title: Extended flexible linker structures in the complement chimaeric conjugate CR2-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy.
abstract: Complement receptor 2 (CR2; CD21) is a membrane-bound regulator of complement activation, being comprised of 15 or 16 short complement repeat (SCR) domains. A recombinant glycosylated human CR2 SCR 1-2 domain pair was engineered with the Fc fragment of a mouse IgG1 antibody to create a chimaera CR2-Ig containing the major ligand binding domains. Such a chimaera has therapeutic potential as a complement inhibitor or immune modulator. X-ray and neutron scattering and analytical ultracentrifugation identified its domain structure in solution, and provided a comparison with controversial folded-back crystal structures for deglycosylated CR2 SCR 1-2. The radius of gyration R(G) of CR2-Ig was determined to be 5.39(+/-0.14) nm and 5.29(+/-0.01) nm by X-ray and neutron scattering, respectively. The maximum dimension of CR2-Ig was determined to be 17 nm. The molecular mass of CR2-Ig ranged between 101,000 Da and 107,000 Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600 Da. Sedimentation velocity gave a sedimentation coefficient of 4.49(+/-0.11) S. Stereochemically complete models for CR2-Ig were constructed from crystal structures for the CR2 SCR 1-2 and mouse IgG1 Fc fragments. The two SCR domains and the Fc fragment were joined by randomised conformational peptides. The analysis of 35,000 possible CR2-Ig models showed that only those models in which the two SCR domains were arranged in an open V-shape in random orientations about the Fc fragment accounted for the scattering and sedimentation data. It was not possible to define one single conformational family of Fab-like fragment relative to the Fc fragment. This flexibility is attributed to the relatively long linker sequence and the absence of the antibody light chain from CR2-Ig. The modelling also confirmed that the structure of CR2 SCR 1-2 is more extended in solution than in its crystal structure.
authors.affiliation: Department of Biochemistry and Molecular Biology, Darwin Building, University College London, UK.
authors.forename: Hannah E
authors.initials: HE
authors.lastname: Gilbert
authors.forename: Mohammed
authors.initials: M
authors.lastname: Aslam
authors.forename: Joel M
authors.initials: JM
authors.lastname: Guthridge
authors.forename: V Michael
authors.initials: VM
authors.lastname: Holers
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Perkins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017464:Receptors, Complement 3d / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D012542:Scattering, Radiation; D016415:Sequence Alignment; D014461:Ultracentrifugation
chemical_list: D007074:Immunoglobulin G; D017464:Receptors, Complement 3d; D011993:Recombinant Fusion Proteins