A combined computational-experimental approach to define the structural origin of antibody recognition of sialyl-Tn, a tumor-associated carbohydrate antigen.

Anti-carbohydrate monoclonal antibodies (mAbs) hold great promise as cancer therapeutics and diagnostics. However, their specificity can be mixed, and detailed characterization is problematic, because antibody-glycan complexes are challenging to crystallize. Here, we developed a generalizable approach employing high-throughput techniques for characterizing the structure and specificity of such mAbs, and applied it to the mAb TKH2 developed against the tumor-associated carbohydrate antigen sialyl-Tn (STn). The mAb specificity was defined by apparent KD values determined by quantitative glycan microarray screening. Key residues in the antibody combining site were identified by site-directed mutagenesis, and the glycan-antigen contact surface was defined using saturation transfer difference NMR (STD-NMR). These features were then employed as metrics for selecting the optimal 3D-model of the antibody-glycan complex, out of thousands plausible options generated by automated docking and molecular dynamics simulation. STn-specificity was further validated by computationally screening of the selected antibody 3D-model against the human sialyl-Tn-glycome. This computational-experimental approach would allow rational design of potent antibodies targeting carbohydrates

Using molecular dynamics trajectories to predict nuclear spin relaxation behaviour in large spin systems

Molecular dynamics (MD) trajectories provide useful insights into molecular structure and dynamics. However, questions persist about the quantitative accuracy of those insights. Experimental NMR spin relaxation rates can be used as tests, but only if relaxation superoperators can be efficiently computed from MD trajectories – no mean feat for the quantum Liouville space formalism where matrix dimensions quadruple with each added spin 1/2. Here we report a module for the Spinach software framework that computes Bloch-Redfield-Wangsness relaxation superoperators (including non-secular terms and cross-correlations) from MD trajectories. Predicted initial slopes of nuclear Overhauser effects for sucrose trajectories using advanced water models and a force field optimised for glycans are within 25% of experimental values

Spin Diffusion Editing for Structural Fingerprints of Therapeutic Antibodies

The growing importance of biologics and biosimilars as therapeutic and diagnostic agents is giving rise to new demands for analytical methodology that can quickly and accurately assess the chemical and physical state of protein-based products. A particular challenge exists in physical characterization where the proper fold and extent of disorder of a protein is a major concern. The ability of NMR to reflect structural and dynamic properties of proteins is well recognized, but sensitivity limitations and high levels of interference from excipients in typical biologic formulations have prevented widespread applications to quality assessment. Here we demonstrate applicability of a simple one-dimensional proton NMR method that exploits enhanced spin diffusion among protons in well-structured areas of a protein. We show that it is possible to reduce excipient signals and allow focus on structural characteristics of the protein. Additional decomposition of the resulting spectra based on rotating frame spin relaxation allows separate examination of components from aggregates and disordered regions. Application to a comparison of two different monoclonal antibodies and to detection of partial pH denaturation of a monoclonal antibody illustrates the procedure

An unprecedented function for a tungsten-containing oxidoreductase

Five tungstopterin-containing oxidoreductases were characterized from the hyperthermophile Pyrococcus furiosus. Each enzyme catalyzes the reversible conversion of one or more aldehydes to the corresponding carboxylic acid, but they have different specificities. The physiological functions of only two of these enzymes are known: one, termed GAPOR, is a glycolytic enzyme that oxidizes glyceraldehyde-3-phosphate, while the other, termed AOR, oxidizes multiple aldehydes generated during peptide fermentation. Two of the enzymes have known structures (AOR and FOR). Herein, we focus on WOR5, the fifth tungstopterin enzyme to be discovered in P. furiosus. Expression of WOR5 was previously shown to be increased during cold shock (growth at 72 ℃), although the physiological substrate is not known. To gain insight into WOR5 function, we sought to determine both its structure and identify its intracellular substrate. Crystallization experiments were performed with a concentrated cytoplasmic extract of P. furiosus grown at 72 ℃ and the structure of WOR5 was deduced from the crystals that were obtained. In contrast to a previous report, WOR5 is heterodimeric containing an additional polyferredoxin-like subunit with four [4Fe-4S] clusters. The active site structure of WOR5 is substantially different from that of AOR and FOR and the significant electron density observed adjacent to the tungsten cofactor of WOR5 was modeled as an aliphatic sulfonate. Biochemical assays and product analysis confirmed that WOR5 is an aliphatic sulfonate ferredoxin oxidoreductase (ASOR). A catalytic mechanism for ASOR is proposed based on the structural information and the potential role of ASOR in the cold-shock response is discussed