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. 2014 Feb 7;343(6171):656-661.
doi: 10.1126/science.1246135.

A Structurally Distinct Human Mycoplasma Protein That Generically Blocks Antigen-Antibody Union

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Free PMC article

A Structurally Distinct Human Mycoplasma Protein That Generically Blocks Antigen-Antibody Union

Rajesh K Grover et al. Science. .
Free PMC article

Abstract

We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the κ and λ light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field.

Figures

Fig. 1
Fig. 1. Immunoglobulins selectively bind to proteins in human mycoplasma
(A) (Left panel) Western blot analysis of the reactivity of plasma from multiple myeloma patient 13PL with cell extracts from Mycoplasma alligatoris, Mycoplasma crocodyli, Mycoplasma fermentans, M. genitalium, Acholeplasma Mycoplasma laidlawii, Mycoplasma mycoides, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma pulmonis. All mycoplasma cells were grown in appropriate media. Cells were lysed according to manufacturer's protocol using lysis buffer from Sigma Aldrich. Nucleic acids were degraded by treatment with DNAase and RNAase. A protease inhibitor cocktail (Roche) was added to prevent proteolytic degradation. The extracts from the same number of cells were separated on SDS-PAGE gels and transferred to nitrocellulose membranes for Western blot analysis. (Right panel) Ponceau red-stained protein bands of the cell extracts. (B) Crystals of 13PL Fab' from a multiple myeloma patient's monoclonal immunoglobulin. (C) Western blot analysis of the reactivity of 13PL Fab' from the plasma of a multiple myeloma patient with the same cell extracts in A. The 13PL Fab' was purified by crystallization. The extracts were separated on SDS-PAGE gels as described in (A). (D) Crystal structure of 13PL Fab' shown in ribbon diagram with the light and heavy chains colored in light and dark gray, respectively. The loops corresponding to CDRs L1, L2, and L3 are colored blue, whereas CDRs H1, H2, and H3 are colored red. The relatively rare disulfide in human CDR3's is colored green.
Fig. 2
Fig. 2. Crystal structures of recombinant protein M TD in complex with PGT135 Fab and with CR9114 Fab
(A) Overall structure of Protein M TD from its complex with PGT135 Fab in ribbon representation. Protein M TD appears to have two main domains: a larger domain I in green (residues 78 to 440), which includes a leucine-rich repeat-like structure in purple (LRR; residues 240 to 330) and a smaller domain II in cyan (residues 441 to 468). (B) Overall structure of Protein M TD in complex with PGT135 Fab. Protein M TD is colored in green, and PGT135 Fab is colored in light gray for the light chain and dark gray for the heavy chain. Protein M TD predominantly binds VL of PGT135 Fab, but also interacts to a lesser extent with VH, CL and CH1. (C) Overall structure of Protein M TD in complex with CR9114 Fab. The coloring scheme is similar to that of (B). Residues 455–468 in domain II of Protein M were flexible and not modeled. Protein M TD predominantly binds VL of CR9114 Fab. (D) Molecular surface representation of PGT135 Fab with the paratope of Protein M TD in red, Fab light chain in light gray and Fab heavy chain in dark gray. (E) Molecular surface representation of CR9144 Fab with the paratope of Protein M TD and colored as in (D). (F) The common paratope residue locations (in red) of Protein M TD for PGT135 Fab and CR9114 Fab are shown on PGT135 Fab, including VL residues 14–18 of FR1; 37, 45, 47 and 49 of FR2; 53–56 of CDR2; 57–61, 76, 77, 79 and 81 of FR3; residues 107 and 109 connecting VL and CL; as well as residues 168 and 170 of CL and residue 168 of CH1.
Fig 3
Fig 3. Protein-M blocks antigen-antibody union
(A) Binding of CR9114 IgG, a human broadly neutralizing antibody against influenza virus to one of its antigens, H5 hemagglutinin (influenza virus strain A/Viet Nam/1203/2004 (H5N1)) was evaluated using ELISA after precomplexing with recombinant Protein M (red) and Protein M TD (blue). Binding of the IgG to the HA in the absence of protein M was used as control (purple). The extent of binding was analyzed by a colorimetric assay. The curves were obtained by a nonlinear regression analysis where the data were fit to a four-parameter logistic equation based on a simple binding model. Error bars represent standard deviation of duplicate measurements. (B) Binding of PGT135 IgG, a human broadly neutralizing antibody against its antigen HIV-1 gp120 (JR-FL gp120 core construct) was evaluated (purple) after precomplexing PGT135 IgG with Protein M (red) and Protein M TD (blue) (at a 1:8 molar ratio). ELISA assay was performed as in (A). (C) Binding of KZ52 IgG, a human broadly neutralizing antibody against its Ebola antigen glycoprotein (purple) was evaluated as in B. ELISA assay was performed as in (A). (D) Binding of 13C6 IgG, a mouse broadly neutralizing antibody against its Ebola glycoprotein antigen (purple) was evaluated as in B. (E) Binding of anti-COL4A3 human polyclonal serum from a patient with Goodpasture's disease to its antigen COL4A3 (purple) was evaluated as in B. (F) Binding of anti-DNA polyclonal serum a mouse with lupus to its antigen chromatin (purple) was evaluated as in B.
Fig. 4
Fig. 4. Structural comparison of the antibody binding sites of Protein M and other immunoglobulin binding proteins
All antibody Fabs or Fcs are shown in ribbon representation with light chain in light gray and one heavy chain in dark. (A) Protein M TD is depicted in green ribbon representation in its complex with PGT135 Fab. Protein M mainly binds to the VL domain. (B) One of the Ig-binding domains of Protein L (in orange) bound to VL of an antibody Fab (PDB code 1HEZ). Noticeably, although the Protein L domain can binds to two Fabs with similar paratope sites (23), only one binding site is shown here. (C) One of the Ig-binding domains of Protein A (in skyblue) bound to VH (weaker secondary site) of an antibody Fab (PDB code 1DEE). (D) One of the Ig-binding domains of Protein G (in pink) bound to CH1 (weaker secondary site) of an antibody Fab (PDB code 1IGC). (E) One of the Ig-binding domains of Protein A (in skyblue) bound to the antibody Fc region (primary site) near the CH2 and CH3 domain interface (PDB code 1FC2). (F) One of the Ig-binding domains of Protein G (in pink) also bound to antibody Fc region (primary site) near CH2 and CH3 domain interface (PDB code 1FCC). Although Protein M and Protein L predominantly bind the VL domain, their binding sites are very different with only one common residue (position 18 of VL). These Ig-binding proteins appear to have different ranges of affinities for antibodies. Generally for human antibodies, Protein M binds strongly to all the types, Protein G binds strongly to the antibody Fc region and weakly to the CH1 domain, Protein A binds strongly to antibody Fc region (except to the IgG3 subtype) and weakly to VH of VH3 gene family, and Protein L binds strongly to VL of κ light-chains, except the VκII subgroup.

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