Lynne J. Lawrence

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors.

Apolipoprotein C-II Amyloid Fibrils Assemble via a Reversible Pathway that Includes Fibril Breaking and Rejoining

Alzheimers and several other diseases are characterized by the misfolding and assembly of protein subunits into amyloid fibrils. Current models propose that amyloid fibril formation proceeds via the self-association of several monomers to form a nucleus, which then elongates by the addition of monomer to form mature fibrils. We have examined the concentration-dependent kinetics of apolipoprotein C-II amyloid fibril formation and correlated this with the final size distribution of the fibrils determined by sedimentation velocity experiments. In contrast to predictions of the nucleation-elongation model, the final size distribution of the fibrils was found to be relatively independent of the starting monomer concentration. To explain these results, we extended the nucleation-elongation model to include fibril breaking and rejoining as integral parts of the amyloid fibril assembly mechanism. The system was examined under conditions that affected the stability of the mature fibrils including the effect of dilution on the free pool of monomeric apolipoprotein C-II and the time-dependent recovery of fibril size following sonication. Antibody-labelling transmission electron microscopy studies provided direct evidence for spontaneous fibril breaking and rejoining. These studies establish the importance of breaking and rejoining in amyloid fibril formation and identify prospective new therapeutic targets in the assembly pathway.

Orientation of antigen binding sites in dimeric and trimeric single chain Fv antibody fragments

Electron microscopy of dimeric and trimeric single chain antibody Fv fragments (scFvs) complexed with anti‐idiotype Fab fragments was used to reveal the orientation of antigen binding sites. This is the first structural analysis that discloses the multivalent binding orientation of scFv trimers (triabodies). Three different scFv molecules were used for the imaging analysis; NC10 scFv‐5 and scFv‐0, with five‐ and zero‐residue linkers respectively between the VH and VL domains, were complexed with 3‐2G12 anti‐idiotype Fab fragments and 11‐1G10 scFv‐0 was complexed with NC41 anti‐idiotype Fab fragments. The scFv‐5 molecules formed bivalent dimers (diabodies) and the zero‐linker scFv‐0 molecules formed trivalent trimers (triabodies). The images of the NC10 diabody‐Fab complex appear as boomerangs, not as a linear molecule, with a variable angle between the two Fab arms and the triabody‐Fab complexes appear as tripods.

Sub‐micellar phospholipid accelerates amyloid formation by apolipoprotein C‐II

Lipid‐free human apolipoprotein C‐II (apoC‐II) forms amyloid fibrils with characteristic β‐structure. This conformation is distinct from the α‐helical fold of lipid‐bound apoC‐II. We have investigated the effect of the short‐chain phospholipid, dihexanoylphosphatidylcholine (DHPC) on amyloid formation by apoC‐II. The α‐helical content of apoC‐II increases in the presence of micellar DHPC (16 mM) and amyloid formation is inhibited. However, at sub‐micellar DHPC concentrations (below 8 mM) amyloid formation is accelerated 6 fold. These results suggest that individual phospholipid molecules in vivo may exert significant effects on amyloid folding pathways.

Sedimentation Velocity Analysis of Flexible Macromolecules: Self-Association and Tangling of Amyloid Fibrils

A novel bead modeling technique has been developed for the analysis of the sedimentation velocity behavior of flexible fibrils. The method involves the generation of a family of bead models representing a sample of the conformations available to the molecule and the calculation of the sedimentation coefficients of these models by established techniques. This approach has been used to investigate the size distribution of amyloid fibrils formed by human apolipoprotein C-II (apoC-II). ApoC-II fibrils have a simple and homogeneous ribbon morphology with no evidence of amorphous aggregation. Freshly prepared apoC-II forms fibrils with systematically larger sedimentation coefficients upon increasing protein concentration (modes of 100, 300, and 800 for apoC-II concentrations of 0.3, 0.7, and 1.0 mg/mL, respectively). The sedimentation coefficient distributions are not affected by rotor speed, and are not significantly changed by dilution once the fibrils are formed. The kinetics of aggregation (1 mg/mL apoC-II) as assessed using thioflavin T and preparative pelleting assays reveal that monomeric apoC-II is depleted after approximately 12 h incubation at room temperature. In contrast, the sedimentation coefficient distribution of fibrils continues to grow larger over a period of 48 h to an average value of 800 S. Calculations using the bead modeling procedure suggest maximum sedimentation coefficients for individual apoC-II fibrils to be around 100 S. The larger experimentally observed sedimentation coefficients for apoC-II fibrils indicate an extensive and time-dependent tangling or association of the fibrils to form specific networks.