2 research outputs found
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. <sup>1</sup>H–<sup>15</sup>N
heteronuclear single-quantum coherence NMR titrations were then used
to probe binding of individual <sup>15</sup>N-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<sup>Kip1</sup>, 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