Thermodynamics of Binding of 2-Methoxy-3-isopropylpyrazine and 2-Methoxy-3-isobutylpyrazine to the Major Urinary Protein

Abstract

In the present study we examine the thermodynamics of binding of two related pyrazine-derived ligands to the major urinary protein, MUP-I, using a combination of isothermal titration calorimetry (ITC), X-ray crystallography, and NMR backbone (15)N and methyl side-chain (2)H relaxation measurements. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than the classical entropy-driven hydrophobic effect. Unfavorable entropic contributions from the protein backbone and side-chain residues in the vicinity of the binding pocket are partially offset by favorable entropic contributions at adjacent positions, suggesting a "conformational relay" mechanism whereby increased rigidity of residues on ligand binding are accompanied by increased conformational freedom of side chains in adjacent positions. The principal driving force governing ligand affinity and specificity can be attributed to solvent-driven enthalpic effects from desolvation of the protein binding pocket.

High resolution X‐ray structures of mouse major urinary protein nasal isoform in complex with pheromones

“…This is consistent with the findings of other groups, where significant difference electron density peaks are observed in the MUP binding pocket even in the absence of exogenously introduced ligand. 21,24,26 Data collection and refinement statistics are presented in Table I.…”

“…24 z We note that all MUP structures, including those presented here, have been determined at pH 4.8-7.2. 21,23,24,[26][27][28][29][30][31][32] The pK a of the conjugate acid of thiazole and pyrazine derivatives is around pH 2, 38,39 thus, the sp2 hybridized nitrogen is less likely to be protonated at the pH of the experiments than is a Glu residue. To satisfy hydrogen bonding potential, we show E118 as protonated at OE2 in the figures.…”

“…26 An alignment of these MUP-I complexes with our DMP-bound MUP-IV structure shows that in MUP-I, the pyrazine ring is shifted into the space occupied by E118 in MUP-IV [ Fig. 7(A)].…”

“…7(C)]. 26 Hence, it appears that the binding mode of pyrazine derivatives also differs between MUP-IV and MUP-I. However, Y120 does play a role in ligand coordination in each of these structures: participating in either a direct or a water-mediated hydrogen bond with the ligand in MUP-I or stabilizing the orientation of E118 in MUP-IV.…”

“…22 X-ray and NMR structures of MUP-I and MUP-II have been determined in the presence of SBT 23,24 as well as other ligands. [24][25][26][27][28][29][30][31][32] However, until now, there have been no published structures of a group 4 isoform. We have determined the X-ray crystal structures of MUP-IV in complex with three mouse pheromones to examine the structural basis of ligand binding specificity.…”