An evolutionarily conserved mechanism controls reversible amyloids of pyruvate kinase via pH-sensing regions.

Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.

Multi-Dimensional Structure and Dynamics Landscape of Proteins in Mammalian Cells Revealed by In-Cell NMR.

Governing function, half-life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in-cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co-existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.

Structural insights into α-synuclein monomer-fibril interactions.

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson's disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels.

The Parkinson's disease protein α-synuclein (αSyn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of αSyn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate PM localization of endogenous pools of αSyn in A2780, HeLa, SK-MEL-2, and differentiated and undifferentiated neuronal SH-SY5Y cells. We demonstrate that αSyn binds to reconstituted PIP2 membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute αSyn in cells. We further delineate that αSyn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to αSyn's function(s) at the plasma membrane.

Mass Photometry of Membrane Proteins.

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers-in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

Nuclear Magnetic Resonance Solution Structure and Functional Behavior of the Human Proton Channel.

The human voltage-gated proton channel [Hv1(1) or VSDO(2)] plays an important role in the human innate immune system. Its structure differs considerably from those of other cation channels. It is built solely of a voltage-sensing domain and thus lacks the central pore domain, which is essential for other cation channels. Here, we determined the solution structure of an N- and C-terminally truncated human Hv1 (Δ-Hv1) in the resting state by nuclear magnetic resonance (NMR) spectroscopy. Δ-Hv1 comprises the typical voltage-sensing antiparallel four-helix bundle (S1-S4) preceded by an amphipathic helix (S0). The solution structure corresponds to an intermediate state between resting and activated forms of voltage-sensing domains. Furthermore, Zn2+-induced closing of proton channel Δ-Hv1 was studied with two-dimensional NMR spectroscopy, which showed that characteristic large scale dynamics of open Δ-Hv1 are absent in the closed state of the channel. Additionally, pH titration studies demonstrated that a higher H+ concentration is required for the protonation of side chains in the Zn2+-induced closed state than in the open state. These observations demonstrate both structural and dynamical changes involved in the process of voltage gating of the Hv1 channel and, in the future, may help to explain the unique properties of unidirectional conductance and the exceptional ion selectivity of the channel.

Probing Ion Binding in the Selectivity Filter of the KcsA Potassium Channel.

In potassium (K+) channels, permeation, selectivity, and gating at the selectivity filter are all governed by the thermodynamics and kinetics of the ion-protein interactions. Specific contacts between the carbonyl groups from the Thr-Val-Gly-Tyr-Gly signature filter sequence and the permeant ions generate four equidistant K+ binding sites, thereby defining the high ion selectivity and controlling the transport rate of K+ channels. Here, we used 15N-labeled ammonium (15NH4+) as a proxy for K+ to study ion interaction with the selectivity filter of the prototypical full-length K+ channel KcsA by solution state NMR spectroscopy in order to obtain detailed insights into the physicochemical basis of K+ gating. We found that in the closed inactive state of KcsA (at pH 7) four K+ binding sites are occupied over a wide range of 15NH4+ concentrations, while in intermediate closed-open conformations (at pH ∼6) the number and occupancy of K+ binding sites are reduced to two. However, in the presence of the scorpion toxin agitoxin II a total loss of 15NH4+ binding is observed. 15NH4+ titration studies allowed us to determine the dissociation constants of the four binding sites with values around 10 mM in the closed state of KcsA. Moreover, kinetic NMR experiments measured in the steady state equilibrium detected an off- and on-rate for 15NH4+ of ca. 102 s-1 and 103 s-1 between KcsA-bound 15NH4+ and the bulk. These findings reveal both the thermodynamics and kinetics of the ion binding sites and thus contribute to our understanding of the action of K+ channels.

Time-Resolved NMR Analysis of Proteolytic α-Synuclein Processing in vitro and in cellulo.

Targeted proteolysis of the disordered Parkinson's disease protein alpha-synuclein (αSyn) constitutes an important event under physiological and pathological cell conditions. In this work, site-specific αSyn cleavage by different endopeptidases in vitro and by endogenous proteases in extracts of challenged and unchallenged cells was studied by time-resolved NMR spectroscopy. Specifically, proteolytic processing was monitored under neutral and low pH conditions and in response to Rotenone-induced oxidative stress. Further, time-dependent degradation of electroporation-delivered αSyn in intact SH-SY5Y and A2780 cells was analyzed. Results presented here delineate a general framework for NMR-based proteolysis studies in vitro and in cellulo, and confirm earlier reports pertaining to the exceptional proteolytic stability of αSyn under physiological cell conditions. However, experimental findings also reveal altered protease susceptibilities in selected mammalian cell lines and upon induced cell stress.

Quantitative mass imaging of single biological macromolecules.

The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.

High-density lipoprotein-like particle formation of Synuclein variants.

α-Synuclein (α-Syn) is an intrinsically disordered protein in solution whose fibrillar aggregates are the hallmark of Parkinson's disease (PD). Although the specific function of α-Syn is still unclear, its high structural plasticity is key for the interactions of α-Syn with biological membranes. Recently, it has been observed that α-Syn is able to form high-density lipoprotein-like (HDL-like) particles that are reminiscent of self-assembling phospholipid bilayer nanodiscs. Here, we extended our preparation method for the production of α-Syn lipoprotein particles to the ß- and γ-Syn variants, and the PD-related familial α-Syn mutants. We show that all human Syns can form stable and homogeneous populations of HDL-like particles with distinct morphologies. Our results characterize the impact of the individual Syns on the formation capacity of these particles and indicate that Syn HDL-like particles are neither causing toxicity nor a toxicity-related loss of α-Syn in PD.

Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I.

High-density lipoprotein (HDL) particles are cholesterol and lipid transport containers. Mature HDL particles destined for the liver develop through the formation of intermediate discoidal HDL particles, which are the primary acceptors for cholesterol. Here we present the three-dimensional structure of reconstituted discoidal HDL (rdHDL) particles, using a shortened construct of human apolipoprotein A-I, determined from a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) data. The rdHDL particles feature a protein double belt surrounding a lipid bilayer patch in an antiparallel fashion. The integrity of this structure is maintained by up to 28 salt bridges and a zipper-like pattern of cation-π interactions between helices 4 and 6. To accommodate a hydrophobic interior, a gross 'right-to-right' rotation of the helices after lipidation is necessary. The structure reflects the complexity required for a shuttling container to hold a fluid lipid or cholesterol interior at a protein:lipid ratio of 1:50.

S-Nitrosylation Induces Structural and Dynamical Changes in a Rhodanese Family Protein.

S-Nitrosylation is well established as an important post-translational regulator in protein function and signaling. However, relatively little is known about its structural and dynamical consequences. We have investigated the effects of S-nitrosylation on the rhodanese domain of the Escherichia coli integral membrane protein YgaP by NMR, X-ray crystallography, and mass spectrometry. The results show that the active cysteine in the rhodanese domain of YgaP is subjected to two competing modifications: S-nitrosylation and S-sulfhydration, which are naturally occurring in vivo. It has been observed that in addition to inhibition of the sulfur transfer activity, S-nitrosylation of the active site residue Cys63 causes an increase in slow motion and a displacement of helix 5 due to a weakening of the interaction between the active site and the helix dipole. These findings provide an example of how nitrosative stress can exert action at the atomic level.

Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles.

Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ∼23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of ∼8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles.

Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

Solution NMR structure and functional analysis of the integral membrane protein YgaP from Escherichia coli.

The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo.

The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.

Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer polypeptide product lowered the yield of the target peptide and complicated its purification. The side product contained the N-terminal 6His-tag together with the thioredoxin fusion partner and the specific enzymatic cleavage site-containing linker followed by an unknown peptide starting with the first 7N-terminal amino acid residues of SS14, as revealed by the Edman degradation. The combination of DNA sequence analysis, the Edman degradation, and high-resolution mass spectrometry allowed to identify the amino acid sequence of the unknown peptide. The appearance of the side product was attributed to inefficient termination of mRNA translation. The stop codon and its downstream sequence optimization allowed eliminating the side product synthesis. The optimized expression system, purification protocol, and post-translational modification procedure yielded 1.5mg of SS14 per liter of minimal medium. Nearly 99% incorporation of (13)C and (15)N isotopes was achieved, as demonstrated by high-resolution mass spectrometry.

Detergent/nanodisc screening for high-resolution NMR studies of an integral membrane protein containing a cytoplasmic domain.

Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [(15)N,(1)H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [(15)N,(1)H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.

Facile backbone structure determination of human membrane proteins by NMR spectroscopy.

Although nearly half of today's major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight <30 kDa yielded 38 hIMPs suitable for structural characterization by solution NMR spectroscopy without additional optimization.

A novel neurotrophic drug for cognitive enhancement and Alzheimer's disease.

Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer's disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer's disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.

The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from alpha-spectrin at sequential stages of elongation via in vivo ribosome-arrested (15)N,(13)C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like beta-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding.