Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I

Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (NMR) titrations to monitor development of ABE I using peptides based on protease-activated receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44–56) and more weakly binding PAR3G (44–56) could already interact with pro-ABE I on prothrombin. ¹H–¹⁵N heteronuclear single-quantum coherence NMR titrations were then used to probe binding of individual ¹⁵N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in the chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures

A Small Molecule Causes a Population Shift in the Conformational Landscape of an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) have roles in myriad biological processes and numerous human diseases. However, kinetic and amplitude information regarding their ground-state conformational fluctuations has remained elusive. We demonstrate using nuclear magnetic resonance (NMR)-based relaxation dispersion that the D2 domain of p27^Kip1, a prototypical IDP, samples multiple discrete, rapidly exchanging conformational states. By combining NMR with mutagenesis and small-angle X-ray scattering (SAXS), we show that these states involve aromatic residue clustering through long-range hydrophobic interactions. Theoretical studies have proposed that small molecules bind promiscuously to IDPs, causing expansion of their conformational landscapes. However, on the basis of previous NMR-based screening results, we show here that compound binding only shifts the populations of states that existed within the ground state of apo p27-D2 without changing the barriers between states. Our results provide atomic resolution insight into how a small molecule binds an IDP and emphasize the need to examine motions on the low microsecond time scale when probing these types of interactions