The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor

Abstract

The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (K d 0.05 nm) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 A Ê resolution by X-ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen-binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 A Ê 2 . Analysis of the electrostatic surface potential of the antigen-binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.

Structural Mimicry of CD4 by a Cross-reactive HIV-1 Neutralizing Antibody with CDR-H2 and H3 Containing Unique Motifs

“…The H3 motif contains residues 95-102 with an insertion of six residues after position 100 and contains hydrophobic residues Phe99 and Leu100, which are exposed at the tip. The Ramachandran plot 37 shows that the 4/j torsion angle distributions are similar to those observed in other wellrefined structures, including two exceptions (Ala51 and Gln29 in the CDR L2 region) generally observed in many immunoglobulin structures, [38][39][40] and Ser15 in the framework of one of the heavy chains F. All the outliers have well-defined electron density. Not observed are the N-terminal residue of each chain, three C-terminal residues of chain B, and five C-terminal residues of chains D and E. Also observed are 499 water molecules and three sulfate ions from the crystallization buffer system.…”

Structural insights into the molecular mechanisms of myasthenia gravis and their therapeutic implications

“…To address this question, we compared the structure of Fab35 with that of two other MG mAbs (Fab198: PDB ID, 1FN4 and Fab192: PDB ID, 1C5D) (Kontou et al, 2000; Poulas et al, 2001). Interestingly, superposition of the structure of Fab198 onto that of Fab35 in the ternary complex shows that these two Fabs share not only a conserved immunoglobulin fold but also a similar antigen-binding site (Figure 4a).…”

“…The CDR-H3 loop of Fab198 (purple) is substantially shorter than that of Fab35 (yellow), as indicated by arrows. ( c ) Structure-based sequence alignment of the nAChR α1-binding loops (CDR-H2, CDR-H3 and CDR-L3) between Fab35, Fab198 and Fab192 (Kontou et al, 2000). Residues shaded in light green are involved in nAChR α1 binding in Fab35, some of these (in bold font and colored in red) are conserved in Fab198 or Fab192.…”

“…Since then, extensive efforts have been put into characterizing the interactions between MG antibodies and nAChR using biochemical (Barkas et al, 1988; Tzartos et al, 1988; Das and Lindstrom, 1989; Saedi et al, 1990; Papadouli et al, 1990, 1993; Luo et al, 2009; Morell et al, 2014), structural (Dellisanti et al, 2007a; Beroukhim and Unwin, 1995; Kontou et al, 2000; Poulas et al, 2001), and modeling approaches (Kleinjung et al, 2000). These studies aimed to understand the basic mechanisms of MG and also the structure/function of nAChR in order to develop effective diagnosis and treatment for MG.…”

Autoimmune diseases involving nicotinic receptors

“…The rat and human immune systems must form antibodies of somewhat different specificity to this region which, nonetheless, compete for binding. The crystal structures of the binding sites of two rat mAbs derived from the same rat to the MIR on human ␣1 AChR have been determined (Kontou et al, 2000;Poulas et al, 2001). The two binding sites are significantly different in shape, providing no clear negative image of the MIR and illustrating the differences in binding properties of antibodies whose binding sites overlap.…”