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.