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Triggering receptor expressed on myeloid cells 2 (TREM2) is a single-pass transmembrane receptor of the immunoglobulin superfamily that is secreted in a soluble (sTREM2) form. Mutations in TREM2 have been linked to increased risk of Alzheimer's disease (AD). A prominent neuropathological component of AD is deposition of the amyloid-β (Aβ) into plaques, particularly Aβ40 and Aβ42. While the membrane-bound form of TREM2 is known to facilitate uptake of Aβ fibrils and the polarization of microglial processes toward amyloid plaques, the role of its soluble ectodomain, particularly in interactions with monomeric or fibrillar Aβ, has been less clear. Our results demonstrate that sTREM2 does not bind to monomeric Aβ40 and Aβ42, even at a high micromolar concentration, while it does bind to fibrillar Aβ42 and Aβ40 with equal affinities (2.6 ± 0.3 µM and 2.3 ± 0.4 µM). Kinetic analysis shows that sTREM2 inhibits the secondary nucleation step in the fibrillization of Aβ, while having little effect on the primary nucleation pathway. Furthermore, binding of sTREM2 to fibrils markedly enhanced uptake of fibrils into human microglial and neuroglioma derived cell lines. The disease-associated sTREM2 mutant, R47H, displayed little to no effect on fibril nucleation and binding, but it decreased uptake and functional responses markedly. We also probed the structure of the WT sTREM2-Aβ fibril complex using integrative molecular modeling based primarily on the cross-linking mass spectrometry data. The model shows that sTREM2 binds fibrils along one face of the structure, leaving a second, mutation-sensitive site free to mediate cellular binding and uptake.

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single β-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit β-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

Infrared (IR) spectroscopy has provided considerable insight into the structures, dynamics, and formation mechanisms of amyloid fibrils. IR probes, such as main chain 13C═18O, have been widely employed to obtain site-specific structural information, yet only secondary structures and strand-to-strand arrangements can be probed. Very few nonperturbative IR probes are available to report on the side-chain conformation and environments, which are critical to determining sheet-to-sheet arrangements in steric zippers within amyloids. Polar residues, such as glutamine, contribute significantly to the stability of amyloids and thus are frequently found in core regions of amyloid peptides/proteins. Furthermore, polyglutamine (polyQ) repeats form toxic aggregates in several neurodegenerative diseases. Here we report the synthesis and application of a new nonperturbative IR probe-glutamine side chain 13C═18O. We use side chain 13C═18O labeling and isotope dilution to detect the presence of intermolecularly hydrogen-bonded arrays of glutamine side chains (Gln ladders) in amyloid-forming peptides. Moreover, the line width of the 13C═18O peak is highly sensitive to its local hydration environment. The IR data from side chain labeling allows us to unambiguously determine the sheet-to-sheet arrangement in a short amyloid-forming peptide, GNNQQNY, providing insight that was otherwise inaccessible through main chain labeling. With several different fibril samples, we also show the versatility of this IR probe in studying the structures and aggregation kinetics of amyloids. Finally, we demonstrate the capability of modeling amyloid structures with IR data using the integrative modeling platform (IMP) and the potential of integrating IR with other biophysical methods for more accurate structural modeling. Together, we believe that side chain 13C═18O will complement main chain isotope labeling in future IR studies of amyloids and integrative modeling using IR data will significantly expand the power of IR spectroscopy to elucidate amyloid assemblies.

A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila. Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.

One of the fundamental events in protein folding is α-helix formation, which involves sequential development of a series of helical hydrogen bonds between the backbone C═O group of residues i and the -NH group of residues i + 4. While we now know a great deal about α-helix folding dynamics, a key question that remains to be answered is where the productive helical nucleation event occurs. Statistically, a helical nucleus (or the first helical hydrogen-bond) can form anywhere within the peptide sequence in question; however, the one that leads to productive folding may only form at a preferred location. This consideration is based on the fact that the α-helical structure is inherently asymmetric, due to the specific alignment of the helical hydrogen bonds. While this hypothesis is plausible, validating it is challenging because there is not an experimental observable that can be used to directly pinpoint the location of the productive nucleation process. Therefore, in this study we combine several techniques, including peptide cross-linking, laser-induced temperature-jump infrared spectroscopy, and molecular dynamics simulations, to tackle this challenge. Taken together, our experimental and simulation results support an α-helix folding mechanism wherein the productive nucleus is formed at the N-terminus, which propagates toward the C-terminal end of the peptide to yield the folded structure. In addition, our results show that incorporation of a cross-linker can lead to formation of differently folded conformations, underscoring the need for all-atom simulations to quantitatively assess the proposed cross-linking design.

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues "planting" the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then "cast" across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

Expansion of intronic GGGGCC repeats in the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Transcription of the expanded repeats results in the formation of RNA-containing nuclear foci and altered RNA metabolism. In addition, repeat-associated non-AUG (RAN) translation of the expanded GGGGCC-repeat sequence results in the production of highly toxic dipeptide-repeat (DPR) proteins. GGGGCC repeat-containing transcripts form G-quadruplexes, which are associated with formation of RNA foci and RAN translation. Zfp106, an RNA-binding protein essential for motor neuron survival in mice, suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Here, we show that Zfp106 inhibits formation of RNA foci and significantly reduces RAN translation caused by GGGGCC repeats in cultured mammalian cells, and we demonstrate that Zfp106 coexpression reduces the levels of DPRs in C9orf72 patient-derived cells. Further, we show that Zfp106 binds to RNA G-quadruplexes and causes a conformational change in the G-quadruplex structure formed by GGGGCC repeats. Together, these data demonstrate that Zfp106 suppresses the formation of RNA foci and DPRs caused by GGGGCC repeats and suggest that the G-quadruplex RNA-binding function of Zfp106 contributes to its suppression of GGGGCC repeat-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

Analogous to reversible post-translational protein modifications, the ability to attach and subsequently remove modifications on proteins would be valuable for protein and biological research. Although bioorthogonal functionalities have been developed to conjugate or cleave protein modifications, they are introduced into proteins on separate residues and often with bulky side chains, limiting their use to one type of control and primarily protein surface. Here we achieved dual control on one residue by genetically encoding S-propargyl-cysteine (SprC), which has bioorthogonal alkyne and propargyl groups in a compact structure, permitting usage in protein interior in addition to surface. We demonstrated its incorporation at the dimer interface of glutathione transferase for in vivo crosslinking via thiol-yne click chemistry, and at the active site of human rhinovirus 3C protease for masking and then turning on enzyme activity via Pd-cleavage of SprC into Cys. In addition, we installed biotin onto EGFP via Sonogashira coupling of SprC and then tracelessly removed it via Pd cleavage. SprC is small in size, commercially available, nontoxic, and allows for bond building and breaking on a single residue. Genetically encoded SprC will be valuable for chemically controlling proteins with an essential Cys and for reversible protein modifications.

Although helices play key roles in peptide-protein and protein-protein interactions, the helical conformation is generally unstable for short peptides (10-15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain-to-side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non-hydrogen-bonded amide groups to solvent. Here, we develop a "capped-strapped" peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end-capping interactions. We have designed a ten-residue capped-strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and Tm ≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design.

The folding of natural proteins typically relies on hydrophobic packing, metal binding, or disulfide bond formation in the protein core. Alternatively, a 3D structure can be defined by incorporating a multivalent cross-linking agent, and this approach has been successfully developed for the selection of bicyclic peptides from large random-sequence libraries. By contrast, there is no general method for the de novo computational design of multicross-linked proteins with predictable and well-defined folds, including ones not found in nature. Here we use Rosetta and Tertiary Motifs (TERMs) to design small proteins that fold around multivalent cross-linkers. The hydrophobic cross-linkers stabilize the fold by macrocyclic restraints, and they also form an integral part of a small apolar core. The designed CovCore proteins were prepared by chemical synthesis, and their structures were determined by solution NMR or X-ray crystallography. These mesosized proteins, lying between conventional proteins and small peptides, are easily accessible either through biosynthetic precursors or chemical synthesis. The unique tertiary structures and ease of synthesis of CovCore proteins indicate that they should provide versatile templates for developing inhibitors of protein-protein interactions.

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to self-purify and catalyze chemical reactions. They also provide attractive backbones for advanced materials. When β-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel β-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn2+-binding histidines with binding geometries unusual in proteins. Each Zn2+ has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn2+ for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn2.