Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3

Abstract

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 (15)N 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 (15)N 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.

Will molecular dynamics simulations of proteins ever reach equilibrium?

“…from the solvent). 39 Such studies have been performed several times before 16,21,22,23,24,25,26,27,28,29 and we followed the protocol developed in those studies: We performed 500 ns simulations of two ligand-bound states of the Gal3 protein and 380 ns simulations of two ligand-bound states of MMP12. The protein conformational entropies were then estimated by the DDH approach.…”

“…16,17,19,20 Therefore, MD simulations are often used to supplement the NMR measurements to provide the total conformational entropy of the protein. 16,21,22,23,24,25,26,27,28,29 Protein conformational entropies are the main topic of this study, but it should be remembered that there are other important contributions to the total entropy as well, e.g. from the solvent.…”

“…Previous calculations have shown that entropy contributions from the bond and angle fluctuations are negligible. 27 The distribution for each dihedral angle, i, was then approximated by a discrete histogram with 72 bins (other numbers of bins have also been tested, but they gave similar results) and the entropy was calculated from…”

The Normal-Mode Entropy in the MM/GBSA Method: Effect of System Truncation, Buffer Region, and Dielectric Constant

“…counting the number of rotable bonds in the ligand, dihedral-distribution histogramming, or quasi-harmonic analysis. 8,9,10,11,12 The latter two estimates seem to be very hard to converge to any useful precision, whereas NMA estimates of entropies converge much better. 10,11,12 In practice, the NMA calculations are performed by first stripping off all water molecules.…”

“…Therefore, other methods to calculate the entropy may give significantly different results. 9,11 Acknowledgements …”