orals 5th Asia-Pacific NMR Symposium 2013

NMR Analysis of a Thermodynamic Cycle: Fast Binding Coupled to Slow Dimerisation (#70)

Martin J Stone 1 , Zil E Huma 1 , Justin P Ludeman 1 , Joshua HY Tan 1 , Jamie Wedderburn 1 , Richard J Payne 2
  1. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia
  2. School of Chemistry, University of Sydney, Sydney, NSW, Australia

NMR spectroscopy is a powerful method for characterising chemical and conformational equilibria. We have characterised a system in which the dimerisation of a protein occurs on the slow exchange time scale but is thermodynamically coupled to ligand binding occurring on a fast exchange time scale.

Chemokines are small proteins that regulate the trafficking of leukocytes to sites of injury or infection. They achieve leukocyte activation by binding to regions of chemokine receptors containing sulfated tyrosine residues. In addition, chemokines weakly dimerise, thereby enhancing their interactions with cell-surface glycosaminoglycans and promoting the formation of chemokine gradients at the endothelial surfaces.

The chemokine monocyte chemoattractant protein-1 (MCP-1) targets the monocyte-expressed receptor CCR2. Although only the monomeric form of MCP-1 can activate CCR21 , we have found that both the monomeric and dimeric forms can bind to CCR2-derived peptides containing sulfotyrosine2 . The monomeric and dimeric forms of MCP-1 give separate peaks in 2D spectra, indicating that they are in slow exchange on the chemical shift time scale. Upon addition of CCR2-derived sulfopeptides, both monomer and dimer peaks undergo chemical shift changes, indicating that both species bind to the sulfopeptides, and the relative intensities of monomer and dimer peaks change, indicating that binding to sulfopeptide affects the monomer-dimer equilibrium. We have modelled this system as a thermodynamic cycle. Computer simulations were used to predict the relationships of binding affinities and dimerisation equilibrium constants in this model to observable NMR parameters - chemical shift changes and peak intensities. By fitting the observed NMR data to this model, we can determine the strength of thermodynamic coupling between ligand binding and dimerisation. Our approach is generally applicable to any system in which coupled fast and slow exchange processes are observable in NMR spectra.

  1. Tan JHY, Canals M, Ludeman JP, Wedderburn J, Boston C, Butler SJ, Carrick AM, Parody TR, Taleski D, Christopoulos A, Payne RJ and Stone MJ (2012). Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1). J Biol Chem 287:14692-14702.
  2. Tan JH, Ludeman JP, Wedderburn J, Canals M, Hall P, Butler SJ, Taleski D, Christopoulos A, Hickey MJ, Payne RJ and Stone MJ (2013). Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1). J Biol Chem. 288:10024–10034.