title: Engineering and characterization of a single chain surrogate light chain variable domain.
abstract: The surrogate light chain (SLC) is a key regulator of B cell development in the bone marrow, resulting in mature B cells that produce antibodies that are capable of interacting with antigens. The SLC comprises two noncovalently interacting proteins: VpreB and 14.1. We engineered a construct to represent the complete immunoglobulin-like domain of the SLC variable domain in a single protein chain that could be bacterially expressed. In this construct, the incomplete immunoglobulin domain of VpreB (residues 1-102) was linked to the J-segment of 14.1 (residues 40-53), which provided one beta-strand to complete the V-like domain (VpreBJ). Because VpreBJ has the interface to VH chains, but lacks the unique region of 14.1, which is important for SLC signaling, we predict that a properly folded VpreBJ would have the potential to act as a dominant negative mutant of the surrogate light chain. X-ray crystallography of VpreBJ at 2.0 A resolution showed that the engineering was successful. With its two beta-pleated sheets, packed face-to-face, the single chain VpreBJ resembles a mature light chain immunoglobulin V-domain (VL). The surface that would normally interact with the VH chain interacts with a crystallographically related VpreBJ molecule. The presence of dimeric species in solution was verified by analytical ultracentrifugation. VpreBJ is easily overexpressed in bacteria, while retaining the native conformation of an immunoglobulin domain, and thus may serve as an important reagent for future studies in B-cell development.
authors.affiliation: Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: Lucia
authors.initials: L
authors.lastname: Morstadt
authors.forename: Andrew
authors.initials: A
authors.lastname: Bohm
authors.forename: Deniz
authors.initials: D
authors.lastname: Yüksel
authors.forename: Krishna
authors.initials: K
authors.lastname: Kumar
authors.forename: B David
authors.initials: BD
authors.lastname: Stollar
authors.forename: James D
authors.initials: JD
authors.lastname: Baleja
mesh_terms: D000595:Amino Acid Sequence; D022801:Complementarity Determining Regions / Q000737:chemistry; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D054444:Immunoglobulin Light Chains, Surrogate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D022801:Complementarity Determining Regions; D054444:Immunoglobulin Light Chains, Surrogate; D007135:Immunoglobulin Variable Region