Mannobiose Binding Induces Changes in Hydrogen Bonding and Protonation States of Acidic Residues in Concanavalin A As Revealed by Neutron Crystallography

Abstract

Plant lectins are carbohydrate-binding proteins with various biomedical applications. Concanavalin A (Con A) holds promise in treating cancerous tumors. To better understand the Con A carbohydrate binding specificity, we obtained a room-temperature neutron structure of this legume lectin in complex with a disaccharide Manα1-2Man, mannobiose. The neutron structure afforded direct visualization of the hydrogen bonding between the protein and ligand, showing that the ligand is able to alter both protonation states and interactions for residues located close to and distant from the binding site. An unprecedented low-barrier hydrogen bond was observed forming between the carboxylic side chains of Asp28 and Glu8, with the D atom positioned equidistant from the oxygen atoms having an O···D···O angle of 101.5°.

Figure 1

Hydrogen bonding and water-mediated interactions made by mannobiose with ConA residues. Carbohydrate-binding site residues are color-coded with carbons colored yellow. The residues from the two symmetry-related ConA molecules are color-coded with carbons colored gray and magenta. D⋯O and D⋯N distances are shown as blue dashed lines and are given in units of angstroms.

Figure 2

Water chain hydrating mannobiose spans a curve from Arg228 to O3 of Man2. The 2F_O – F_C neutron scattering length density map (teal mesh) and the 2F_O – F_C electron density map (orange mesh) for water molecules are both shown at 1.2σ contour levels. Hydrogen bonds are shown as blue dashed lines, and D⋯O distances are given in units of angstroms. W1 is the same water as shown in Figure 1 (for the sake of clarity, W2 and W3_sym were omitted).

Figure 3

Interactions between the protonated Asp71_sym from the symmetry-related ConA and mannobiose. The 2F_O – F_C neutron scattering length density map (teal mesh) is shown at the 1.2σ contour level. The omit difference F_O – F_C neutron scattering length density map (red mesh) for the carboxyl D atom is shown at the 3σ contour level. Hydrogen bonds are shown as blue dashed lines, and D⋯O distances are given in units of angstroms.

Figure 4

Mn²⁺ (violet sphere)-binding site and protonation of the Asp28–Glu8 pair. The 2F_O – F_C neutron scattering length density map (teal mesh) is shown at the 1.5σ contour level. The omit difference F_O –F_C neutron scattering length density map (red mesh) for the D atom in the low-barrier hydrogen bond is shown at the 3σ contour level. Hydrogen bonds are shown as blue dashed lines, and D⋯O distances are given in units of angstroms. The side chain of Val32 was omitted for the sake of clarity.

Scheme 1

Chemical Structure of Manα1–2Man