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.

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.

Eraldo Antonini Lectures, 1983-2019.

"Can order spring from Chaos?" is the title of an extensive Report on Italian science published by NATURE on 12 May 1983 and written by Robert Walgate, the Chief European Correspondent. It is a twenty pages complete paper touching all aspects of the struggle of Italian scientists to work in the "curious amalgam of ingenuity and muddle, a reflection of the political system". (Nature, 1983; 303: 109-128). To read it after four decades is interesting but somewhat depressing since the main problems unfolded in the paper have not been solved, starting with the largely insufficient support of fundamental curiosity driven research. At page 114 you could find a item called: ITALY's TOP SCIENTISTS: Four in the top one thousand. The Author refers to the data reported by the ISI (Institute of Scientific Information) that took two years to scan 3,000 major journals over the period 1965-78 and covered 5 millions articles and 67 millions references. The four top Italian scientists working in Italy were: Eraldo Antonini (3127 citations), Enrico Clementi (4001), Silvio Garattini (2833), and Giorgio Giacomelli (2483); 3 out of four were 52 years old, and one 55. Antonini did not see the Report since he passed away on March 18, 1983. However the information leaked before the publication of Nature because I remember the Messaggero of Rome reporting a whole page with the ranking of the four Italians, and even a picture of Eraldo. The students of the first year Medical course, his Class, welcomed the Professor with a standing ovation. After a short time the Board of the SIB (Società Italiana di Biochimica) casted a unanimous vote in favour of the motion of President Noris Siliprandi to begin the annual Congress with an Antonini Lecture, forever. As reported below, the tradition began immediately at the Congress in Saint-Vicent, Italy, and is continuing. In this paper I report an account of the Eraldo Antonini Lectures that I attended over the years and until September 2019, a few months before the pandemics lock down.

An Outlook on the Complexity of Protein Morphogenesis in Health and Disease.

The study of the mechanisms whereby proteins achieve their native functionally competent conformation has been a key issue in molecular biosciences over the last 6 decades. Nevertheless, there are several debated issues and open problems concerning some aspects of this fundamental problem. By considering the emerging complexity of the so-called "native state," we attempt hereby to propose a personal account on some of the key topics in the field, ranging from the relationships between misfolding and diseases to the significance of protein disorder. Finally, we briefly describe the recent and exciting advances in predicting protein structures from their amino acid sequence.

From Kuru to Alzheimer: A personal outlook.

Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein morphogenesis is a spontaneous reversible process leading from an ensemble of disordered structures to the ordered functionally competent protein; conforming to Aristotle's definition of substance, the synolon of matter and form. The overall process of folding is generally consistent with a two state transition between the native and the denatured protein: not only the denatured state is an ensemble of several structures, but also the native protein populates distinct functionally relevant conformational (sub)states. This two-state view should be revised, given that any globular protein can populate a peculiar third state called amyloid, characterized by an overall architecture that at variance with the native state, is by-and-large independent of the primary structure. In a nut shell, we should accept that beside the folded and unfolded states, any protein can populate a third state called amyloid which gained center stage being the hallmark of incurable neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases as well as others. These fatal diseases are characterized by clear-cut clinical differences, yet display some commonalities such as the presence in the brain of amyloid deposits constituted by one misfolded protein specific for each disease. Some aspects of this complex problem are summarized here as an excursus from the prion's fibrils observed in the brain of aborigines who died of Kuru to the amyloid detectable in the cortex of Alzheimer's patients.

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.

Control of Oxygen Affinity in Mammalian Hemoglobins: Implications for a System Biology Description of the Respiratory Properties of the Red Blood Cell.

Hemoglobin and myoglobin have been considered for a long time the paradigmatic model systems for protein function, to the point of being defined the "hydrogen atom[s] of biology". Given this privileged position and the huge amount of quantitative information available on these proteins, the red blood cell might appear as the model system and"hydrogen atom" of system biology. Indeed, since the red cell's main function is O2 transport by hemoglobin, the gap between the protein and the cell may appear quite small. Yet, a surprisingly large amount of detailed biochemical information is required for the modelization of the respiratory properties of the erythrocyte. This problem is compounded if modelization aims at uncovering or explaining evolutionarily selected functional properties of hemoglobin. The foremost difficulty lies in the fact that hemoglobins having different intrinsic properties and relatively ancient evolutionary divergence may behave similarly in the complex milieu of blood, whereas very similar hemoglobins sharing a substantial sequence similarity may present important functional differences because of the mutation of a few key residues. Thus, the functional properties of hemoglobin and blood may reflect more closely the recent environmental challenges than the remote evolutionary history of the animal. We summarize in this review the case of hemoglobins from mammals, in an attempt to provide a reasoned summary of their complexity that, we hope, may be of help to scientists interested in the quantitative exploration of the evolutionary physiology of respiration. Indeed the basis of a meaningful modelization of the red cell requires a large amount of information collected in painstaking and often forgotten studies of the biochemical properties of hemoglobin carried out over more than a century.

Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV-visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15-40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

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.

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.

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.

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.

Identification of a novel nucleophosmin-interaction motif in the tumor suppressor p14arf.

The tumor suppressor p14arf interacts, in response to oncogenic signals, with the p53 E3-ubiquitin ligase HDM2, thereby resulting in p53 stabilization and activation. In addition, it also exerts tumor-suppressive functions in p53-independent contexts. The activities of p14arf are regulated by the nucleolar chaperone nucleophosmin (NPM1), which controls its levels and cellular localization. In acute myeloid leukemia with mutations in the NPM1 gene, mutated NPM1 aberrantly translocates in the cytosol carrying with itself p14arf that is subsequently degraded, thus impairing the p14arf-HDM2-p53 axis. In this work we investigated the complex between these two proteins by means of NMR and other techniques. We identified a novel NPM1-interacting motif in the C-terminal region of p14arf, which corresponds to its predicted nucleolar localization signal. This motif recognizes a specific region of the NPM1 N-terminal domain and, upon binding, the two proteins form soluble high molecular weight complexes. By NMR, we identified critical residues on both proteins involved in the interaction. Collectively, our data provide a structural framework to rationalize the overall assembly of the p14arf-NPM1 supramolecular complexes. A number of p14arf cancer-associated mutations cluster in this motif and their effect on the interaction with NPM1 was also analyzed.

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".

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.

Neuroglobin: From structure to function in health and disease.

In 2000, the third member of the globin family was discovered in human and mouse brain and named neuroglobin (Ngb). Ngb is a monomeric 3/3 globin structurally similar to myoglobin and to the α- and ß-chains of hemoglobin, however it displays a bis-histidyl six-coordinate heme-Fe atom. Therefore, ligand binding to the Ngb metal center is limited from the dissociation of the distal His(E7)64-Fe bond. From its discovery, more than 500 papers on Ngb structure, expression, reactivity, and localization have been published to highlight its biochemical properties and its role(s) in health and disease. In vivo experiments have shown that increased levels of Ngb significantly protect both heart and brain from hypoxic/ischemic and oxidative stress-related insults, whereas decreased Ngb levels lead to an exacerbation of tissue injuries. Although some contradictory data emerged, human Ngb overexpression has been hypothesized to protect neurons from mitochondrial dysfunctions and neurodegenerative disorders such as Alzheimer's disease, and to play a shielding role in cancer cells. Recently, the recognition of Ngb interactors and inducers enlarges the functions of this stress-inducible globin, opening new therapeutic approaches to prevent neuronal cell death. Here, structural and functional aspects of human Ngb are examined critically to highlight its roles in health and disease.

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.

Molecular medicine - To be or not to be.

Molecular medicine is founded on the synergy between Chemistry, Physics, Biology and Medicine, with the ambitious goal of tackling diseases from a molecular perspective. This Review aims at retracing a personal outlook of the birth and development of molecular medicine, as well as at highlighting some of the most urgent challenges linked to aging and represented by incurable neurodegenerative diseases caused by protein misfolding. Furthermore, we emphasize the emerging role of the retromer dysfunctions and improper protein sorting in Alzheimer's disease and other important neurological disordered.