Andres Binolfi

Structural disorder of monomeric α-synuclein persists in mammalian cells

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson’s disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

It has long been axiomatic that a protein’s structure determines its function. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDRs) defy this structure–function paradigm. They do not exhibit stable secondary and/or tertiary structures and exist as dynamic ensembles of interconverting conformers with preferred, nonrandom orientations.1−4 The concept of IDPs and IDRs as functional biological units was initially met with skepticism. For a long time, disorder, intuitively implying chaos, had no place in our perception of orchestrated molecular events controlling cell biology. Over the past years, however, this notion has changed. Aided by findings that structural disorder constitutes an ubiquitous and abundant biological phenomenon in organisms of all phyla,5−7 and that it is often synonymous with function,8−11 disorder has become an integral part of modern protein biochemistry. Disorder thrives in eukaryotic signaling pathways12 and functions as a prominent player in many regulatory processes.13−15 Disordered proteins and protein regions determine the underlying causes of many neurodegenerative disorders and constitute the main components of amyloid fibrils.16 They further contribute to many forms of cancer, diabetes and to cardiovascular and metabolic diseases.17,18 Research into disordered proteins produced significant findings and established important new concepts. On the structural side, novel experimental and computational approaches identified and described disordered protein ensembles3,19,20 and led to terms such as secondary structure propensities, residual structural features, and transient long-range contacts.1,21 The discovery of coupled folding-and-binding reactions defined the paradigm of disorder-to-order transitions22 and high-resolution insights into the architectures of amyloid fibrils were obtained.23,24 On the biological side, we learned about the unexpected intracellular stability of disordered proteins, their roles in integrating post-translational protein modifications in cell signaling and about their functions in regulatory processes ranging from transcription to cell fate decisions.15,25,26 One open question remaining to be addressed is how these in vitro structural insights relate to biological in vivo effects. How do complex intracellular environments modulate the in vivo properties of disordered proteins and what are the implications for their biological functions (Figure ​(Figure11)?27−29 Figure 1 Intracellular complexity. (A) Left: Cryo-electron tomography slice of a mammalian cell. Middle: Close-up view of cellular structures colored according to their identities: Right: Three-dimensional surface representation of the same region. Yellow, endoplasmic ...

Site-specific interactions of Cu(II) with α and β-synuclein: bridging the molecular gap between metal binding and aggregation.

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinsons disease (PD) and other neurodegenerative synucleinopathies. Protein-metal interactions play a critical role in AS aggregation and might represent the link between the pathological processes of protein aggregation and oxidative damage. Our previous studies established a hierarchy in AS-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. In this work, we have addressed unresolved structural details related to the binding specificity of Cu(II) through the design of site-directed and domain-truncated mutants of AS and by the characterization of the metal-binding features of its natural homologue beta-synuclein (BS). The structural properties of the Cu(II) complexes were determined by the combined application of nuclear magnetic resonance, electron paramagnetic resonance, UV-vis, circular dichroism spectroscopy, and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Two independent, noninteracting copper-binding sites with significantly different affinities for the metal ion were detected in the N-terminal regions of AS and BS. MALDI MS provided unique evidence for the direct involvement of Met1 as the primary anchoring residue for Cu(II) in both proteins. Comparative spectroscopic analysis of the two proteins allowed us to deconvolute the Cu(II) binding modes and unequivocally assign the higher-affinity site to the N-terminal amino group of Met1 and the lower-affinity site to the imidazol ring of the sole His residue. Through the use of competitive chelators, the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the submicromolar range for both AS and BS. Our results prove that Cu(II) binding in the C-terminal region of synucleins represents a nonspecific, very low affinity process. These new insights into the bioinorganic chemistry of PD are central to an understanding of the role of Cu(II) in the fibrillization process of AS and have implications for the molecular mechanism by which BS might inhibit AS amyloid assembly.

Bacterial in-cell NMR of human α-synuclein: a disordered monomer by nature?

The notion that human α-synuclein is an intrinsically disordered monomeric protein was recently challenged by a postulated α-helical tetramer as the physiologically relevant protein structure. The fact that this alleged conformation had evaded detection for so many years was primarily attributed to a widely used denaturation protocol to purify recombinant α-synuclein. In the present paper, we provide in-cell NMR evidence obtained directly in intact Escherichia coli cells that challenges a tetrameric conformation under native in vivo conditions. Although our data cannot rule out the existence of other intracellular protein states, especially in cells of higher organisms, they indicate clearly that inside E. coli α-synuclein is mostly monomeric and disordered.

Bioinorganic Chemistry of Parkinson's Disease: Structural Determinants for the Copper-Mediated Amyloid Formation of Alpha-Synuclein

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinsons disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.

Thermodynamics of protein destabilization in live cells

Significance A key question in structural biology is how protein properties mapped out under simplified conditions in vitro transfer to the complex environment in live cells. The answer, it appears, varies. Defying predictions from steric crowding effects, experimental data have shown that cells in some cases stabilize and in other cases destabilize the native protein structures. In this study, we reconcile these seemingly conflicting results by showing that the in-cell effect on protein thermodynamics is sequence specific: The outcome depends both on the individual target protein and on its detailed host-cell environment. Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior.

Structural and mechanistic basis behind the inhibitory interaction of PcTS on alpha-synuclein amyloid fibril formation.

The identification of aggregation inhibitors and the investigation of their mechanism of action are fundamental in the quest to mitigate the pathological consequences of amyloid formation. Here, characterization of the structural and mechanistic basis for the antiamyloidogenic effect of phthalocyanine tetrasulfonate (PcTS) on α-synuclein (AS) allowed us to demonstrate that specific aromatic interactions are central for ligand-mediated inhibition of amyloid formation. We provide evidence indicating that the mechanism behind the antiamyloidogenic effect of PcTS is correlated with the trapping of prefibrillar AS species during the early stages of the assembly process. By using NMR spectroscopy, we have located the primary binding region for PcTS to a specific site in the N terminus of AS, involving the amino acid Tyr-39 as the anchoring residue. Moreover, the residue-specific structural characterization of the AS-PcTS complex provided the basis for the rational design of nonamyloidogenic species of AS, highlighting the role of aromatic interactions in driving AS amyloid assembly. A comparative analysis with other proteins involved in neurodegenerative disorders reveals that aromatic recognition interfaces might constitute a key structural element to target their aggregation pathways. These findings emphasize the use of aggregation inhibitors as molecular probes to assess structural and toxic mechanisms related to amyloid formation and the potential of small molecules as therapeutics for amyloid-related pathologies.

Site-Specific Copper-Catalyzed Oxidation of α-Synuclein: Tightening the Link between Metal Binding and Protein Oxidative Damage in Parkinson’s Disease

Amyloid aggregation of α-synuclein (AS) has been linked to the pathological effects associated with Parkinsons disease (PD). Cu(II) binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific Cu(I)-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, Cu(I) binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif (1)MDVFM(5) constitute key structural determinants for the high-affinity binding of Cu(I) to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for Cu(I), whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-Cu(I) complex, whereas Met-116 and Met-127 in the C-terminal region remain unaffected. Met-1 displays higher susceptibility to oxidative damage compared to Met-5 because it is directly involved in both Cu(II) and Cu(I) coordination, resulting in closer exposure to the reactive oxygen species that may be generated by the redox cycling of copper. Our findings support a mechanism where the interaction of AS with copper ions leads to site-specific metal-catalyzed oxidation in the protein under physiologically relevant conditions. In light of recent biological findings, these results support a role for AS-copper interactions in neurodegeneration in PD.

Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts

We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the 15N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D 1H-15N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.

Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites

Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinsons disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinsons disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells.