Molecular recognition: structural and energetic aspects of preorganization, substrate specificity, and oligomerization

Abstract

Understanding the structural and energetic characteristics of the interactions between biomolecules is a center of focus in the world of drug design. The approach taken here is to analyze three fairly different molecular recognition phenomenons. First, the energetics of binding by a Shc based peptide to monomeric and domain-swapped dimeric Grb2-SH2 is analyzed. The peptide bound to monomer with over 15-fold higher affinity than to domain-swapped dimeric Grb2-SH2. Two crystal structures were solved which suggest the molecular basis for the observed binding thermodynamic differences are alternate conformations of the EF loop in monomer and domain-swapped dimer Grb2-SH2. Secondly, the impact of introducing a cyclopropane ring, to act as a vii conformational constraint, into Shc based peptides that bind the Grb2-SH2 domain is studied. Isothermal titration calorimetry experiments showed the constrained ligands bound with higher affinity compared to flexible controls. Interestingly, the higher affinity for the constrained compounds was the result of an enthalpic advantaged, in contrast to an expected entropic advantage for the restriction of rotors. Several crystal structures were solved, which suggest the conformationally constrained ligands likely bound as a more rigid complex compared to the flexible control. Lastly, the structural basis for the substrate specificity profiles of three PC-PLCBc protein variants is investigated. The crystal structures of these protein variants were solved, and structures of complexes between a phosphatidylserine analogue and wild-type PC-PLCBc and two phosphatidylserine specific protein variants were also obtained. Those structures suggest active site water molecules and the side chain conformations of residues at the 4th and 55th positions play a crucial role in the substrate specificity profile of the protein variants studied.