Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain

Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (∼10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured state. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein–protein interactions

Demonstration of a Folding after Binding Mechanism in the Recognition between the Measles Virus N_TAIL and X Domains

In the past decade, a wealth of experimental data has demonstrated that a large fraction of proteins, while functional, are intrinsically disordered at physiological conditions. Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their biological targets, a phenomenon known as induced folding. Induced folding may occur through two extreme mechanisms, namely conformational selection and folding after binding. Although the pre-existence of ordered structures in IDPs is a prerequisite for conformational selection, it does not necessarily commit to this latter mechanism, and kinetic studies are needed to discriminate between the two possible scenarios. So far, relatively few studies have addressed this issue from an experimental perspective. Here, we analyze the interaction kinetics between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N_TAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that N_TAIL recognizes XD by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step; i.e., binding precedes folding. The implications of our kinetic results, in the context of previously reported equilibrium data, are discussed. These results contribute to enhancing our understanding of the molecular mechanisms by which IDPs recognize their partners and represent a paradigmatic example of the need of kinetic methods to discriminate between reaction mechanisms

Analysis of the two different phases in kinetic folding experiments.

<p>Chevron plots of cp- and pwtSAP97 PDZ2 in 50 mM potassium phosphate, pH 7.5, showing the rate constants corresponding to the two observed phases. The black continuous line shows an on-pathway fit to the kobs values for cpSAP97 PDZ2. The fits to off-pathway and triangular schemes were equally good and are not shown. For cpSAP97 PDZ2 the phase with the largest amplitude is always the fastest one, while for pwtSAP97 PDZ2 the phase with the largest amplitude is the fastest one between urea concentrations 0–2.5 M and then it switches to be the slower one, hence, what is referred to as the main phase in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050055#pone-0050055-g005" target="_blank">Figure 5</a> is represented by both kobs1 and kobs2.</p

A Complex Equilibrium among Partially Unfolded Conformations in Monomeric Transthyretin

Aggregation of transthyretin (TTR) is known to be linked to the development of systemic and localized amyloidoses. It also appears that TTR exerts a protective role against aggregation of the Aβ peptide, a process linked to Alzheimer’s disease. <i>In vitro</i>, both processes correlate with the ability of TTR to populate a monomeric state, yet a complete description of the possible conformational states populated by monomeric TTR <i>in vitro</i> at physiological pH is missing. Using an array of biophysical methods and kinetic tests, we show that once monomers of transthyretin are released from the tetramer, equilibrium is established between a set of conformational states possessing different degrees of disorder. A molten globular state appears in equilibrium with the fully folded monomer, whereas an off-pathway species accumulates transiently during refolding of TTR. These two conformational ensembles are distinct in terms of structure, kinetics, and their pathways of formation. Further subpopulations of the protein fold differently because of the occurrence of proline isomerism. The identification of conformational states unrevealed in previous studies opens the way for further characterization of the amyloidogenicity of TTR and its protective role in Alzheimer’s disease

Data collection and refinement statistics.

1<p>Values in parentheses represent the highest resolution bin.</p

Chevron plots of the main phases of cp- and pwtSAP97 PDZ2 under different conditions.

<p>The main phase is the <i>k</i>_obs value with the largest amplitude. Rollovers in the refolding and unfolding arm of the chevron plots can be detected when altering between stabilizing and destabilizing buffers, respectively. These rollovers illustrate switches between the rate limiting transition states of the (un)folding reaction. Fitting was done using <i>β</i>_T–values obtained from a curve fit with 6 different PDZ domains in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050055#pone.0050055-Gianni2" target="_blank">[19]</a> and the good fit to the data for the circular permutant illustrates that the positions of the folding transition states along the reaction coordinate is similar for all PDZ domains, including the circular permutant. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050055#pone.0050055.s001" target="_blank">Table S1</a> for the best fit parameters. The 0.6 M Na₂SO₄ buffer also contained 50 mM potassium phosphate, pH 7.5, while the 50 mM potassium acetate buffer, pH 5.6, contained KCl to keep the ionic strength at the same value for all experiments.</p

Structure of the circularly permuted SAP97 PDZ2 (cpSAP97 PDZ2).

<p><b>A.</b> Schematic picture of the rearrangement of secondary structural elements in cpSAP97 PDZ2. The secondary structure arrangement is naturally occurring in a PDZ domain in green alga <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050055#pone.0050055-Liao1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050055#pone.0050055-Ivarsson3" target="_blank">[18]</a> and even though it seems modest, had a significant effect on the folding of PTP-BL PDZ2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050055#pone.0050055-Ivarsson1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050055#pone.0050055-Ivarsson2" target="_blank">[7]</a>. <b>B.</b> Ribbon representation of the cpSAP97 PDZ2 structure showing the new N and C termini. <b>C.</b> Superposition of the two cpSAP97 PDZ2 molecules in the crystal structure, A (green) and B (blue), shown as C_α trace. <b>D.</b> Superposition of cpSAP97 PDZ2 (green) and pwtSAP97 PDZ2 (pink) shown as C_α trace.</p

Probing the Role of Backbone Hydrogen Bonds in Protein–Peptide Interactions by Amide-to-Ester Mutations

One of the most frequent protein–protein interaction modules in mammalian cells is the postsynaptic density 95/discs large/zonula occludens 1 (PDZ) domain, involved in scaffolding and signaling and emerging as an important drug target for several diseases. Like many other protein–protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as β-strands to form an extended antiparallel β-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the amino acid side chains of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, ΔΔ<i>G</i> = 1.3 to >3.8 kcal mol^–1). This decrease in affinity was mainly due to an increase in the dissociation rate constant, but a significant decrease in the association rate constant was found for some amide-to-ester mutations suggesting that native hydrogen bonds have begun to form in the transition state of the binding reaction. This study provides a general framework for studying the role of backbone hydrogen bonds in protein–peptide interactions and for the first time specifically addresses these for PDZ domain–peptide interactions

NOAKI trial survey data.

(XLSX)</p

Equilibrium parameters for the stability of cpSAP97 PDZ2 and kinetic parameters for its association with the peptide LQRRRETQV.

1<p>Shared <i>m</i>_D-N –value in the curve fitting.</p>2<p>Free fitting.</p>3<p>From ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050055#pone.0050055-Haq2" target="_blank">[51]</a>.</p