Contributions of Amino Acid Side Chains to the Kinetics and Thermodynamics of the Bivalent Binding of Protein L to Ig Light Chain<xref ref-type="fn" rid="bi034873sAF2"></xref>

Protein L is a bacterial surface protein with 4-5 immunoglobulin (Ig)-binding domains (B1B5), each of which appears to have two binding sites for Ig, corresponding to the two edges of its â-sheet. To verify these sites biochemically and to probe their relative contributions to the protein L-Ig κ light chain (κ) interaction, we compared the binding of PLW (the Y47W mutant of the B1 domain) to that of mutants designed to disrupt binding to sites 1 and 2, using gel filtration, BIAcore surface plasmon resonance, fluorescence titration, and solid-phase radioimmunoassays. Gel filtration experiments show that PLW binds κ both in 1:1 complexes and multivalently, consistent with two binding sites. Covalent dimers of the A20C and V51C mutants of PLW were prepared to eliminate site 1 and site 2 binding, respectively; both the A20C and V51C dimers bind κ in 1:1 complexes and multivalently, indicating that neither site 1 nor site 2 is solely responsible for κ binding. The A20R mutant was designed computationally to eliminate site 1 binding while preserving site 2 binding; consistent with this design, the A20R mutant binds κ in 1:1 complexes but not multivalently. To probe the contributions of amino acid side chains to binding, we prepared 75 point mutants spanning nearly every residue of PLW; BIAcore studies of these mutants revealed two binding-energy “hot spots” consistent with sites 1 and 2. These data indicate that PLW binds κ at both sites with similar affinities (high nanomolar), with the strongest contributions to the binding energy from Tyr34 (site 2) and Tyr36 (site 1). Compared to other protein-protein complexes, the binding is insensitive to amino acid substitutions at these sites, consistent with the large number of main chain interactions relative to side chain interactions. The strong binding of protein L to Ig κ light chains of various species may result from the ambidextrous binding of the B1-B5 domains and the unimportance of specific side chain interactions. Protein L is an immunoglobulin (Ig)1-binding protein found on the surface of some strains of the anaerobic bacterium Peptostreptococcus magnus (1). Protein L correlates with the virulence of such bacteria (2, 3) and can induce histamine release from human basophils and mast cells by binding and cross-linking IgE on the surface of these cells (4, 5). Protein L from P. magnus strain 312 has five homologous Ig-binding domains (denoted as B1-B5) of 72-76 residues each (6). Although the initial residues of B1 are disordered (7), its C-terminal 62 residues form a very stable domain (8) (Tm ≈ 70 °C), consisting of a single R-helix roughly parallel to a four-stranded â-sheet with an unusual, ubiquitin-like topology 2-1-4-3 (9, 10) (Figure 1). This structure closely resembles the structure of the corresponding domain in protein G from group C and G streptococci (1113), although there is no detectable sequence homology and the two domains differ in their binding specificity. Whereas the Ig-binding domains in protein G and in protein A from Staphylococcus aureus (14) bind to only Ig heavy chains, the protein L domains bind to only Ig κ light chains (κ) from a variety of mammalian species (1, 15, 16). Specifically, protein L binds to the framework region of the variable domain of the VκI, VκIII, and VκIV subgroups but does not † This work was supported by grants from the Swedish Medical Research Council (project #9926), the Swedish Society for Medical Research, the Royal Physiographic Society in Lund, the Kock, Wiberg, and Österlund Foundations, King Gustav V’s 80-year Foundation, and the Howard Hughes Medical Institute. T.K. was supported by the Human Frontier Science Program and the European Molecular Biology Organisation. * To whom correspondence should be addressed. Present address: Division of Cell Biology and Immunology, University of Dundee, Dundee, DD1 5EH, UK. Telephone: +44-1382-345096. Fax: +441382-345783. ‡ Lund University. § University of Washington. # University of California San Francisco. 1 Abbreviations: PpL, a generic immunoglobulin-binding domain of protein L; PLW, the Y47W mutant of the B1 domain of protein L; IgG and IgE, immunoglobulins G and E; VL and CL, the variable and constant domains of Ig light chain; VH and CH, the variable and constant domains of Ig heavy chain; Vκ, the variable domain of Ig κ light chain; SPR, surface plasmon resonance; SPRIA, solid-phase radioimmunoassay; PBS, phosphate-buffered saline. 2445 Biochemistry 2004, 43, 2445-2457 10.1021/bi034873s CCC: $27.50 © 2004 American Chemical Society Published on Web 02/13/2004 appear to bind to heavy chains, Ig λ light chains, the CL domains of κ, or the variable domain of the VκII subgroup (17). In recent X-ray structures (18-20), a single site on the κ was observed to bind at two separate sites on the B1 domain of protein L, denoted here as site 1 and site 2 (Figure 1a,b, respectively). At site 1, the second â-strand of B1 binds antiparallel to the second â-strand of the Vκ domain (Figure 1c), whereas, at site 2, the third â-strand of B1 binds parallel to the same â-strand of the Vκ domain (Figure 1d). Previous biochemical studies, however, have detected only a single binding site on PpL (6, 21), consistent with the absence of precipitation between the two isolated domains (6) and with NMR studies that detected binding at site 1 but no definitive evidence for binding to site 2 (19, 22). A more recent binding study (23) found that the site 1 mutation Y36F lowers the affinity 50-fold (from roughly 100 nM to 3 μM), suggesting that binding occurs predominantly at site 1 on B1. These observations suggest that the site 2 binding may be an artifact of crystallization. To verify these sites biochemically and to probe their relative contributions to protein L-κ binding in solution, we compared the binding of PLW (the Y47W mutant of the B1 domain) to that of mutants designed to disrupt binding to sites 1 and 2, using gel filtration, BIAcore surface plasmon resonance, fluorescence titration, and solid-phase radioimmunoassays. We find that both sites contribute to binding in solution. Specifically, the A20C and V51C covalent dimers bind multivalently (despite the respective disruption of sites 1 and 2), while the A20R mutant (designed to eliminate site 1 binding) forms only 1:1 complexes with κ in solution. We further obtain a detailed map of side chain contributions in both sites by characterizing the effects of 75 mutations in PLW on both the kinetics and thermodynamics of κ binding. BIAcore studies of these mutants revealed two bindingenergy “hot spots” consistent with sites 1 and 2. MATERIALS AND METHODS Materials. Most of the 75 PLW variants have been described previously (24); the additional site-specific mutations were made with the QuikChange site-directed mutagenesis kit (Stratagene) using the protocol suggested by the manufacturer. A pET15b-derived construct expressing PLW (25) was used as a template. All mutations were verified by DNA sequencing using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems). Expression and purification of PLW and its mutants were carried out as described previously (8). Human polyclonal IgG was purchased from Pierce and used without further purification. The human Ig κ light chain 4479 was a generous gift from Dr. Lars Björck of Lund University in Sweden. Gel Filtration Experiments. Varying concentrations of PLW (or a mutant thereof) and Ig κ light chain were run on two different Pharmacia Biotech Superdex 75 HR 10/30 analytical gel filtration columns. The molecular weight standards used on column 1 were aprotinin, ribonuclease A, chymotrypsinogen, ovalbumin, and albumin, whereas the molecular weight standards used on column 2 were aprotinin, FIGURE 1: Ribbon diagrams of the two binding sites on the B1 domain of protein L alone and in complex with Ig κ light chain, taken from a high-resolution X-ray crystal structures (PDB accession code 1hz5 for a and b and 1hez for c and d). (a) Site 1; the positions of Gln-18, Ala-20, Phe-22, and Tyr-36 are indicated. (b) Site 2; the positions of Phe-26, Glu-27, Thr-30, Tyr-34, Asp-38, Trp-47, Val-49, and Val-51 are indicated. (c) Site 1 on protein L binds to the second â-strand of the variable domain of Ig κ light chain with antiparallel â-strands. (d) Site 2 on protein L binds to the same site on Ig κ light chain, but with parallel â-strands. The figures were created using the program MOLMOL (45). 2446 Biochemistry, Vol. 43, No. 9, 2004 Svensson et al.