title: Solution structure of TT30, a novel complement therapeutic agent, provides insight into its joint binding to complement C3b and C3d.
abstract: A novel therapeutic reagent TT30 was designed to be effective in diseases of the alternative pathway of complement such as paroxysmal nocturnal hemoglobinuria and other diseases. TT30 is constructed from the first four short complement regulator (SCR) domains of complement receptor type 2 (CR2) that bind to complement C3d, followed by the first five SCR domains of complement factor H that bind to complement C3b. In order to assess how TT30 binds to C3d and C3b, we determined the TT30 solution structure by a combination of analytical ultracentrifugation, X-ray scattering and constrained modeling. The sedimentation coefficients and radius of gyration of TT30 were unaffected by citrate or phosphate-buffered saline buffers and indicate an elongated monomeric structure with a sedimentation coefficient of 3.1 S and a radius of gyration R(G) of 6.9 nm. Molecular modeling starting from 3000 randomized TT30 conformations showed that high-quality X-ray curve fits were obtained with extended SCR arrangements, showing that TT30 has a limited degree of inter-SCR flexibility in its solution structure. The best-fit TT30 structural models are readily merged with the crystal structure of C3b to show that the four CR2 domains extend freely into solution when the five complement factor H domains are bound within C3b. We reevaluated the solution structure of the CR2-C3d complex that confirmed its recent crystal structure. This recent CR2-C3d crystal structure showed that TT30 is able to interact readily with C3d ligands in many orientations when TT30 is bound to C3b.
authors.affiliation: Department of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: Keying
authors.initials: K
authors.lastname: Li
authors.forename: Jayesh
authors.initials: J
authors.lastname: Gor
authors.forename: V Michael
authors.initials: VM
authors.lastname: Holers
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Storek
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Perkins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D003179:Complement C3b / Q000737:chemistry* / Q000378:metabolism; D015933:Complement C3d / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D018360:Crystallography, X-Ray; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017464:Receptors, Complement 3d / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D014461:Ultracentrifugation
chemical_list: D008024:Ligands; D017464:Receptors, Complement 3d; D011994:Recombinant Proteins; D012996:Solutions; D003179:Complement C3b; D015933:Complement C3d