A domain from streptococcal protein G has already been used to obtain crystals of a Fab–peptide complex (Derrick et al., 1999 ). SpG binds to the C H1 domain and hence does not interact with Fv fragment, the smallest immunoglobulin portion that can bind antigen, nor with single-chain antibodies (scFv). However, PpL can be used for the crystallization of Fabs such as SpG and SpA, each providing an interaction with a functionally distinct region on the Fab portion of particular immunoglobulin families or subclasses (Graille et al., 2001 Derrick & Wigley, 1992 Graille et al., 2000 ). While, SpA and SpG domains recognize a common site at the interface between the C H2 and C H3 domains on the Fc of IgG (Fc γ Deisenhofer, 1981 Sauer-Eriksson et al., 1995 ) and can thus be used in the crystallization of Fc-fusion proteins (Stura, Taussig et al., 2002 ) PpL does not. Single domains of these immunoglobulin-binding proteins (IBP), PpL, SpA and SpG, can aid the crystallization of antibodies and their complexes because they provide alternative ways of forming a crystal lattice and thus they modify the chances of crystallizing the Fab and/or its complex (Stura et al., 2001 Stura, Graille et al., 2002 ). They are composed of two to five homologous repeats of immunoglobulin-binding domains that recognize distinct regions on a wide repertoire of immunoglobulins. The best studied are Staphylococcus aureus protein A (SpA Langone, 1982 ), streptococcal protein G (SpG Björck & Kronvall, 1984 ) and Peptostreptococcus magnus protein L (PpL Björck, 1988 ). Several Gram-positive bacterial proteins interact with immunoglobulins. In both mutants two different tryptophan residues participate in crystal-packing interactions, suggesting that this residue may be particularly interesting for enhancing crystal-contact formation. Here, a comparison of two single-site mutants that differ at three different positions is reported. Such mutants will have a free surface that can participate in crystal contacts and that can be modified to improve its crystal contact-forming properties. However, it is possible to design mutants that can bind at only one site by making use of the crystal structures obtained so far. In this context the small PpL domain is sandwiched between two Fab and cannot participate in crystal contacts, thus mutants are unlikely to increase the chances of crystallizing a particular complex. Each wild-type PpL domain has two light-chain binding sites that target the same region of the light chain and can thus bring together two Fab–antigen complexes within the crystal lattice. Thus, a single domain of this protein can be used to aid the crystallization of Fab, free or complexed to their antigen when it is not possible to obtain crystals without it. Protein L from Peptostreptococcus magnus (PpL) is a multidomain protein composed of four or five immunoglobulin-binding domains that target the κ light chain of a large repertoire of human and murine antibodies.
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