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.

[Risk assessment and its appropriateness supporting the physician in the certification of a suspected occupational disease.] / La valutazione dei rischi e la sua appropriatezza a supporto del medico nella certificazione di sospetta tecnopatia.

OBJECTIVES: The topic of appropriateness was defined as the new frontier of development of health interventions. RH Brook in an Editorial published in the BMJ in 1994 defines the appropriateness "… an intervention for which the expected benefits are greater (with a sufficient margin) the possible negative consequences … excluding economic considerations". In workplaces the goal of appropriateness should cover not only the actions of health surveillance and health protocols, but also and primarily all stages of the risk assessment process. METHODS: Only through an assessment of occupational risks that aims to identify, evaluate and measure the real professional risk factors in specific working environments, it is possible to meet the requirements of effectiveness, efficiency and protection of ethical principles in the identification of priorities (according to values of person, community and society) that represent the size of the appropriateness of an intervention. At the same time, the risk assessment should provide to the occupational pyisician instruments to study with scientific, justifiable and reproducible criteria the possible correlation between damage to the worker's health and the occupational risk factors. RESULTS: In the process of reporting of suspected technopathy, both for prevention purposes (according to art. 139 of Presidential Decree 1165/68, art. 10 of Legislative Decree 38/00 and DM 10 June 2014), both for insurance purposes (according to art. 53 of Presidential Decree 1165/68 and Ministerial Decree 9 April 2008), it should be considered two indispensable judgment elements to study the correlation between the disease and the work: on the one hand the level (measured or estimated) of the occupational risk factors; on the other hand the appropriateness of the risks assessment compared to the best and most current scientific evidence (Evidence Based Medicine - EBM), according to technical standards and specific guidelines. CONCLUSIONS: Our study underlines the importance of appropriateness in the risk assessment process to analyze the correlation between the exposure to specific occupational hazards and the suspected technopathy.

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 validity of proofs in demonstrating risk and in research into the causal connection of occupational diseases]. / La validità delle prove nella dimostrazione del rischio e nella ricerca del nesso causale delle malattie professionali.

The verification of the occupational origin of a disease is a forensic medical activity requiring: the confirmation of the existence and of the exact nosographic identification of the disease, as well as the type of work really performed, and the actual exposure to an effective occupational hazard during an adequate time, and finally a reconstruction of the causal relationship based on objective data. Checking the disease is essentially documentary, corroborated by direct survey by medical examination. The assessment of exposure to the occupational hazard must be scrupulous also, not being acceptable the medical history alone: that is, it does require documentary evidence. Finally, the logical process of recognition of causation requires the application of rigorous forensic medical methodology, with references to current scientific knowledge, and the application of legal criteriology from the legal field of law in which you are moving. Indeed, forensic medical methodology is not the same of epidemiological one: probability of occurrence of an event is not a proof, but only a circumstantial element. A forensic medical doctor organizes every evidence and circumstantial evidence in a unique decision-making process, as a result of a logical process, and probabilistic data can be among circumstantial evidence, but they must suit the case in details, in order to reach the so called "logic probability". But this doesn't mean that you have "proven" the occupational origin of a disease. In the "demonstration" of a fact you use the same forensic medical methodology (thus referring to classic criteria: temporality, biological gradient and plausibility, topographical, exclusion, and phenomenal continuity if suitable, too), and also the same general scientific references, nevertheless the law can be different in causality principles admitted (the principles governing the causal link are the same in Criminal Code and Civil law both, but they differ in private insurance), and besides there is a different rule of evidence, that is the "quantum of evidence" required (the amount of evidence needed) and, secondarily, also a different quality of proof required (how reliable such evidence should be considered, and which types of evidence admitted). Therefore in Civil law you can reach a "procedural truth" different from the one in Criminal law, and various from the one needed to prevail in litigation with social security insurance or with private insurance.

Understanding Intramolecular Crosstalk in an Intrinsically Disordered Protein.

The interaction between NTAIL and XD from the measles virus represents a paradigmatic example of molecular recognition between an intrinsically disordered protein and a folded partner. By binding to XD, a small portion of NTAIL (classically denoted as MoRE) undergoes a disorder-to-order transition, populating an α-helical structure, while the reminder of the protein remains disordered. Here, we demonstrate an unexpected crosstalk between such a disordered region and the adjacent molecular recognition element (MoRE). This result was obtained by producing a series of truncation and site-directed variants of NTAIL while measuring the effects on the kinetics of folding and binding. We show that the disordered region of NTAIL exerts its inhibitory role by slowing the folding step of the MoRE, thereby tuning the affinity of the interaction.

Mapping the allosteric network within a SH3 domain.

SH3 domains are very abundant protein-protein interactions modules, involved in the regulation of several cellular processes. Whilst they have been associated to allosteric communication pathways between contiguous domains in multi-domain proteins, there is lack of information regarding the intra-domain allosteric cross-talk within the SH3 moiety. Here we scrutinize the presence of an allosteric network in the C-terminal SH3 domain of Grb2 protein, upon binding the Grb2-associated binding 2 protein. To explore allostery, we performed double mutant cycle analysis, a powerful quantitative approach based on mutagenesis in conjunction with kinetic experiments. Data reveal the presence of an unexpected allosteric sparse network that modulates the affinity between the SH3 domain and its physiological partner.

Mechanism of Folding and Binding of the N-Terminal SH2 Domain from SHP2.

SHP2 is a phosphatase protein, involved in many cellular pathways, comprising two SH2 domains (namely N-SH2 and C-SH2) and a phosphatase domain. Among others, the interaction between SHP2 and Gab2 (Grb2 associated binder) is critical in cell death and differentiation. SHP2 binds to Gab2 through its SH2 domains, which recognize specific regions of Gab2 characterized by the presence of a phosphorylated tyrosine. In order to shed light on the dynamic and functional properties of this protein-protein interaction, we studied the mechanism of folding of N-SH2 and the binding process to a peptide mimicking a region of Gab2. The data presented represent the first description by stopped-flow of the kinetics of binding of an SH2 domain in solution. By performing experiments at different ionic strengths, we elucidate the electrostatic nature of the interaction, highlighting a key role of the negative charge of the phosphotyrosine in the recognition event of the reaction. Furthermore, by analyzing the equilibrium and kinetics of folding of N-SH2 folding we demonstrate the presence of an intermediate along the folding pathway. These results are discussed in the light of previous works on another SH2 domain.

Folding Mechanism of the SH3 Domain from Grb2.

SH3 domains are small protein modules involved in the regulation of important cellular pathways. These domains mediate protein-protein interactions recognizing motifs rich in proline on the target protein. The SH3 domain from Grb2 (Grb2-SH3) presents the typical structure of an SH3 domain composed of two three-stranded antiparallel ß-sheets orthogonally packed onto each other, to form a single hydrophobic core. Grb2 interacts, via SH3 domain, with Gab2, a scaffolding disordered protein, triggering some key metabolic pathways involved in cell death and differentiation. In this work we report a mutational analysis (Φ value analysis) of the folding pathway of Grb2-SH3 that, coupled with molecular dynamic simulations, allows us to assess the structure of the transition state and the mechanism of folding of this domain. Data suggest that Grb2-SH3 folds via a native-like, diffused transition state with a concurrent formation of native-like secondary and tertiary structure (nucleation-condensation mechanism) and without the accumulation of folding intermediates. The comparison between our data and previous folding studies on SH3 domains belonging to other proteins highlights that proteins of this class may fold via alternative pathways, stabilized by different nuclei leading or not to accumulation of folding intermediates. This comparative analysis suggests that the alternative folding pathways for this class of SH3 domains can be selectively regulated by the specific amino acid sequences.

ß-catenin knockdown promotes NHERF1-mediated survival of colorectal cancer cells: implications for a double-targeted therapy.

Nuclear activated ß-catenin plays a causative role in colorectal cancers (CRC) but remains an elusive therapeutic target. Using human CRC cells harboring different Wnt/ß-catenin pathway mutations in APC/KRAS or ß-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic ß-catenin signaling negatively regulates the expression of NHERF1 (Na+/H+ exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear ß-catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that ß-catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon ß-catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single ß-catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/ß-catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with ß-catenin knockdown alone. Collectively, our data unveil novel ß-catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining ß-catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/ß-catenin-targeted therapeutics.

Understanding the mechanism of binding between Gab2 and the C terminal SH3 domain from Grb2.

Gab2 is a large disordered protein that regulates several cellular signalling pathways and is overexpressed in different forms of cancer. Because of its disordered nature, a detailed characterization of the mechanisms of recognition between Gab2 and its physiological partners is particularly difficult. Here we provide a detailed kinetic characterization of the binding reaction between Gab2 and the C-terminal SH3 domain of the growth factor receptor-bound protein 2 (Grb2). We demonstrate that Gab2 folds upon binding following an induced fit type mechanism, whereby recognition is characterized by the formation of an intermediate, in which Gab2 is primarily disordered. In this scenario, folding of Gab2 into the bound conformation occurs only after binding. However, an alanine scanning of the proline residues of Gab2 suggests that the intermediate contains some degree of native-like structure, which might play a role for the recognition event to take place. The results, which represent a fundamental step forward in the understanding of this functional protein-protein interaction, are discussed on the light of previous structural works on these proteins.

Unveiling the folding mechanism of the Bromodomains.

Bromodomains (BRDs) are small protein domains often present in large multidomain proteins involved in transcriptional regulation in eukaryotic cells. They currently represent valuable targets for the development of inhibitors of aberrant transcriptional processes in a variety of human diseases. Here we report urea-induced equilibrium unfolding experiments monitored by circular dichroism (CD) and fluorescence on two structurally similar BRDs: BRD2(2) and BRD4(1), showing that BRD4(1) is more stable than BRD2(2). Moreover, we report a description of their kinetic folding mechanism, as obtained by careful analysis of stopped-flow and temperature-jump data. The presence of a high energy intermediate for both proteins, suggested by the non-linear dependence of the folding rate on denaturant concentration in the millisec time regime, has been experimentally observed by temperature-jump experiments. Quantitative global analysis of all the rate constants obtained over a wide range of urea concentrations, allowed us to propose a common, three-state, folding mechanism for these two BRDs. Interestingly, the intermediate of BRD4(1) appears to be more stable and structurally native-like than that populated by BRD2(2). Our results underscore the role played by structural topology and sequence in determining and tuning the folding mechanism.

The Folding Pathway of the KIX Domain.

The KIX domain is an 89-residues globular domain with an important role in mediating protein-protein interactions. The presence of two distinct binding sites in such a small domain makes KIX a suitable candidate to investigate the effect of the potentially divergent demands between folding and function. Here, we report an extensive mutational analysis of the folding pathway of the KIX domain, based on 30 site-directed mutants, which allow us to assess the structures of both the transition and denatured states. Data reveal that, while the transition state presents mostly native-like interactions, the denatured state is somewhat misfolded. We mapped some of the non-native contacts in the denatured state using a second round of mutagenesis, based on double mutant cycles on 15 double mutants. Interestingly, such a misfolding arises from non-native interactions involving the residues critical for the function of the protein. The results described in this work appear to highlight the diverging demands between folding and function that may lead to misfolding, which may be observed in the early stages of folding.

Towards a structural biology of the hydrophobic effect in protein folding.

The hydrophobic effect is a major driving force in protein folding. A complete understanding of this effect requires the description of the conformational states of water and protein molecules at different temperatures. Towards this goal, we characterise the cold and hot denatured states of a protein by modelling NMR chemical shifts using restrained molecular dynamics simulations. A detailed analysis of the resulting structures reveals that water molecules in the bulk and at the protein interface form on average the same number of hydrogen bonds. Thus, even if proteins are 'large' particles (in terms of the hydrophobic effect, i.e. larger than 1 nm), because of the presence of complex surface patterns of polar and non-polar residues their behaviour can be compared to that of 'small' particles (i.e. smaller than 1 nm). We thus find that the hot denatured state is more compact and richer in secondary structure than the cold denatured state, since water at lower temperatures can form more hydrogen bonds than at high temperatures. Then, using Φ-value analysis we show that the structural differences between the hot and cold denatured states result in two alternative folding mechanisms. These findings thus illustrate how the analysis of water-protein hydrogen bonds can reveal the molecular origins of protein behaviours associated with the hydrophobic effect.

Fuzzy regions in an intrinsically disordered protein impair protein-protein interactions.

Despite the partial disorder-to-order transition that intrinsically disordered proteins often undergo upon binding to their partners, a considerable amount of residual disorder may be retained in the bound form, resulting in a fuzzy complex. Fuzzy regions flanking molecular recognition elements may enable partner fishing through non-specific, transient contacts, thereby facilitating binding, but may also disfavor binding through various mechanisms. So far, few computational or experimental studies have addressed the effect of fuzzy appendages on partner recognition by intrinsically disordered proteins. In order to shed light onto this issue, we used the interaction between the intrinsically disordered C-terminal domain of the measles virus (MeV) nucleoprotein (NTAIL ) and the X domain (XD) of the viral phosphoprotein as model system. After binding to XD, the N-terminal region of NTAIL remains conspicuously disordered, with α-helical folding taking place only within a short molecular recognition element. To study the effect of the N-terminal fuzzy region on NTAIL /XD binding, we generated N-terminal truncation variants of NTAIL , and assessed their binding abilities towards XD. The results revealed that binding increases with shortening of the N-terminal fuzzy region, with this also being observed with hsp70 (another MeV NTAIL binding partner), and for the homologous NTAIL /XD pairs from the Nipah and Hendra viruses. Finally, similar results were obtained when the MeV NTAIL fuzzy region was replaced with a highly dissimilar artificial disordered sequence, supporting a sequence-independent inhibitory effect of the fuzzy region.

Understanding the effect of alternative splicing in the folding and function of the second PDZ from protein tyrosine phosphatase-BL.

PDZ domains are the most prominent biological structural domains involved in protein-protein interactions in the human cell. The second PDZ domain of the protein tyrosine phosphatase BL (PDZ2) interacts and binds the C-termini of the tumour suppressor protein APC and of the LIM domain-containing protein RIL. One isoform of PDZ2 (PDZ2as) involves an alternative spliced form that exhibits an insertion of 5 residues in a loop. PDZ2as abrogates binding to its partners, even if the insertion is directly located in its binding pocket. Here, we investigate the folding and function of PDZ2as, in comparison to the previously characterized PDZ2 domain. Data reveal that, whilst the thermodynamic stability of PDZ2as appears as nearly identical to that of PDZ2, the insertion of 5 amino acids induces formation of some weak transient non-native interactions in the folding transition state, as mirrored by a concomitant increase of both the folding and unfolding rate constants. From a functional perspective, we show that the decrease in affinity is caused by a pronounced decrease of the association rate constants (by nearly ten fold), with no effect on the microscopic dissociation rate constants. The results are briefly discussed in the context of previous work on PDZ domains.

Demonstration of a folding after binding mechanism in the recognition between the measles virus NTAIL 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 (NTAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that NTAIL 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.