Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This "antigen clasping" produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody-antigen recognition and suggests a strategy for developing extremely specific antibodies.

BCL::Fold--protein topology determination from limited NMR restraints.

When experimental protein NMR data are too sparse to apply traditional structure determination techniques, de novo protein structure prediction methods can be leveraged. Here, we describe the incorporation of NMR restraints into the protein structure prediction algorithm BCL::Fold. The method assembles discreet secondary structure elements using a Monte Carlo sampling algorithm with a consensus knowledge-based energy function. New components were introduced into the energy function to accommodate chemical shift, nuclear Overhauser effect, and residual dipolar coupling data. In particular, since side chains are not explicitly modeled during the minimization process, a knowledge based potential was created to relate experimental side chain proton-proton distances to Cß -Cß distances. In a benchmark test of 67 proteins of known structure with the incorporation of sparse NMR restraints, the correct topology was sampled in 65 cases, with an average best model RMSD100 of 3.4 ± 1.3 Å versus 6.0 ± 2.0 Å produced with the de novo method. Additionally, the correct topology is present in the best scoring 1% of models in 61 cases. The benchmark set includes both soluble and membrane proteins with up to 565 residues, indicating the method is robust and applicable to large and membrane proteins that are less likely to produce rich NMR datasets.