Research Highlights

Simultaneous determination of protein structure and dynamics using cryo-electron microscopy. Biophysical Journal (2018).

Cryo-electron microscopy is rapidly emerging as a powerful technique to determine the structures of complex macromolecular systems elusive to other techniques. Since many of these systems are highly dynamical, characterising also their movements is a crucial step to unravel their biological functions. To achieve this goal, we report an integrative modelling approach to simultaneously determine structure and dynamics from cryo-electron microscopy density maps. By quantifying the level of noise in the data and dealing with their ensemble-averaged nature, this approach enables the integration of multiple sources of information to model ensembles of structures and infer their populations. We illustrate the method by characterising structure and dynamics of the integral membrane receptor STRA6, thus providing insights into the mechanisms by which it interacts with retinol binding protein and translocates retinol across the membrane.

Selective targeting of primary and secondary nucleation pathways in Abeta42 aggregation using a rational antibody scanning method. Science Advances (2017).

Antibodies targeting Abeta42 are under intense scrutiny because of their therapeutic potential for Alzheimer's disease. To enable systematic searches, we present an “antibody scanning” strategy for the generation of a panel of antibodies against Abeta42. Each antibody in the panel is rationally designed to target a specific linear epitope, with the selected epitopes scanning the Abeta42 sequence. By screening in vitro the panel to identify the specific microscopic steps in the Abeta42 aggregation process influenced by each antibody, we identify two antibodies that target specifically the primary and the secondary nucleation steps, which are key for the production of Abeta42 oligomers. These two antibodies act, respectively, to delay the onset of aggregation and to block the proliferation of aggregates, and correspondingly reduce the toxicity in a Caenorhabditis elegans model overexpressing Abeta42. These results illustrate how the antibody scanning method described here can be used to readily obtain very small antibody libraries with extensive coverage of the sequences of target proteins.

Systematic development of small molecules to inhibit specific microscopic steps of Abeta42 aggregation in Alzheimer's disease. Proc. Natl. Acad. Sci. USA (2017).

The aggregation of the 42-residue form of the amyloid-beta peptide (Abeta42) is a pivotal event in Alzheimer's disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Abeta42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Abeta42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Abeta42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.

A protein homeostasis signature in healthy brains recapitulates tissue vulnerability to Alzheimer's disease. Science Advances (2016).

In Alzheimer's disease, aggregates of Abeta and tau in amyloid plaques and neurofibrillary tangles spread progressively across brain tissues following a characteristic pattern, implying a tissue-specific vulnerability to the disease. We report a transcriptional analysis of healthy brains and identify an expression signature that predicts - at ages well before the typical onset - the tissue-specific progression of the disease. We obtain this result by finding a quantitative correlation between the histopathological staging of the disease and the expression patterns of the proteins that coaggregate in amyloid plaques and neurofibrillary tangles, together with those of the protein homeostasis components that regulate Abeta and tau. Because this expression signature is evident in healthy brains, our analysis provides an explanatory link between a tissue-specific environmental risk of protein aggregation and a corresponding vulnerability to Alzheimer's disease.

An anti-cancer drug suppresses the primary nucleation reaction that initiates the formation of toxic Abeta aggregates linked with Alzheimer's disease. Science Advances (2016).

The conversion of the Abeta peptide into pathogenic aggregates is linked to the onset and progression of Alzheimer's disease. Although this observation has prompted an extensive search for therapeutic agents to modulate the concentration of Abeta or inhibit its aggregation, all clinical trials with these objectives have so far failed, at least in part because of a lack of understanding of the molecular mechanisms underlying the process of aggregation and its inhibition. To address this problem we describe a chemical kinetics approach for rational drug discovery, in which the effects of small molecules on the rates of specific microscopic steps in Abeta aggregation are analysed quantitatively. By applying this approach we report that bexarotene, an FDA-approved anti-cancer drug, targets selectively the primary nucleation step in Abeta aggregation, delays the formation of toxic species in neuroblastoma cells and completely suppresses Abeta deposition and its consequences in a C. elegans model of Abeta-mediated toxicity. These results suggest that the prevention of the primary nucleation of Abeta by compounds such as bexarotene could potentially reduce the risk of onset of Alzheimer's disease, and more generally that our strategy provides a general framework for the rational identification of a range of candidate drugs directed against neurodegenerative disorders.

Metainference: A Bayesian inference method for heterogeneous systems. Science Advances (2016).

Modelling a complex system is almost invariably a challenging task. The incorporation of experimental observations can be used to improve the quality of a model, and thus to obtain better predictions about the behavior of the corresponding system. This approach, however, is affected by a variety of different errors, especially when a system populates simultaneously an ensemble of different states and experimental data are measured as averages over such states. To address this problem we present a Bayesian inference method, called 'metainference', that is able to deal with errors in experimental measurements as well as with experimental measurements averaged over multiple states. To achieve this goal, metainference models a finite sample of the distribution of models using a replica approach, in the spirit of the replica-averaging modelling based on the maximum entropy principle. To illustrate the method we present its application to a heterogeneous model system and to the determination of an ensemble of structures corresponding to the thermal fluctuations of a protein molecule. Metainference thus provides an approach to model complex systems with heterogeneous components and interconverting between different states by taking into account all possible sources of errors.

A rational design strategy for the selective activity enhancement of a molecular chaperone toward a target substrate. Biochemistry (2015)

Molecular chaperones facilitate the folding and assembly of proteins and inhibit their aberrant aggregation. They thus offer several opportunities for biomedical and biotechnological applications, as for example they can often prevent protein aggregation more effectively than other therapeutic molecules, including small molecules and antibodies. Here we present a method of designing molecular chaperones with enhanced activity against specific amyloidogenic substrates while leaving unaltered their functions toward other substrates. The method consists of grafting onto a molecular chaperone a peptide designed to bind specifically an epitope in the target substrate. We illustrate this strategy by describing Hsp70 variants with increased affinities for alpha-synuclein and Abeta42 but otherwise unaltered affinities for other substrates. These designed variants inhibit protein aggregation and disaggregate preformed fibrils significantly more effectively than wild-type Hsp70 indicating that the strategy presented here provides a possible route for tailoring rationally molecular chaperones for specific purposes.

Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins. Proc. Natl. Acad. Sci. USA (2015)

Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (alpha-synuclein, Abeta42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of alpha-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions.

Structure of a low-population intermediate state in the release of an enzyme product. eLife (2015)

Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilize it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and the product.

A molecular chaperone breaks the catalytic cycle that generates toxic Abeta oligomers. Nat. Struct. Mol. Biol. (2015)

Alzheimer's disease is a highly debilitating and increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with the aggregation of the amyloid-beta peptide (Abeta42). Recent studies have revealed that the formation of highly neurotoxic oligomers during Abeta aggregation is strongly catalysed by the surfaces of larger aggregates. These findings indicate that a particularly effective way to limit Abeta42 toxicity would be through agents that inhibit this catalytic cycle. Here we show that a molecular chaperone, a Brichos domain, has the ability to specifically block the catalytic production of oligomers. We further demonstrate by a series of biophysical techniques that the Brichos domain achieves this inhibition in vitro by binding on the surface of the fibrils and redirecting the reactive flux from the monomeric to the fibrillar states via a pathway that involves a minimal formation of toxic oligomeric intermediates. We then verify by means of cytotoxicity and electrophysiology experiments using brain tissue that this mechanism also occurs in vivo. These results suggest that biological systems have evolved to achieve effective and efficient suppression of the toxic effects of protein misfolding and aggregation by targeting the individual microscopic pathways that create toxic oligomers, rather than perturbing the overall aggregation reaction.

Cyclophilin A catalyses proline isomerization by an electrostatic handle mechanism. Proc. Natl. Acad. Sci. USA 111, 10203-10208 (2014).

One of the most widespread molecular switches in biochemical pathways is based on the isomerization of the amino acid proline, a process that normally is facilitated by enzymes known as 'proline isomerases'. We show that cyclophilin A, one of the most common proline isomerases, acts by a simple mechanism, which we describe as an 'electrostatic handle'. In this mechanism, the enzyme creates an electrostatic environment in its catalytic site that rotates a peptide bond in the substrate by pulling the electric dipole associated with the carbonyl group preceding the peptide bond itself. Our results thus identify a specific mechanism by which electrostatics is exploited in enzyme catalysis.

Statistical mechanics of the denatured state of a protein using replica-averaged metadynamics. J. Am. Chem. Soc. 136, 8982-8991 (2014).

The characterization of denatured states of proteins is challenging because the lack of permanent structure in these states makes it difficult to apply to them standard methods of structural biology. In this work we use all-atom replica-averaged metadynamics (RAM) simulations with NMR chemical shift restraints to determine an ensemble of structures representing an acid-denatured state of the 86-residue protein ACBP. This approach has enabled us to reach convergence in the free energy landscape calculations, obtaining an ensemble of structures in relatively accurate agreement with independent experimental data used for validation. By observing at atomistic resolution the transient formation of native and non-native structures in this acid-denatured state of ACBP, we rationalize the effects of single-point mutations on the folding rate, stability, and transition-state structures of this protein, thus characterizing the role of the unfolded state in determining the folding process.

Widespread aggregation and neurodegenerative diseases are associated with supersaturated proteins. Cell Reports 5, 781-790 (2013).

The maintenance of protein solubility is a fundamental aspect of cellular homeostasis because protein aggregation is associated with a wide variety of human diseases. Numerous proteins unrelated in sequence and structure, however, can misfold and aggregate, and widespread aggregation can occur in living systems under stress or aging. A crucial question in this context is why only certain proteins appear to aggregate readily in vivo, whereas others do not. We identify here the proteins most vulnerable to aggregation as those whose cellular concentrations are high relative to their solubilities. We find that these supersaturated proteins represent a metastable subproteome involved in pathological aggregation during stress and aging and are overrepresented in biochemical processes associated with neurodegenerative disorders. Consequently, such cellular processes become dysfunctional when the ability to keep intrinsically supersaturated proteins soluble is compromised. Thus, the simultaneous analysis of abundance and solubility can rationalize the diverse cellular pathologies linked to neurodegenerative diseases and aging.

Atomic structure and hierarchical assembly of a cross-beta amyloid fibril. PNAS 110, 5468-5473 (2013).

The cross-beta amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of beta-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-beta amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale - including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryo-electron microscopy, scanning transmission electron microscopy, and atomic force microscopy - we report the atomic-resolution (0.5 A) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent beta-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

Using chemical shifts as replica-averaged structural restraints in molecular dynamics simulations to characterise the dynamics of proteins. J. Am. Chem. Soc. 134, 3968-3971 (2012).

Following the recognition that NMR chemical shifts can be used for protein structure determination, rapid advances have recently been made in methods for extending this strategy for proteins and protein complexes of increasing size and complexity. A remaining major challenge is to develop approaches to exploit the information contained in the chemical shifts about conformational fluctuations in native states of proteins. In this work we show that it is possible to determine an ensemble of conformations representing the free energy surface of RNase A using chemical shifts as replica-averaged restraints in molecular dynamics simulations. Analysis of this surface indicates that chemical shifts can be used to characterize the conformational equilibrium.

Structure of an intermediate state in protein folding and aggregation. Science 336, 362-368 (2012).

Protein-folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. To gain insight, we used relaxation dispersion nuclear magnetic resonance spectroscopy to determine the structure of a low-populated, on-pathway folding intermediate of the A39V/N53P/V55L (A, Ala; V, Val; N, Asn; P, Pro; L, Leu) Fyn SH3 domain. The carboxyl terminus remains disordered in this intermediate, thereby exposing the aggregation-prone amino-terminal β strand. Accordingly, mutants lacking the carboxyl terminus and thus mimicking the intermediate fail to safeguard the folding route and spontaneously form fibrillar aggregates. The structure provides a detailed characterization of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.

Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions. Cell 144, 67-78 (2011).

Protein aggregation is linked with neurodegeneration and numerous other diseases by mechanisms that are not well understood. Here, we have analyzed the gain-of-function toxicity of artificial beta sheet proteins that were designed to form amyloid-like fibrils. Using quantitative proteomics, we found that the toxicity of these proteins in human cells correlates with the capacity of their aggregates to promote aberrant protein interactions and to deregulate the cytosolic stress response. The endogenous proteins that are sequestered by the aggregates share distinct physicochemical properties: They are relatively large in size and significantly enriched in predicted unstructured regions, features that are strongly linked with multifunctionality. Many of the interacting proteins occupy essential hub positions in cellular protein networks, with key roles in chromatin organization, transcription, translation, maintenance of cell architecture and protein quality control. We suggest that amyloidogenic aggregation targets a metastable subproteome, thereby causing multifactorial toxicity and, eventually, the collapse of essential cellular functions.

Characterization of the structure of a misfolded intermediate populated during the folding process of a PDZ domain. Nature Struct. Mol. Biol. 17, 1431-1437 (2010).

Incorrectly folded states transiently populated during the protein folding process are potentially prone to aggregation and have been implicated in a range of misfolding disorders that include Alzheimer's and Parkinson's diseases. Despite their importance, however, the structures of these states and the mechanism of their formation have largely escaped detailed characterization because of their short-lived nature. Here we present the structures of all the major states involved in the folding process of a PDZ domain, which include an off-pathway misfolded intermediate. By using a combination of kinetic, protein engineering, biophysical and computational techniques, we show that the misfolded intermediate is characterized by an alternative packing of the N-terminal β-hairpin onto an otherwise native-like scaffold. Our results suggest a mechanism of formation of incorrectly folded transient compact states by which misfolded structural elements are assembled together with more extended native-like regions.

An analytical solution to the kinetics of filament assembly. Science, 326, 1533-1537 (2009).

We present an analytical treatment of a set of coupled kinetic equations that governs the self-assembly of filamentous molecular structures. Application to the case of protein aggregation demonstrates that the kinetics of amyloid growth can often be dominated by secondary rather than by primary nucleation events. Our results further reveal a range of general features of the growth kinetics of fragmenting filamentous structures, including the existence of generic scaling laws that provide mechanistic information in contexts ranging from in vitro amyloid growth to the in vivo development of mammalian prion diseases.

Prediction of aggregation-prone regions of structured proteins. J. Mol. Biol. 380, 425-436 (2008).

We present a method for predicting the regions of the sequences of peptides and proteins that are most important in promoting their aggregation and amyloid formation. The method extends previous approaches by allowing such predictions to be carried out for conditions under which the molecules concerned can be folded or contain a significant degree of persistent structure. In order to achieve this result, the method uses only knowledge of the sequence of amino acids to estimate simultaneously both the propensity for folding and aggregation and the way in which these two types of propensity compete. We illustrate the approach by its application to a set of peptides and proteins both associated and not associated with disease. Our results show not only that the regions of a protein with a high intrinsic aggregation propensity can be identified in a robust manner but also that the structural context of such regions in the monomeric form is crucial for determining their actual role in the aggregation process.

Structure determination of protein complexes using NMR chemical shifts. JACS 143, 15990-15996 (2008).

Nuclear magnetic resonance (NMR) spectroscopy provides a range of powerful techniques for determining the structures and the dynamics of proteins. The high-resolution determination of the structures of protein-protein complexes, however, is still a challenging problem for this approach, since it can normally provide only a limited amount of structural information at protein-protein interfaces. We present here the determination using NMR chemical shifts of the structure (PDB code 2K5X) of the cytotoxic endonuclease domain from bacterial toxin colicin (E9) in complex with its cognate immunity protein (Im9). In order to achieve this result, we introduce the CamDock method, which combines a flexible docking procedure with a refinement that exploits the structural information provided by chemical shifts. The results that we report thus indicate that chemical shifts can be used as structural restraints for the determination of the conformations of protein complexes that are difficult to obtain by more standard NMR approaches.

Protein structure determination from NMR chemical shifts. PNAS 104, 9615-9620 (2007).

NMR spectroscopy plays a major role in the determination of the structures and dynamics of proteins and other biological macromolecules. Chemical shifts are the most readily and accurately measurable NMR parameters, and they reflect with great specificity the conformations of native and nonnative states of proteins. We show, using 11 examples of proteins representative of the major structural classes and containing up to 123 residues, that it is possible to use chemical shifts as structural restraints in combination with a conventional molecular mechanics force field to determine the conformations of proteins at a resolution of 2A or better. This strategy should be widely applicable and, subject to further development, will enable quantitative structural analysis to be carried out to address a range of complex biological problems not accessible to current structural techniques.

Correlation between expression levels and aggregation rates of proteins. TiBS 32, 204-206 (2007).

We have found that expression levels of human genes in vivo are remarkably anti-correlated with the aggregation rates of the corresponding proteins measured in vitro by experiment. This result indicates that human proteins have evolved to resist aggregation and to function efficiently, but with almost no margin of safety to respond to genetic and environmental factors that decrease their solubility or increase their concentration in vivo. We speculate that this result, which we call the 'life on the edge' hypothesis, provides a compelling reason for the existence of disorders that are associated with protein aggregation, such as Alzheimer's and Parkinson's diseases.

Simultaneous determination of protein structure and dynamics. Nature 433, 128-132 (2005).

We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy - for obtaining experimental information at the atomic level about the structural and dynamical features of proteins - with the ability of molecular dynamics simulations to explore a wide range of prot ein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant advances in our ability to understand and utilize the structures of native proteins.