Observation and Characterization of the Interaction between a Single Immunoglobulin Binding Domain of Protein L and Two Equivalents of Human κ Light Chains

Abstract

Protein L is a multidomain cell wall bound protein found in ϳ10% of the Peptostreptococcal isolates (1) and contains a series of repeated domains, some of which are able to bind to immunoglobulins without initiating an immune response. Expression of this protein has been correlated to the virulence of these opportunistic pathogenic bacteria (1) that are found in the gastrointestinal and urogenital tracts (2). Its presence has been found to cause cellular responses such as histamine release from basophils and mast cells (3) presumably by crosslinking IgE molecules bound to surface Ig receptors.1 H NMR spectroscopy of a single Ig-binding domain of protein L (isolated from strain 312, PpL 312 ) 1 revealed that PpL 312 is a rigid structure consisting of a ␤-sheet, formed by two pairs of antiparallel ␤-strands, lying on top of a single ␣-helical section (4), with a flexible N terminus (5). Further NMR studies (6) led to the proposal that the binding site of PpL 312 for -chain involves residues from the second ␤-strand and from the loop between the third ␤-strand and the ␣-helix and located the binding site for PpL 312 on the -chain to the second ␤-strand and the two ␤-strands located on the outer surface of the framework region of the V L domain (7).Parallel studies on the binding interaction of a single Igbinding domain of protein L from strain 3316 (PpL 3316 ) found that it forms a high affinity complex with -chains with a K d of 112 nM (8, 9). The x-ray crystallographic structure of this domain has been determined in complex with the human antibody fragment, Fab 2A2, and revealed that two Fab 2A2 fragments can in fact bind to sites on opposite faces of PpL 3316 (10). Site 1 of PpL 3316 is that characterized by Beckingham et al. (9) and equivalent to the site studied on PpL 312 using NMR by Wikström et al. (5). Site 2 of PpL 3316 , involving part of the helix and strands 3 and 4, was identified for the first time by the crystallographic study (10). This site has not yet been characterized although preliminary studies suggested that the affinity of site 2 is lower than that of site 1 (10).By use of a program of site directed mutagenesis we have been able to derive the relative binding affinities of sites 1 and 2 and propose a mechanism by which PpL 3316 binds light chains that is consistent with published data. The hydroxyl group of Tyr 53 is important for the formation of a high affinity complex at site 1. Previous enzyme-linked immunosorbent assay experiments (11) have shown that nitration of Tyr 53 or its mutation to Phe dramatically increases the K d of the -chainPpL 3316 interaction. Another important residue at site 1 is Leu 57 . No binding of the 1 -chain used in these studies can be detected at site 1 of the mutant Y53F/L57H by fluorimetry or isothermal titration calorimetry, and this mutant is thus ideal for measuring the affinity at site 2.Important residues for binding at site 2 are Asp 55 and Ala 66 . The mutation D55A eliminates a salt bridge and dramatically weakens or eliminates binding (12), and co...

Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers

“…In addition, in the B cell receptor of hairy cell leukemia chains has been shown to be preferentially used (23). Yet another well-established functional difference between and antibodies is the interaction of chains with the bacterial surface protein L (20,24). It has also been demonstrated that the isotype of the IGL chain can influence the kinetics of the intracellular assembly of antibodies and the susceptibility of the interchain disulfide bonds to reducing agents (25).…”

Engineering High Affinity Superantigens by Phage Display

“…6,9 Its existence has recently been confirmed by a combination of mutagenesis, stopped-flow fluorescence (SFF) and isothermal titration calorimetry (ITC). 10 Both methods, SFF and ITC reveal the site 2 affinity for huVkI in C* to be approximately 100-fold lower than site 1. However, it is unclear if that differential is maintained for different B and C domains and their binding to different Vk subtypes.…”

“…These studies also hinted at the existence of a second, low affinity Ig binding site on PpL, which has now been confirmed by other methods. 10,11 Recombinant PpL (4-domains) is commercially available (Affitech, Norway) and a highly sensitive reagent for the detection of human Vk chains, superior to for example commonly used antipeptide tag antibodies. 12 However, higher affinity Ig binding domains would be desirable reagents for many biotechnological applications including for increased sensitivity in immunoassays such as enzyme-linked immunosorbant assays (ELISAs) and Western blots or as capture reagents for antibody arrays.…”

Different crystal packing in Fab–protein L semi-disordered peptide complex

“…Only two mutants gave usable data even if crystals were also obtained with other mutants. The single-site mutant L57H that was shown by calorimetry and stopped-flow fluorescence to have reduced site 1 affinity (Housden et al, 2004) gives peptide-complexed crystals at low pH, while PpL mutant H74C, that can bind at both site 1 and site 2, gives the same packing but at high pH with NaCl added. This may be due to the His to Cys mutation, but in order to separate out all the possibilities more trials would be needed.…”

“…1a). The additional L57H mutation weakens PpL site 1 binding (Housden et al, 2004) and thus it may help slow down the crystallization. A reduction in MPEG concentration is also implemented to achieve the same goal.…”