Angelo Toto

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

Significance A considerable fraction of proteins, although functional, does not display a well-ordered native state, posing the structure–function dogma into question. Although different models have been described, the role of protein disorder is still shrouded in mystery. Here, we investigate the mechanism of recognition between an intrinsically unstructured protein and its partner. Unexpectedly, we found the binding to occur with a very high degree of geometrical precision, suggesting that a potential value of disorder is not, in this case, to be searched in the speeding up of the reaction thanks to an increased capture radius. Given that in the cell disordered polypeptides may be more vulnerable than folded domains, the advantage in being natively unfolded remains a conundrum. 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.

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

Significance The amino acid sequence of a protein encodes a wide range of different properties, including function, folding, and avoidance of aggregation. The resulting requirements on the sequence may be in conflict with each other, thus creating the possibility of misfolding and dysfunction. To investigate possible mechanisms whereby such unwanted outcomes can be prevented, we study the folding process of frataxin, a protein whose aberrant folding is associated with Friedreich’s ataxia. Our results indicate that the regions prone to misfolding are highly protected against aggregation along the folding pathway of this 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.

Molecular Recognition by Templated Folding of an Intrinsically Disordered Protein.

Intrinsically disordered proteins often become structured upon interacting with their partners. The mechanism of this ‘folding upon binding’ process, however, has not been fully characterised yet. Here we present a study of the folding of the intrinsically disordered transactivation domain of c-Myb (c-Myb) upon binding its partner KIX. By determining the structure of the folding transition state for the binding of wild-type and three mutational variants of KIX, we found a remarkable plasticity of the folding pathway of c-Myb. To explain this phenomenon, we show that the folding of c-Myb is templated by the structure of KIX. This adaptive folding behaviour, which occurs by heterogeneous nucleation, differs from the robust homogeneous nucleation typically observed for globular proteins. We suggest that this templated folding mechanism may enable intrinsically disordered proteins to achieve specific and reliable binding with multiple partners while avoiding aberrant interactions.

The mechanism of binding of the KIX domain to the mixed lineage leukemia protein and its allosteric role in the recognition of c-Myb

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.

Mutational Analysis of the Binding-Induced Folding Reaction of the Mixed-Lineage Leukemia Protein to the KIX Domain

Intrinsically disordered proteins represent a large class of proteins that lack a well-defined three-dimensional structure in isolation but can undergo a disorder to order transition upon binding to their physiological ligands. Understanding the mechanism by which these proteins fold upon binding represents a challenge. Here we present a detailed mutational study of the kinetics of the binding reaction between the transactivation domain of the mixed-lineage leukemia protein, an intrinsically disordered protein, and the KIX domain, performed under different experimental conditions. The experimental data allow us to infer the mechanism of folding upon binding and to pinpoint the key interactions present in the transition state. Furthermore, we identify a peculiar malleability of the observed mechanism upon changes in reaction conditions. This finding, which is in opposition to the robustness typically observed in the folding of globular proteins, is discussed in the context of previous work on intrinsically disordered proteins.

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 Friedreichs 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 binding of the second PDZ domain from the Protein Tyrosine Phosphatase-BL to the Adenomatous Polyposis Coli tumor suppressor

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.

Regulation of the Human Phosphatase PTPN4 by the inter-domain linker connecting the PDZ and the phosphatase domains

Human protein tyrosine phosphatase non-receptor type 4 (PTPN4) has been shown to prevent cell death. The active form of human PTPN4 consists of two globular domains, a PDZ (PSD-95/Dlg/ZO-1) domain and a phosphatase domain, tethered by a flexible linker. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We previously demonstrated that the PDZ domain inhibits the phosphatase activity of PTPN4 and that the mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We demonstrate here that the linker connecting the PDZ domain and the phosphatase domain is involved in the regulation of the phosphatase activity in both PDZ-related inhibition and PDZ ligand-related activation events. We combined bioinformatics and kinetic studies to decipher the role of the linker in the PTPN4 activity. By comparing orthologous sequences, we identified a conserved patch of hydrophobic residues in the linker. We showed that mutations in this patch affect the regulation of the PTPN4 bidomain indicating that the PDZ-PDZ ligand regulation of PTPN4 is a linker-mediated mechanism. However, the mutations do not alter the binding of the PDZ ligand. This study strengthens the notion that inter-domain linker can be of functional importance in enzyme regulation of large multi-domain proteins.

Understanding the role of phosphorylation in the binding mechanism of a PDZ domain

The PDZ domain is one of the most common protein–protein interaction domains in mammalian species. While several studies have demonstrated the importance of phosphorylation in interactions involving PDZ domains, there is a paucity of detailed mechanistic data addressing how the PDZ interaction is affected by phosphorylation. Here, we address this question by equilibrium and kinetic binding experiments using PDZ2 from protein tyrosine phosphatase L1 and its interaction with a peptide from the natural ligand RIL. The results show that phosphorylation of a serine residue in the RIL peptide has dual and opposing effects: it increases both the association and dissociation rate constants, which leads to an overall weakening of binding. Furthermore, we performed binding experiments with a RIL peptide in which the serine was replaced by a glutamate, a commonly used method to mimic phosphorylation in proteins. Strikingly, both the affinity and the ionic strength dependence of the affinity differed markedly for the phosphoserine and glutamate peptides. These results show that, in this particular case, glutamate is a poor mimic of serine phosphorylation.

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.