[Show abstract][Hide abstract] ABSTRACT: Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron-sulfur clusters but whose mutations are associated with Friedreich's ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.
Proceedings of the National Academy of Sciences of the United States of America. 09/2014;
[Show abstract][Hide abstract] ABSTRACT: 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. In vitro 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 in vitro 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 due to 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 of its protective role in Alzheimer's disease.
[Show abstract][Hide abstract] ABSTRACT: Many biological processes are regulated by the interaction between protein domains and their corresponding binding partners. The PDZ domain is one of the most common protein-protein interaction modules in mammalian cells, whose role is to bind C-terminal sequences of specific targets. The second PDZ domain from the Protein Tyrosine Phosphatase-BL (PDZ2) binds to the C-terminal of Adenomatous Polyposis Coli protein (APC), one of the major tumor suppressor whose task is to regulate cell adhesion and proliferation. Here, we present a detailed kinetics analysis of the interaction between PDZ2 domain and a peptide mimicking the PDZ binding motif of APC. By analyzing data obtained at different experimental conditions, we propose a plausible mechanism for binding. Furthermore, a comparison between the dissociation rate constant measured by different methodologies allow us to identify an additional kinetic step, which is likely to arise from a conformational change of PDZ2 occurring after binding. The data are discussed on the light of previous work on PDZ domains.
Protein engineering, design & selection : PEDS. 06/2014;
[Show abstract][Hide abstract] ABSTRACT: The KIX domain is a mediator of the interaction between different transcription factors. This complex function is carried out via two distinct binding sites located on opposite sides of the protein, namely the ‘c-Myb site' and the ‘MLL site', named after their characteristic ligands - the transactivation domain of c-Myb and the Mixed Lineage Leukemia protein (MLL). Both these ligands are unstructured in isolation and fold only upon binding, posing the KIX domain as an ideal candidate to explore the binding induced folding reaction of intrinsically unstructured proteins. Here we complement the recent kinetic description on the interaction between KIX and c-Myb, by characterizing the binding kinetics between KIX and MLL, at different pH and ionic strength conditions. Furthermore, we analyze quantitatively the mechanism of allosteric communication between the topologically distinct c-Myb and MLL sites. The implications of our results are discussed in the light of previous work on other intrinsically unstructured systems.
[Show abstract][Hide abstract] ABSTRACT: The interactions between proteins and ligands often involve a conformational change in the protein. This conformational change can occur before (conformational selection) or after (induced fit) the association with ligand. It is often very difficult to distinguish induced fit from conformational selection when hyperbolic binding kinetics are observed. In light of a recent paper in this journal (Vogt et al., Biophys. Chem., 186, 2014, 13-21) and the current interest in binding mechanisms emerging from observed sampling of distinct conformations in protein domains, as well as from the field of intrinsically disordered proteins, we here describe a kinetic method that, at least in some cases, unequivocally distinguishes induced fit from conformational selection. The method relies on measuring the observed rate constant λ for binding and varying both the protein and the ligand in separate experiments. Whereas induced fit always yields a hyperbolic dependence of increasing λ values, the conformational selection mechanism gives rise to distinct kinetics when the ligand and protein (displaying the conformational change) concentration is varied in separate experiments. We provide examples from the literature and discuss the limitations of the approach.
[Show abstract][Hide abstract] ABSTRACT: The role of the denatured state in protein folding represents a key issue for the proper evaluation of folding kinetics and mechanisms. The yeast ortholog of the human frataxin, a mitochondrial protein essential for iron homeostasis and responsible for Friedreich's ataxia, has been shown to undergo cold denaturation above 0 °C, in the absence of chemical denaturants. This interesting property provides the unique opportunity to explore experimentally the molecular mechanism of both the hot and cold denaturation. In this work, we present the characterization of the temperature and urea dependence of the folding kinetics of yeast frataxin, and show that while at neutral pH and in the absence of a denaturant a simple two-state model may satisfactorily describe the temperature dependence of the folding and unfolding rate constants, the results obtained in urea over a wide range of pH reveal an intriguing complexity, suggesting that folding of frataxin involves a broad smooth free energy barrier.
Physical Chemistry Chemical Physics 01/2014; · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins are very common and instrumental for cellular signaling. Recently, a number of studies have investigated the kinetic binding mechanisms of IDPs and IDRs. These results allow us to draw conclusions about the energy landscape for the coupled binding and folding of disordered proteins. The association rate constants of IDPs cover a wide range (10(5)-10(9) M(-1) s(-1)) and are largely governed by long-range charge-charge interactions, similarly to interactions between well-folded proteins. Off-rate constants also differ significantly among IDPs (with half-lives of up to several minutes) but are usually around 0.1-1000 s(-1), allowing for rapid dissociation of complexes. Likewise, affinities span from pM to μM suggesting that the low-affinity high-specificity concept for IDPs is not straightforward. Overall, it appears that binding precedes global folding although secondary structure elements such as helices may form before the protein-protein interaction. Short IDPs bind in apparent two-state reactions whereas larger IDPs often display complex multi-step binding reactions. While the two extreme cases of two-step binding (conformational selection and induced fit) or their combination into a square mechanism is an attractive model in theory, it is too simplistic in practice. Experiment and simulation suggest a more complex energy landscape in which IDPs bind targets through a combination of conformational selection before binding (e.g., secondary structure formation) and induced fit after binding (global folding and formation of short-range intermolecular interactions).
Physical Chemistry Chemical Physics 12/2013; · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A classical dogma of molecular biology dictates that the 3D structure of a protein is necessary for its function. However, a considerable fraction of the human proteome, although functional, does not adopt a defined folded state under physiological conditions. These intrinsically disordered proteins tend to fold upon binding to their partners with a molecular mechanism that is elusive to experimental characterization. Indeed, although many hypotheses have been put forward, the functional role (if any) of disorder in these intrinsically denatured systems is still shrouded in mystery. Here, we characterize the structure of the transition state of the binding-induced folding in the reaction between the KIX domain of the CREB-binding protein and the transactivation domain of c-Myb. The analysis, based on the characterization of a series of conservative site-directed mutants, reveals a very high content of native-like structure in the transition state and indicates that the recognition between KIX and c-Myb is geometrically precise. The implications of our results in the light of previous work on intrinsically unstructured systems are discussed.
Proceedings of the National Academy of Sciences 08/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The affinity and specificity of protein-ligand interactions are influenced by energetic crosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interaction pathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands.
[Show abstract][Hide abstract] ABSTRACT: 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 anti-parallel 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, ΔΔG = 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.
Journal of the American Chemical Society 05/2013; · 10.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable of the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.
Biochemical and Biophysical Research Communications 04/2013; · 2.28 Impact Factor