Angela Morrone

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.

A folding-after-binding mechanism describes the recognition between the transactivation domain of c-Myb and the KIX domain of the CREB-binding protein

A large body of evidence suggests that a considerable fraction of the human proteome may be at least in part intrinsically unstructured. While disordered, intrinsically unstructured proteins are nevertheless functional and mediate many interactions. Despite their significant role in regulation, however, little is known about the molecular mechanism whereby intrinsically unstructured proteins exert their function. This basic problem is critical to establish the role, if any, of disorder in cellular systems. Here we present kinetic experiments supporting a mechanism of binding-induced-folding when the KIX domain of the CREB-binding protein binds the transactivation domain of c-Myb, an intrinsically unstructured domain. The high-resolution structure of this physiologically important complex was previously determined by NMR spectroscopy. Our data reveal that c-Myb recognizes KIX 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. On the basis of the pH dependence of the observed combination and dissociation rate constants we propose a plausible mechanism for complex formation. The implications of our results in the light of previous work on intrinsically unstructured systems are discussed.

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 GA88 and GB88, 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 GA88 and GB88. We show that, contrary to expectation, GB88, characterized by a native α+β fold, displays in the denatured state a content of native-like helical structure greater than GA88, 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.

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.

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.

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 GA (a 3-α helix fold) and GB (an α/β 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 GA 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 GBs, 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.

GB1 Is Not a Two-State Folder: Identification and Characterization of an On-Pathway Intermediate

The folding pathway of the small α/β protein GB1 has been extensively studied during the past two decades using both theoretical and experimental approaches. These studies provided a consensus view that the protein folds in a two-state manner. Here, we reassessed the folding of GB1, both by experiments and simulations, and detected the presence of an on-pathway intermediate. This intermediate has eluded earlier experimental characterization and is distinct from the collapsed state previously identified using ultrarapid mixing. Failure to identify the presence of an intermediate affects some of the conclusions that have been drawn for GB1, a popular model for protein folding studies.

Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain

Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.

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.

Morphogenesis of a protein: folding pathways and the energy landscape.

Current knowledge on the reaction whereby a protein acquires its native three-dimensional structure was obtained by and large through characterization of the folding mechanism of simple systems. Given the multiplicity of amino acid sequences and unique folds, it is not so easy, however, to draw general rules by comparing folding pathways of different proteins. In fact, quantitative comparison may be jeopardized not only because of the vast repertoire of sequences but also in view of a multiplicity of structures of the native and denatured states. We have tackled the problem of the relationships between the sequence information and the folding pathway of a protein, using a combination of kinetics, protein engineering and computational methods, applied to relatively simple systems. Our strategy has been to investigate the folding mechanism determinants using two complementary approaches, i.e. (i) the study of members of the same family characterized by a common fold, but substantial differences in amino acid sequence, or (ii) heteromorphic pairs characterized by largely identical sequences but with different folds. We discuss some recent data on protein-folding mechanisms by presenting experiments on different members of the PDZ domain family and their circularly permuted variants. Characterization of the energetics and structures of intermediates and TSs (transition states), obtained by Φ-value analysis and restrained MD (molecular dynamics) simulations, provides a glimpse of the malleability of the dynamic states and of the role of the topology of the native states and of the denatured states in dictating folding and misfolding pathways.