Mycoplasma Arthritidis-derived Mitogen Research Articles

Crystal Structure of the Mycoplasma arthritidis-Derived Mitogen in Apo Form Reveals a 3D Domain-Swapped Dimer

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular Vβ elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-Å resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the “reconstituted” MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAMwt molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.

Structure of Crohn'S Disease-Related Proteins and their Binding to Class II MHC

T cell response to enteric bacteria is important in inflammatory bowel disease. pfiT is a T-cell superantigen associated with human Crohn's disease. The bacterial superantigens are a class of protein toxins that share the capacity to induce massive activation of the human immune system. These molecules simultaneously bind to major histocompatibility complex class II molecules on the surface of antigen-presenting cells and T-cell receptors (TCRs) on T cells to stimulate large numbers of T cells. The aim of this study is to analyze the molecular mechanism of superantigen recognition by host receptors. Here, we report the crystal structure of pfiT. This protein was overexpressed in Escherichia coli and purified though GST-affinity and size exclusion chromatography. The protein is selenomethionine labeled and single wavelength anomalous dispersion method was used for determination of the crystal structure. The superantigen crystallizes in the monoclinic space group P21, with two molecules in asymmetric unit cell. The structure was determined to 2.5A resolution. In addition, we performed radiolabeled competitive binding assays between three superantigens: pfiT, Mycoplasma arthritidis-derived mitogen (MAM), PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome. Analyses showed that both the microbial homologuepfit and PA2885, just as potent superantigen MAM, are capable of binding to target mammalian cells. Moreover, we labeled these superantigens with FITC and analyzed them by FACS in PBMC. The statistic results show that antibody against HLA-DR has strong effect to block these SAg' binding ability with PBMC, and antibodies against HLA-DQ and DP can also compete binding site in a much weaker manner. These findings support the concept that pfiT, PA2885, MAM are superantigens and can bind to class II MHC molecule .Research is funded by the Crohn’ & Colitis Foundation of America.

Zinc Induces Dimerization of the Class II Major Histocompatibility Complex Molecule That Leads to Cooperative Binding to a Superantigen

Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vbeta elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306-318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn(2+) is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn(2+). However, in the presence of Zn(2+), a dimerized MAM/HLA-DR1/HA complex can arise through the Zn(2+)-induced DR1 dimer. In the presence of Zn(2+), cooperative binding of MAM to the DR1 dimer was also observed.

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen (SAg) that can activate large fractions of T cells bearing particular TCR Vβ elements. Here we report the mutagenesis, biochemical and biophysical studies on the dimerization of MAM in solution. Our studies showed that although MAM mainly exists as a monomer in solution, a small percentage of MAM molecules form homodimer at high protein concentration, regardless of the presence of Zn 2+. A distinct peak corresponding to a MAM homodimer was detected in the presence of EDTA, using both chemical cross-linking and analytical ultracentrifugation methods. Further mutagenesis studies revealed that single mutation of residues at the interface of the crystallographic dimer of MAM does not significantly affect the dimerization of MAM in solution. Circular dichroism (CD) analysis indicated that addition of Zn 2+ does not induce conformational changes of MAM from its apo-state. Thermal denaturation experiments indicated that addition of Zn 2+ to MAM solution resulted in a decrease of melting point ( T m), whereas addition of EDTA did not affect the T m of MAM. These results imply that there is no defined Zn 2+-binding site on MAM.

A single point mutation changes the crystallization behavior of Mycoplasma arthritidis-derived mitogen.

Mycoplasma arthritidis-derived mitogen (MAM) functions as a conventional superantigen (SAg). Although recombinant MAM has been crystallized by the hanging-drop vapour-diffusion method, the crystals diffracted poorly to only 5.0 A resolution, with large unit-cell parameters a = 163.8, b = 93.0, c = 210.9 A, beta = 93.7 degrees in the monoclinic space group P2(1). Unit-cell content analysis revealed that as many as 24 molecules could be present in the asymmetric unit. Systematic alanine mutagenesis was applied in order to search for mutants that give crystals of better quality. Two mutants, L50A and K201A, were crystallized under the same conditions as wild-type MAM (MAMwt). Crystals of the L50A mutant are isomorphous with those of MAMwt, while a new crystal form was obtained for the K201 mutant, belonging to the cubic space group P4(1)32 with unit-cell parameters a = b = c = 181.9 A. Diffraction data were collected to 3.6 and 2.8 A resolution from crystals of the MAM L50A and K201A mutants, respectively. Molecular-replacement calculations suggest the presence of two molecules in the asymmetric unit for the MAM K201A mutant crystal, resulting in a VM of 5.0 A Da(-1) and a solvent content of 75%. An interpretable electron-density map for the MAM K201A mutant crystal was produced using the molecular-replacement method.

Crystal Structure of Mycoplasma arthritidis Mitogen Complexed with HLA-DR1 Reveals a Novel Superantigen Fold and a Dimerized Superantigen-MHC Complex

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular TCR Vbeta elements. Here we report the crystal structure of MAM complexed with a major histocompatibility complex (MHC) antigen, HLA-DR1, loaded with haemagglutinin peptide 306-318 (HA). The structure reveals that MAM has a novel fold composed of two alpha-helical domains. This fold is entirely different from that of the pyrogenic superantigens, consisting of a beta-grasped motif and a beta barrel. In the complex, the N-terminal domain of MAM binds orthogonally to the MHC alpha1 domain and the bound HA peptide, and to a lesser extent to the MHC beta1 domain. Two MAM molecules form an asymmetric dimer and cross-link two MHC antigens to form a plausible, dimerized MAM-MHC complex. These data provide the first crystallographic evidence that superantigens can dimerize MHC molecules. Based on our structure, a model of the TCR2MAM2MHC2 complex is proposed.

Crystallization and preliminary crystallographic analysis of Mycoplasma arthritidis-derived mitogen complexed with peptide/MHC class II antigen.

Mycoplasma arthritidis-derived mitogen (MAM), a bacterial superantigen, has been crystallized in complex with its human receptor, major histocompatibility complex (MHC) class II antigen, by the hanging-drop vapor-diffusion method. Crystals were obtained under three conditions, with ammonium sulfate, phosphate salt and PEG 8000 as the precipitant. The crystals grown under these conditions all belong to space group I222, with the same unit-cell parameters: a = 137.4, b = 178.2, c = 179.6 A. Diffraction data were collected to 3.3 and 3.4 A resolution from crystals of native and selenomethionylated MAM-MHC complexes, respectively. Self- and cross-rotation function calculations suggest the presence of two complex molecules in the asymmetric unit, resulting in a V(M) of 4.0 and a solvent content of 69%. An interpretable electron-density map was produced using a combination of molecular replacement and SAD phasing.

Zinc-binding Sites in the N Terminus of Mycoplasma arthritidis-derived Mitogen Permit the Dimer Formation Required for High Affinity Binding to HLA-DR and for T Cell Activation

Zinc-dependent superantigens can be divided into two subfamilies based on how they use zinc ions for interactions with major histocompatibility complex (MHC) class II molecules. Members of the first subfamily use zinc ions for interactions with histidine 81 on the beta-chain of MHC class II molecules, whereas members of the second subfamily use zinc ions for dimer formation. The zinc-binding motif is located in the C terminus of the molecule in both subfamilies. While our recent studies with Mycoplasma arthritidis-derived mitogen (MAM) have provided the first direct evidence demonstrating the binding to MHC class II molecules in a zinc-dependent manner, it still not known how zinc coordinates the interaction. Data presented here show that the zinc ion is mainly required to induce MAM/MAM dimer formation. Residues in the N terminus of MAM are involved in dimer formation and MHC class II binding, while histidine 14 and aspartic acid 31 of the MAM sequence are the major residues mediating MAM/MAM dimerization. Zinc-induced dimer formation is necessary for MAM binding, MHC class II-induced cell-cell adhesion, and efficient T cell activation. Together these results depict the unique mode of interaction of MAM in comparison with other superantigens.

Involvement of zinc in the binding of Mycoplasma arthritidis-derived mitogen to the proximity of the HLA-DR binding groove regardless of histidine 81 of the beta chain.

Although our recent studies have provided the first evidence demonstrating the direct binding of Mycoplasma arthritidis-derived mitogen (MAM) to MHC class II molecules, it is not yet established how MAM interacts with these molecules. Herein, we demonstrate that MAM binds preferentially and with high affinity to HLA-DR molecules in a zinc-dependent manner. MAM's affinity (25 nM) for HLA-DR molecules is comparable to that of staphylococcal superantigens, and is slightly higher than that for murine MHC class II molecules expressed on the A20 B cell line (111 nM). The amino acid residues located between 14 - 31 and 76 - 90 of the MAM N-terminus play a critical role in MAM / HLA-DR interactions. Histidine at position 81 of the HLA-DR beta-chain, known to be critical for binding of zinc-coordinated superantigens, is not necessary for MAM / HLA-DR interactions. The HLA-DR residues involved in MAM binding are located in the proximal binding groove of the HLA-DR molecule, where the nature of the peptide of the binding groove plays an important role in MAM / HLA-DR interaction. This is the first detailed characterization of MAM's interactions with MHC class II molecules showing a mode of interaction with HLA-DR distinct from that of other superantigens.

Binding of Mycoplasma arthritidis-derived mitogen to human MHC class II molecules via its N terminus is modulated by invariant chain expression and its C terminus is required for T cell activation.

Mycoplasma arthritidis-derived mitogen (MAM) is considered to be a member of the super-antigen family despite the fact that there is no evidence until now indicating its binding to MHC class II molecules. To demonstrate its direct binding and to determine the regions involved in MHC class II and TCR interactions, we generated a recombinant wild-type and two truncated forms of the MAM protein. Data obtained in the course of the present investigation show that MAM binds specifically and significantly to human MHC class II molecules. Evidence is also provided that MAM bears two distinct binding regions: one is located within its N terminus and interacts with MHC class II molecules, while the second region which is located in its C terminus mediates its recognition by the TCR. Association of the MHC class II-associated invariant chain peptide with the peptide binding groove on the cell surface completely abolished MAM binding and presentation. This inhibitory effect is restored by the expression of HLA-DM molecules, suggesting that the nature of the peptide within the binding groove and/or the stability of the MHC class II molecules on the cell surface may modulate MAM/MHC class II interactions.

Functional analysis of Mycoplasma arthritidis-derived mitogen interactions with class II molecules.

The ability of superantigens (SAGs) to trigger various cellular events via major histocompatibility complex (MHC) class II molecules is largely mediated by their mode of interaction. Having two MHC class II binding sites, staphylococcal enterotoxin A (SEA) is able to dimerize MHC class II molecules on the cell surface and consequently induces cytokine gene expression in human monocytes. In contrast, cross-linking with specific monoclonal antibodies or T-cell receptor is required for staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST-1) to induce similar responses. In the present study, we report how Mycoplasma arthritidis-derived mitogen (MAM) may interact with MHC class II molecules to induce cytokine gene expression in human monocytes. The data presented indicate that MAM-induced cytokine gene expression in human monocytes is Zn2+ dependent. The MAM-induced response is completely abolished by pretreatment with SEA mutants that have lost their capacity to bind either the MHC class II alpha or beta chain, with wild-type SEB, or with wild-type TSST-1, suggesting that MAM induces cytokine gene expression most probably by inducing dimerization of class II molecules. In addition, it seems that SEA and MAM interact with the same or overlapping binding sites on the MHC class II beta chain and, on the other hand, that they bind to the alpha chain most probably through the regions that are involved in SEB and TSST-1 binding.

Studies on the mechanism of stimulation of T cells by the Mycoplasma arthritidis-derived mitogen. Role of class II IE molecules.

A mitogen derived from the supernatant of broth cultures of Mycoplasma arthritidis (MAS-P) stimulates a proliferative response by normal, unprimed T cells and interleukin 2 production by some, but not all, T cell hybridomas. The response requires an IE-positive accessory cell (AC). The direct participation of IE, and not IA, in this system was confirmed by two sets of experiments. First, L cells transfected with IE, but not IA, provided effective AC function for both normal T cells and the T cell hybridoma DO-11.10. Second, we have taken a more direct approach by showing that purified IE incorporated in liposomes and used to coat glass beads can support the MAS-P response of the DO-11.10 T cell hybridoma in the absence of intact AC or other AC molecules. Although the receptor for IE-MAS-P has not been identified, we have eliminated from consideration two potential T cell recognition structures. Monoclonal antibody to the antigen-major histocompatibility complex specific receptor failed to inhibit the MAS-P response of DO-11.10 or the T cell line LBRM-33. Furthermore, the L3T4 molecule did not appear to be involved since an L3T4-negative variant of DO-11.10 responded well to the mitogen. In addition, we show that both Lyt-2-positive and L3T4-positive T cells respond to this class II-restricted stimulus. Thus, we postulate the existence of a non-T cell receptor, non-L3T4 receptor that recognizes MAS-P in association with a presumed nonpolymorphic region of IE.