Hidden kinetic traps in multidomain folding highlight the presence of a misfolded but functionally competent intermediate.

Although more than 75% of the proteome is composed of multidomain proteins, current knowledge of protein folding is based primarily on studies of isolated domains. In this work, we describe the folding mechanism of a multidomain tandem construct comprising two distinct covalently bound PDZ domains belonging to a protein called Whirlin, a scaffolding protein of the hearing apparatus. In particular, via a synergy between NMR and kinetic experiments, we demonstrate the presence of a misfolded intermediate that competes with productive folding. In agreement with the view that tandem domain swapping is a potential source of transient misfolding, we demonstrate that such a kinetic trap retains native-like functional activity, as shown by the preserved ability to bind its physiological ligand. Thus, despite the general knowledge that protein misfolding is intimately associated with dysfunction and diseases, we provide a direct example of a functionally competent misfolded state. Remarkably, a bioinformatics analysis of the amino acidic sequence of Whirlin from different species suggests that the tendency to perform tandem domain swapping between PDZ1 and PDZ2 is highly conserved, as demonstrated by their unexpectedly high sequence identity. On the basis of these observations, we discuss on a possible physiological role of such misfolded intermediate.

Templated folding of intrinsically disordered proteins.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed "templated folding," whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

How Robust Is the Mechanism of Folding-Upon-Binding for an Intrinsically Disordered Protein?

The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of NTAIL upon binding to XD by measuring the effect on both the folding and binding steps of NTAIL when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of NTAIL of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs.

A Carboxylate to Amide Substitution That Switches Protein Folds.

Metamorphic proteins are biomolecules prone to adopting alternative conformations. Because of this feature, they represent ideal systems to investigate the general rules allowing primary structure to dictate protein topology. A comparative molecular dynamics study was performed on the denatured states of two proteins, sharing nearly identical amino-acid sequences (88 %) but different topologies, namely an all-α-helical bundle protein named GA 88 and an α+ß-protein named GB 88. The analysis allowed successful design of and experimental validation of a site-directed mutant that promotes, at least in part, the switch in folding from GB 88 to GA 88. The mutated position, in which a glutamic acid was replaced by a glutamine, does not make any intramolecular interactions in the native state of GA 88, such that its stabilization can be explained by considering the effects on the denatured state. The results represent a direct demonstration of the role of the denatured state in sculpting native structure.

Analyzing the Folding and Binding Steps of an Intrinsically Disordered Protein by Protein Engineering.

Intrinsically disordered proteins (IDPs) are functionally active despite lacking a well-defined three-dimensional structure. Such proteins often undergo a disorder-to-order transition, or induced folding, when binding to their specific physiological partner. Because of cooperativity, the folding and binding steps typically appear as a single event, and therefore, induced folding is extremely difficult to characterize experimentally. In this perspective, the interaction between the disordered C-terminal domain of the measles virus nucleoprotein NTAIL and the folded X domain of the viral phosphoprotein (XD) is particularly interesting because the inherent complexity of the observed kinetics allows characterization of the binding and folding steps individually. Here we present a detailed structural description of the folding and binding events occurring in the recognition between NTAIL and XD. This result was achieved by measuring the effect of single-amino acid substitutions in NTAIL on the reaction mechanism. Analysis of the experimental data allowed us (i) to identify the key residues involved in the initial recognition between the two molecules and (ii) to depict the general features of the folding pathway of NTAIL. Furthermore, an analysis of the changes in stability obtained for the whole set of variants highlights how the sequence of this IDP has not been selected during evolution to fold efficiently. This feature might be a consequence of the weakly funneled nature of the energy landscape of IDPs in their unbound state and represents a plausible explanation of their highly dynamic nature even in the bound state, typically defined as "fuzziness".

Identification and Structural Characterization of an Intermediate in the Folding of the Measles Virus X Domain.

Although most proteins fold by populating intermediates, the transient nature of such states makes it difficult to characterize their structures. In this work we identified and characterized the structure of an intermediate of the X domain of phosphoprotein (P) of measles virus. We obtained this result by a combination of equilibrium and kinetic measurements and NMR chemical shifts used as structural restraints in replica-averaged metadynamics simulations. The structure of the intermediate was then validated by rationally designing four mutational variants predicted to affect the stability of this state. These results provide a detailed view of an intermediate state and illustrate the opportunities offered by a synergistic use of experimental and computational methods to describe non-native states at atomic resolution.

Understanding the frustration arising from the competition between function, misfolding, and aggregation in a globular protein.

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.

Structure of the transition state for the binding of c-Myb and KIX highlights an unexpected order for a disordered system.

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.

Folding pathways of proteins with increasing degree of sequence identities but different structure and function.

Much experimental work has been devoted in comparing the folding behavior of proteins sharing the same fold but different sequence. The recent design of proteins displaying very high sequence identities but different 3D structure allows the unique opportunity to address the protein-folding problem from a complementary perspective. Here we explored by Φ-value analysis the pathways of folding of three different heteromorphic pairs, displaying increasingly high-sequence identity (namely, 30%, 77%, and 88%), but different structures called G(A) (a 3-α helix fold) and G(B) (an α/ß fold). The analysis, based on 132 site-directed mutants, is fully consistent with the idea that protein topology is committed very early along the pathway of folding. Furthermore, data reveals that when folding approaches a perfect two-state scenario, as in the case of the G(A) domains, the structural features of the transition state appear very robust to changes in sequence composition. On the other hand, when folding is more complex and multistate, as for the G(B)s, there are alternative nuclei or accessible pathways that can be alternatively stabilized by altering the primary structure. The implications of our results in the light of previous work on the folding of different members belonging to the same protein family are discussed.

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin.

The acknowledged success of the Monod-Wyman-Changeux (MWC) allosteric model stems from its efficacy in accounting for the functional behavior of many complex proteins starting with hemoglobin (the paradigmatic case) and extending to channels and receptors. The kinetic aspects of the allosteric model, however, have been often neglected, with the exception of hemoglobin and a few other proteins where conformational relaxations can be triggered by a short and intense laser pulse, and monitored by time-resolved optical spectroscopy. Only recently the application of time-resolved wide-angle X-ray scattering (TR-WAXS), a direct structurally sensitive technique, unveiled the time scale of hemoglobin quaternary structural transition. In order to test the generality of the MWC kinetic model, we carried out a TR-WAXS investigation in parallel on adult human hemoglobin and on a recombinant protein (HbYQ) carrying two mutations at the active site [Leu(B10)Tyr and His(E7)Gln]. HbYQ seemed an ideal test because, although exhibiting allosteric properties, its kinetic and structural properties are different from adult human hemoglobin. The structural dynamics of HbYQ unveiled by TR-WAXS can be quantitatively accounted for by the MWC kinetic model. Interestingly, the main structural change associated with the R-T allosteric transition (i.e., the relative rotation and translation of the dimers) is approximately 10-fold slower in HbYQ, and the drop in the allosteric transition rate with ligand saturation is steeper. Our results extend the general validity of the MWC kinetic model and reveal peculiar thermodynamic properties of HbYQ. A possible structural interpretation of the characteristic kinetic behavior of HbYQ is also discussed.

Frustration Sculpts the Early Stages of Protein Folding.

The funneled energy landscape theory implies that protein structures are minimally frustrated. Yet, because of the divergent demands between folding and function, regions of frustrated patterns are present at the active site of proteins. To understand the effects of such local frustration in dictating the energy landscape of proteins, here we compare the folding mechanisms of the two alternative spliced forms of a PDZ domain (PDZ2 and PDZ2as) that share a nearly identical sequence and structure, while displaying different frustration patterns. The analysis, based on the kinetic characterization of a large number of site-directed mutants, reveals that although the late stages for folding are very robust and biased by native topology, the early stages are more malleable and dominated by local frustration. The results are briefly discussed in the context of the energy-landscape theory.

The kinetics of folding of frataxin.

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.

The Mechanism of Folding of Human Frataxin in Comparison to the Yeast Homologue - Broad Energy Barriers and the General Properties of the Transition State.

The funneled energy landscape theory suggests that the folding pathway of homologous proteins should converge at the late stages of folding. In this respect, proteins displaying a broad energy landscape for folding are particularly instructive, allowing inferring both the early, intermediate and late stages of folding. In this paper we explore the folding mechanisms of human frataxin, an essential mitochondrial protein linked to the neurodegenerative disorder Friedreich's ataxia. Building upon previous studies on the yeast homologue, the folding pathway of human frataxin is thoroughly examined, revealing a mechanism implying the presence of a broad energy barrier, reminiscent of the yeast counterpart. Through an extensive site-directed mutagenesis, we employed a Φ -value analysis to map native-like contacts in the folding transition state. The presence of a broad energy barrier facilitated the exploration of such contacts in both early and late folding events. We compared results from yeast and human frataxin providing insights into the impact of native topology on the folding mechanism and elucidating the properties of the underlying free energy landscape. The findings are discussed in the context of the funneled energy landscape theory of protein folding.

Structure of nucleophosmin DNA-binding domain and analysis of its complex with a G-quadruplex sequence from the c-MYC promoter.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a key role in several cellular functions, including ribosome maturation and export, centrosome duplication, and response to stress stimuli. More than 50 mutations at the terminal exon of the NPM1 gene have been identified so far in acute myeloid leukemia; the mutated proteins are aberrantly and stably localized in the cytoplasm due to high destabilization of the NPM1 C-terminal domain and the appearance of a new nuclear export signal. We have shown previously that the 70-residue NPM1 C-terminal domain (NPM1-C70) is able to bind with high affinity a specific region at the c-MYC gene promoter characterized by parallel G-quadruplex structure. Here we present the solution structure of the NPM1-C70 domain and NMR analysis of its interaction with a c-MYC-derived G-quadruplex. These data were used to calculate an experimentally restrained molecular docking model for the complex. The NPM1-C70 terminal three-helix bundle binds the G-quadruplex DNA at the interface between helices H1 and H2 through electrostatic interactions with the G-quadruplex phosphate backbone. Furthermore, we show that the 17-residue lysine-rich sequence at the N terminus of the three-helix bundle is disordered and, although necessary, does not participate directly in the contact surface in the complex.

The folding pathway of a functionally competent C-terminal domain of nucleophosmin: protein stability and denatured state residual structure.

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 than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was 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.

Deciphering the folding transition state structure and denatured state properties of nucleophosmin C-terminal domain.

Nucleophosmin (NPM1), one of the most abundant nucleolar proteins, is a frequent target of oncogenic mutations in acute myeloid leukaemia (AML). Mutation-induced changes at the C-terminal domain of NPM1 (Cter-NPM1) compromise its stability and cause the aberrant translocation of NPM1 to the cytosol. Hence, this protein represents a suitable candidate to investigate the relations between folding and disease. Since Cter-NPM1 folds via a compact denatured state, stabilization of the folded state of the mutated variants demands detailed structural information on both the native and denatured states. Here, we present the characterization of the complete folding pathway of Cter-NPM1 and provide molecular details for both the transition and the denatured states. The structure of the transition state was assessed by Phi-value analysis, whereas residual structure in the denatured state was mapped by evaluating the effect of mutations as modulated by conditions promoting denatured state compaction. Data reveal that folding of Cter-NPM1 proceeds via an extended nucleus and that the denatured state retains significant malleable structure at the interface between the second and third helices. Our observations constitute the essential prerequisite for structure-based drug-design studies, aimed at identifying molecules that may rescue pathological NPM1 mutants by stabilizing the native-like state.

Modulation of Allosteric Control and Evolution of Hemoglobin.

Allostery arises when a ligand-induced change in shape of a binding site of a protein is coupled to a tertiary/quaternary conformational change with a consequent modulation of functional properties. The two-state allosteric model of Monod, Wyman and Changeux [J. Mol. Biol. 1965; 12, 88-118] is an elegant and effective theory to account for protein regulation and control. Tetrameric hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the ideal system to test for the validity of the MWC theory. The small ligands affecting Hb's behavior (organic phosphates, protons, bicarbonate) are produced by the red blood cell during metabolism. By binding to specific sites, these messengers make Hb sensing the environment and reacting consequently. HbI and HbIV from trout and human HbA are classical cooperative models, being similar yet different. They share many fundamental features, starting with the globin fold and the quaternary assembly, and reversible cooperative O2 binding. Nevertheless, they differ in ligand affinity, binding of allosteric effectors, and stability of the quaternary assembly. Here, we recollect essential functional properties and correlate them to the tertiary and quaternary structures available in the protein databank to infer on the molecular basis of the evolution of oxygen transporters.

Sequence-specific long range networks in PSD-95/discs large/ZO-1 (PDZ) domains tune their binding selectivity.

Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.

The denatured state dictates the topology of two proteins with almost identical sequence but different native structure and function.

The protein folding problem is often studied by comparing the mechanisms of proteins sharing the same structure but different sequence. The recent design of the two proteins G(A)88 and G(B)88, displaying different structures and functions while sharing 88% sequence identity (49 out of 56 amino acids), allows the unique opportunity for a complementary approach. At which stage of its folding pathway does a protein commit to a given topology? Which residues are crucial in directing folding mechanisms to a given structure? By using a combination of biophysical and computational techniques, we have characterized the folding of both G(A)88 and G(B)88. We show that, contrary to expectation, G(B)88, characterized by a native α+ß fold, displays in the denatured state a content of native-like helical structure greater than G(A)88, which is all-α in its native state. Both experiments and simulations indicate that such residual structure may be tuned by changing pH. Thus, despite the high sequence identity, the folding pathways for these two proteins appear to diverge as early as in the denatured state. Our results suggest a mechanism whereby protein topology is committed very early along the folding pathway, being imprinted in the residual structure of the denatured state.

Hemoglobin allostery: variations on the theme.

The two-state allosteric model of Monod, Wyman and Changeux (1965) offers a simple and elegant, yet very powerful and comprehensive, description of the functional behavior of hemoglobin. Although the extensive body of structural and functional information available is by-and-large consistent with this conceptual framework, some discrepancies between theory and experiment have been extensively discussed and considered to demand modifications of the original hypothesis. More recently the role of tertiary structural changes has been re-analyzed leading to extended kinetic models or indicating that powerful heterotropic effectors may be of paramount importance in controlling the function of human hemoglobin. The aim of this review is to analyze, and possibly reconcile, some discrepancies. We always felt that by looking at hemoglobins other than human HbA, the relative role of tertiary and quaternary allosteric effects may be better understood. The model systems illustrated below are the different hemoglobins from trout's blood, since they are characterized by the most striking variability of heterotropic effects, ranging from totally absent to very extreme with dominant contributions of tertiary effects. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.