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Structure Determination of a Galectin-3 - Carbohydrate Complex Using Paramagnetism-based NMR Constraints

¹ Universtiy of Georgia;
² Silla University, Busan, Korea;
³ Massachusetts Institute of Technology;
⁴ University of Georgia

(RECEIVED January 16, 2008; ACCEPTED April 11, 2008)

The determination of the location and conformation of a natural ligand bound to a protein receptor is often a first step in the rational design of molecules that can modulate receptor function. NMR observables, including NOEs, often provide the basis for these determinations. However, when ligands are carbohydrates, interactions mediated by extensive hydrogen-bonding networks often reduce or eliminate NOEs between ligand and protein protons. In these cases it is useful to look to other distance and orientation dependent observables that can constrain the geometry of ligand-protein complexes. Here we illustrate the use of paramagnetism-based NMR constraints, including pseudo contact shifts (PCS) and field induced residual dipolar couplings (RDCs). When a paramagnetic center can be attached to the protein, field induced RDC and PCS reflect only bound-state properties of the ligand, even when averages over small fractions of bound states and large fractions of free states are observed. The effects can also be observed over a long range making it possible to attach a paramagnetic center to a remote part of the protein. The system studied here is a Galectin-3-lactose complex. A lanthanide binding peptide showing minimal flexibility with respect to the protein was integrated into the C-terminus of an expression construct for the Galectin-3 carbohydrate-binding domain. Dysprosium ion, which has a large magnetic susceptibility anisotropy, was complexed to the peptide making it possible to observe both PCSs and field induced RDCs for the protein and the ligand. The structure determined from these constraints shows agreement with a crystal structure of a Galectin-3-N-acetyllactosamine complex.

Keywords: Structure; Active site/binding site/epitope mapping; NMR Spectroscopy; Heteronuclear NMR; New Methods; Docking Proteins

⁵ E-mail: jpresteg{at}ccrc.uga.edu

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