The epidermal growth factor receptor (EGFR) activates cellular pathways controlling cell proliferation, differentiation, migration, and survival. It thus represents a valid therapeutic target for treating solid cancers. Here, we used an electron microscopy-based technique (Protein Tomography) to study the structural rearrangement accompanying activation and inhibition of native, individual, EGFR molecules. Reconstructed tomograms (3D density maps) showed a level of detail that allowed individual domains to be discerned. Monomeric, resting EGFR ectodomains demonstrated large flexibility, and a number of distinct conformations were observed. In contrast, ligand-activated EGFR complexes were detected only as receptor dimers with ring-like conformations. Zalutumumab, a therapeutic inhibitory EGFR antibody directed against domain III, locked EGFR molecules into a very compact, inactive conformation. Biochemical analyses showed bivalent binding of zalutumumab to provide potent inhibition of EGFR signaling. The structure of EGFR-zalutumumab complexes on the cell surface visualized by Protein Tomography indicates that the cross-linking spatially separates the EGFR molecules' intracellular kinase domains to an extent that appears incompatible with the induction of signaling. These insights into the mechanisms of action of receptor inhibition may also apply to other cell-surface tyrosine kinase receptors of the ErbB family.

Visualization of SV2A conformations in situ by the use of Protein Tomography.

The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography. We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicates that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins.

ESR and theoretical studies of trimer radical cations of coronene.

Highly resolved ESR spectra of monomer, dimer and trimer radical cations of coronene (C24H12) were observed at room temperature for a solution of 1,1,1,3,3,3-hexafluoro-2-propan-2-ol (HFP) containing thallium(III) trifluoroacetate as oxidant. The spectra consisting of multiple lines with isotropic 1H-hyperfine splitting (hfs) constants of 0.0766 mT (24H) and 0.013 mT (6H) were attributable to a mixture of the dimer with the trimer radical cations, (C24H12)2+ and (C24H12)3+. For (C24H12)2+, the 1H-hfs constant agreed well with the reported value, 0.077 mT. However, for (C24H12)3+, the values were significantly different from the reported ones, 0.117 mT (12H) and 0.020 mT (24H), by Ohya Nishiguchi et al. [H. Ohya-Nishiguchi, H. Ide, N. Hirota, Chem. Phys. Lett. 66 (1979) 581], but rather similar to those reported by Willigen et al. [H. van Willigen, E. De Boer, J.T. Cooper, W.F. Forbes, J. Chem . Phys. 49 (1968) 1190]. In conflict with Willigen's report, however, no ESR line broadening which has been ascribed to a low stationary concentration of (C24H12)3+ was detected. Based on ab initio MO calculations for benzene as a compact model of C24H12, the structure of (C24H12)3+ was investigated in terms of the observed 1H-hfs constants. A staggered sandwich C(2v) structure was suggested being at the "global" minimum for the benzene trimer cation. In the structure, the unpaired electron spin is predominantly localized to the central ring, which is qualitatively in agreement with the previous ESR results of (C24H12)3+ by Ohya-Nishiguchi et al. In addition, as a "local" minimum, the benzene trimer was indicated to have a slipped sandwich Cs structure, which is less stable by ca. 19 kJ mol(-1) than the "global" minimum. In this structure, the unpaired electron spin was nearly equally distributed on both the central and one of the two side C24H12 molecules. The observed 1H-hfs constants were possibly attributable to the (C24H12)3+ cation with the analogous slipped sandwich Cs structure.