Human Immunoglobulin Complexed with Protein L from Finegoldia magna
Created by Marisa Konishi
Protein L (PpL) is found on the surface of Finegoldia magna cells and binds with the kappa light chains of different mammal immunoglobulin proteins to form an antibody-antigen complex (PDB ID= 1HEZ). These complexes have been found to correspond to the bacteria’s virulence (1). An antibody, or immunoglobulin, is a Y-shaped protein produced by B cells. These proteins are used by the immune system to bind to foreign particles, antigens. Different antibodies are specific in interacting with certain antigens. Binding often leads to identification and a response by the body to neutralize foreign particles (2). In clinical studies, the presence of PpL on bacterial surfaces has been observed as a factor of bacterial vaginosis. Additionally, PpL interaction with mammal immunoglobulins causes basophils and mast cells to release histamine (1). This action is an immune response that helps white blood cells and antibodies enter sites of infection (2). The molecular weight of the antibody-antigen complex consisting of a human antibody and PpL is 101911.72 Da and the isoelectric point (pI) is 8.19 (3).
PpL bound to a human immunoglobulin is an example of how different pairs of antibodies and antigens interact with each other through unique intermolecular bonding. The structure of the immunoglobulin bound to PpL consists of two identical kappa light chains and two identical heavy chains (2). The light chains are comprised of a variable domain (VL) and constant (CL). Each light chain is 214 residues long with the VL consisting of residues 1-107 and CL consisting of residues 108-214. Likewise, the heavy chains also consist of a variable (VH) and constant (CH) domain. Each heavy chain is 224 residues with VH taking up residues 1-121 and CH extending from 122-224 (4). The VL and VH differ between antibody molecules while the CL and CH are the same for all antibodies of the same class. The diversity in the VL and VH are found mainly in three loop regions within each domain called hypervariable regions. Parts of the VL and VH between the hypervariable regions are called framework regions. Variances in these areas are used to differentiate the heavy and light chains of immunoglobulins into classes and subclasses (5).
Each immunoglobulin domain has a similar structure mainly composed of two antiparallel β-sheets tightly packed to form a compressed β-barrel (6). The kappa light chains are composed of 5% helices and 51% of β-sheets. The heavy chains are composed of 4% helices and 47% β-sheets (5). An antibody consists of two functional regions: fragment antigen-binding (Fab) and fragment crystallizable (Fc). The basic antibody has two Fab regions. Fab is the combining site consisting of one VL and one VH. Fc is the most easily crystallized part of the antibody. This region is made up of CH of both heavy chains and is responsible for mediating the effector functions of antibodies. Effector functions are responses by the body to antigen-antibody complexes. Effector functions include actions such as the binding of antibodies to receptors on different types of cells to activate or lyse them (4).
PpL consists of five homologous sequences. The sequence is 61 residues, consisting of two binding sites that interact with VL. The binding domain of PpL comprises subunit E in the antibody-antigen complex. PpL is sought for its ability to bind to the variable regions of many different types of antibodies, which makes it valuable for recognition and purification of recombinant single chain variable fragments (scFv). An scFv is a type of engineered antibody that is being increasingly used in maximizing the specificity of phage display. Each binding site of PpL forms an interface with the VL. There is no conformational change seen with PpL or the immunoglobulin upon binding. The binding of PpL to VL is the first known interaction of bacterial virulence that is not at the conventional combining site. One PpL domain is able to bind to two Fabs simultaneously, interacting with both VL regions of the antibody. Additionally, ligand components of the immunoglobulin-PpL complex include two imidazole rings (1). Imidazole was used to induce crystallization, binding to two 3-residue long sites on the VL (Lys-45, Leu-46, and Leu-47; Arg-96, Thr-97, and Phe-98) and two sites on the VH (Leu-45, Glu-46, and Trp-47; Cys-96, Ala-97, and Lys-98) (5).
The first VL-PpL interface involves interactions with 13 residues of VL. Ten are located in the framework regions. The other three include Lys-107 from region connecting the VL and CL, Glu-143 from the CL, and Arg-24 from the hypervariable region. 12 amino acids of PpL are involved, mainly located on the β2 strand and α helix. Six hydrogen bonds are formed at the first VL-PpL interface: Ser-9 of VL and Glu-38 of PpL, Ser-9 of VL and Lys-40 of PpL, Ser-12 of VL and Thr-36 of PpL, and Thr-20 of VL and Tyr-53 of PpL. Ser-10 of VL and Glu-38 of PpL are involved in two hydrogen bonds. Mutation of Tyr-53 of PpL to Phe showed a 23% drop in the affinity between the immunoglobulin and PpL (1).
The second VL-PpL interface consists of interactions with 15 residues of VL. Ten of these residues overlap with the first interface. However, none of the residues that significantly impact the binding affinity are involved. 14 amino acids from PpL are involved in the interface and come mainly from the β3 strand and α helix. The second interface is characterized by six hydrogen bonds and two salt bridges. Interactions involved are between Ser-7 of VL and Asp-55 of PpL, Ser-10 of VL and Thr-65 of PpL, Ser-12 of VL and Ala-66 of PpL, Ser-12 of VL and Leu-68 of PpL, Arg-18 of VL and Gly 71 of PpL, Arg-24 of VL and Asp-55 of PpL, Lys-107 of VL and Asp-67 of PpL, and Lys-107 of VL and Leu-68 of PpL. Mutation of Asp-55 to a Ala was made to disrupt the salt bridge with Arg-24 of VL and hydrogen bond with the Ser-7 side chain of VL. Although disruption of one hydrogen bond in the first interface significantly altered the dissociation constant of the antibody-antigen complex with PpL, the disruption of the salt bridge and the hydrogen bond of the second interface did not alter the dissociation constant. The result with the mutant indicates that the second binding site is novel and that the binding of PpL to the immunoglobulin is mostly controlled by the first VL-PpL interface (1).
A matrix metalloproteinase is a proteolytic enzyme that uses zinc to catalyze cleaving of proteins (7). Although functions of matrix metalloproteinase and the antigen-antibody complex with PpL are different, both have binding sites that target other proteins. The matrix metalloproteinase is composed of three copies of two types of subunits. One subunit type (subunits A, D, E) consists of 6% helices and 50% β-sheets. The second subunit type (subunits B, C, F) consists of 8% helices and 50% β-sheets. The primary and tertiary structure of the kappa light chain of human immunoglobulin most closely resembles the protein component comprising subunits A, D, and E (5). The conservation suggests that these two subunits are crucial to both proteins’ binding abilities. Use of DALI shows a Z score of 25.6 and protein BLAST shows an E value of 1e-116 with a sequence similarity of 79%. DALI compares the overall physical structures of proteins for similarities by measuring intramolecular distances of the folded protein. A score of 2 or greater indicates similar folding (8). Protein BLAST examines similarities in the primary structure of proteins by lining sequences up to each other. Lower E values indicate higher similarity in the primary structure of the two proteins (9).