Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3
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The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used 15N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the 15N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes.
KeywordsSpin relaxation Order parameters Molecular dynamics simulations Ligand binding Entropy
This work was supported by the Swedish Research Council (MA, UR), The Göran Gustafsson Foundation for Research in Natural Sciences and Medicine (MA), and the FLÄK Research School for Pharmaceutical Sciences at Lund University (MA, UR). Computer resources were provided by Lunarc at Lund University and HPC2N at Umeå University. We thank Hakon Leffler for the plasmid harboring the Gal3-thioredoxin fusion construct, and HL, Ulf Nilsson, and Gunnar Karlström for discussions.
- Case DA, Darden TA, Cheatham TE I, Simmerling CL, Wang J, Duke RE, Luo R, Crowley M, Walker RC, Zhang W, Merz KM, Wang B, Hayik S, Roitberg A, Seabra G, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wu XW, Brozell SR, Steinbrecher T, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Mathews DH, Seetin MG, Sagui C, Babin V, Kollman PA (2008) AMBER 10. University of California, San FranciscoGoogle Scholar
- Cavanagh J, Fairbrother WJ, Palmer AG, Rance M, Skelton NJ (2007) Protein NMR spectroscopy: principles and practice, 2nd edn. Elsevier, San DiegoGoogle Scholar
- Grzesiek S, Bax A (1992) Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. J Magn Reson 96:432–440Google Scholar
- Hill TL (1986) An introduction to statistical thermodynamics. Dover, New YorkGoogle Scholar
- Kay LE, Ikura M, Tschudin R, Bax A (1990) Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514Google Scholar
- Kollman PA, Massova I, Reyes C, Kuhn B, Huo S, Chong L, Lee M, Lee T, Duan Y, Wang W, Donini O, Cieplak P, Srinivasan J, Case DA, Cheatham TE III (2000) Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc Chem Res 33:889–897Google Scholar
- Lundström P, Teilum K, Carstensen T, Bezsonova I, Wiesner S, Hansen F, Religa TL, Akke M, Kay LE (2007) Fractional 13C enrichment of isolated carbons using [1–13C]- or [2–13C]-glucose facilitates the accurate measurement of dynamics at backbone Ca and side-chain methyl positions in proteins. J Biomol NMR 38:199–212CrossRefGoogle Scholar
- Ramamoorthy A, Wu CH, Opella SJ (1997) Magnitudes and orientations of the principal elements of the H-1 chemical shift, H-1-N-15 dipolar coupling, and N-15 chemical shift interaction tensors in N-15(epsilon 1)-tryptophan and N-15(pi)-histidine side chains determined by three-dimensional solid-state NMR spectroscopy of polycrystalline samples. J Am Chem Soc 119:10479–10486CrossRefGoogle Scholar