Boehm, Mark Karl; (1999) Structural studies of multidomain proteins of the immunoglobulin superfamily. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Carcinoembryonic antigen (CEA) is an important tumour marker. It is heavily glycosylated and contains seven immunoglobulin (Ig) domains. Its solution structure was determined by X-ray and neutron scattering. X-rays showed it has a radius of gyration (RG) of 8.0 nm and a cross-sectional radius of gyration (Rxs) of 2.1 nm. Neutron data showed that CEA is monomeric. Models of CEA were built using domains from homologous structures. The models which best-fitted the experimental data had elongated "zig-zag" structures and inter-domain orientations similar to those in CD2. IgA is the most abundant class of human immunoglobulin. IgA1 contains two Fabs joined by hinges to an Fc, and two C-terminal tailpieces. Solution scattering showed that IgA1 has an RG of 6.11-6.20 nm and IgA1 lacking tailpieces has an RG of 5.84-6.16 nm. It was predicted that the hinges are extended and that the tailpieces are compact. Automated curve-fitting modelling confirmed both of these predictions, and showed that IgA1l is "T-shaped", in which the tailpieces fold back against the Fc. This structure is unlike those observed for IgG proteins. MFE-23 is an anti-CEA single chain Fv antibody that is used for targeting tumours. It contains two domains joined by a flexible linker. The MFE-23 crystal structure was solved by molecular replacement, and the final model had an R-factor of 19.0% at 2.4 Å resolution. Its antigen-binding loops have well-defined structures. In the structure, MFE-23 forms dimers. Neutron scattering showed that MFE-23 exists as monomers below 1 mg/ml and oligomers at higher concentrations. The interaction between MFE-23 and CEA was modelled using lattice contacts in the MFE-23 crystal. In this model, the antigen-binding loops formed contacts with the first two domains of CEA, there was good surface complementarity, appropriate electrostatic contacts, and no steric conflicts with CEA carbohydrate. The models will be most useful for studying the functions of these proteins.

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