An electrostatic cluster guides Aß40 fibril formation in sporadic and Dutch-type cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is associated with the accumulation of fibrillar Aß peptides upon and within the cerebral vasculature, which leads to loss of vascular integrity and contributes to disease progression in Alzheimer's disease (AD). We investigate the structure of human-derived Aß40 fibrils obtained from patients diagnosed with sporadic or familial Dutch-type (E22Q) CAA. Using cryo-EM, two primary structures are identified containing elements that have not been observed in in vitro Aß40 fibril structures. One population has an ordered N-terminal fold comprised of two ß-strands stabilized by electrostatic interactions involving D1, E22, D23 and K28. This charged cluster is disrupted in the second population, which exhibits a disordered N-terminus and is favored in fibrils derived from the familial Dutch-type CAA patient. These results illustrate differences between human-derived CAA and AD fibrils, and how familial CAA mutations can guide fibril formation.

Quantitative prediction and measurement of Piezo's membrane footprint.

Piezo proteins are mechanosensitive ion channels that can locally curve the membrane into a dome shape [Y. R. Guo, R. MacKinnon, eLife 6, e33660 (2017)]. The curved shape of the Piezo dome is expected to deform the surrounding lipid bilayer membrane into a membrane footprint, which may serve to amplify Piezo's sensitivity to applied forces [C. A. Haselwandter, R. MacKinnon, eLife 7, e41968 (2018)]. If Piezo proteins are embedded in lipid bilayer vesicles, the membrane shape deformations induced by the Piezo dome depend on the vesicle size. We employ here membrane elasticity theory to predict, with no free parameters, the shape of such Piezo vesicles outside the Piezo dome, and show that the predicted vesicle shapes agree quantitatively with the corresponding measured vesicle shapes obtained through cryoelectron tomography, for a range of vesicle sizes [W. Helfrich, Z. Naturforsch. C 28, 693-703 (1973)]. On this basis, we explore the coupling between Piezo and membrane shape and demonstrate that the features of the Piezo dome affecting Piezo's membrane footprint approximately follow a spherical cap geometry. Our work puts into place the foundation for deducing key elastic properties of the Piezo dome from membrane shape measurements and provides a general framework for quantifying how proteins deform bilayer membranes.

Elastic properties and shape of the Piezo dome underlying its mechanosensory function.

We show in the companion paper that the free membrane shape of lipid bilayer vesicles containing the mechanosensitive ion channel Piezo can be predicted, with no free parameters, from membrane elasticity theory together with measurements of the protein geometry and vesicle size [C. A. Haselwandter, Y. R. Guo, Z. Fu, R. MacKinnon, Proc. Natl. Acad. Sci. U.S.A., 10.1073/pnas.2208027119 (2022)]. Here we use these results to determine the force that the Piezo dome exerts on the free membrane and hence, that the free membrane exerts on the Piezo dome, for a range of vesicle sizes. From vesicle shape measurements alone, we thus obtain a force-distortion relationship for the Piezo dome, from which we deduce the Piezo dome's intrinsic radius of curvature, [Formula: see text] nm, and bending stiffness, [Formula: see text], in freestanding lipid bilayer membranes mimicking cell membranes. Applying these estimates to a spherical cap model of Piezo embedded in a lipid bilayer, we suggest that Piezo's intrinsic curvature, surrounding membrane footprint, small stiffness, and large area are the key properties of Piezo that give rise to low-threshold, high-sensitivity mechanical gating.

Uncovering translation roadblocks during the development of a synthetic tRNA.

Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.

Continued expansion of the genetic code has required the use of synthetic tRNAs for decoding. Some of these synthetic tRNAs have unique structural features that are not observed in canonical tRNAs. Here, the authors applied single-molecule, biochemical and structural methods to determine whether these distinct features were deleterious for efficient protein translation on the ribosome. With a focus on selenocysteine insertion, the authors explored an allo-tRNA with a 9/3 acceptor domain. They observed a translational roadblock that occurred in A to P site tRNA translocation. This block was mediated by a tertiary interaction across the tRNA core, directing the variable arm position into an unfavorable conformation. A single-nucleotide mutation disrupted this interaction, providing flexibility in the variable arm and promoting efficient protein production.

Symmetric activation and modulation of the human calcium-sensing receptor.

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs.

Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

Anti-Parallel ß-Hairpin Structure in Soluble Aß Oligomers of Aß40-Dutch and Aß40-Iowa.

The amyloid-ß (Aß) peptides are associated with two prominent diseases in the brain, Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). Aß42 is the dominant component of cored parenchymal plaques associated with AD, while Aß40 is the predominant component of vascular amyloid associated with CAA. There are familial CAA mutations at positions Glu22 and Asp23 that lead to aggressive Aß aggregation, drive vascular amyloid deposition and result in degradation of vascular membranes. In this study, we compared the transition of the monomeric Aß40-WT peptide into soluble oligomers and fibrils with the corresponding transitions of the Aß40-Dutch (E22Q), Aß40-Iowa (D23N) and Aß40-Dutch, Iowa (E22Q, D23N) mutants. FTIR measurements show that in a fashion similar to Aß40-WT, the familial CAA mutants form transient intermediates with anti-parallel ß-structure. This structure appears before the formation of cross-ß-sheet fibrils as determined by thioflavin T fluorescence and circular dichroism spectroscopy and occurs when AFM images reveal the presence of soluble oligomers and protofibrils. Although the anti-parallel ß-hairpin is a common intermediate on the pathway to Aß fibrils for the four peptides studied, the rate of conversion to cross-ß-sheet fibril structure differs for each.

Author Correction: Structure of human GABAB receptor in an inactive state.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanism of ligand activation of a eukaryotic cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide (CN) binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length Caenorhabditis elegans cyclic GMP-activated channel TAX-4, reconstituted in lipid nanodiscs. These structures, together with computational and functional analyses and a mutant channel structure, reveal a double-barrier hydrophobic gate formed by two S6 amino acids in the central cavity. cGMP binding produces global conformational changes that open the cavity gate located ~52 Å away but do not alter the structure of the selectivity filter-the commonly presumed activation gate. Our work provides mechanistic insights into the allosteric gating and regulation of CN-gated and nucleotide-modulated channels and CNG channel-related channelopathies.

Structure of human GABAB receptor in an inactive state.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Structural and functional characterization of the bestrophin-2 anion channel.

The bestrophin family of calcium (Ca2+)-activated chloride (Cl-) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca2+, comprises four members in mammals (bestrophin 1-4). Here we report cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca2+ at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis reveals that, although the channel is sensitive to Ca2+, it has substantial Ca2+-independent activity for Cl-, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway even when Ca2+ is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating.

The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy.

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

Late steps in bacterial translation initiation visualized using time-resolved cryo-EM.

The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Rapid and accurate formation of the 70S initiation complex is promoted by initiation factors, which must dissociate from the 30S initiation complex before the resulting 70S elongation-competent complex can begin the elongation of translation1. Although comparisons of the structures of the 30S2-5 and 70S4,6-8 initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a structure of the 70S elongation-competent complex formed via an initiation-factor-catalysed reaction has precluded an understanding of the rearrangements to the ribosome, initiation factors and fMet-tRNAfMet that occur during maturation of a 70S initiation complex into a 70S elongation-competent complex. Here, using time-resolved cryogenic electron microscopy9, we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNAfMet positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

Structure and activity of lipid bilayer within a membrane-protein transporter.

Membrane proteins function in native cell membranes, but extraction into isolated particles is needed for many biochemical and structural analyses. Commonly used detergent-extraction methods destroy naturally associated lipid bilayers. Here, we devised a detergent-free method for preparing cell-membrane nanoparticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Å resolution. We discovered a remarkably well-organized lipid-bilayer structure associated with transmembrane domains of the AcrB trimer. This bilayer patch comprises 24 lipid molecules; inner leaflet chains are packed in a hexagonal array, whereas the outer leaflet has highly irregular but ordered packing. Protein side chains interact with both leaflets and participate in the hexagonal pattern. We suggest that the lipid bilayer supports and harmonizes peristaltic motions through AcrB trimers. In AcrB D407A, a putative proton-relay mutant, lipid bilayer buttresses protein interactions lost in crystal structures after detergent-solubilization. Our detergent-free system preserves lipid-protein interactions for visualization and should be broadly applicable.

Time-Resolved Cryo-electron Microscopy Using a Microfluidic Chip.

With the advent of direct electron detectors, cryo-EM has become a popular choice for molecular structure determination. Among its advantages over X-ray crystallography are (1) no need for crystals, (2) much smaller sample volumes, and (3) the ability to determine multiple structures or conformations coexisting in one sample. In principle, kinetic experiments can be done using standard cryo-EM, but mixing and freezing grids require several seconds. However, many biological processes are much faster than that time scale, and the ensuing short-lived states of the molecules cannot be captured. Here, we lay out a detailed protocol for how to capture such intermediate states on the millisecond time scale with time-resolved cryo-EM.

A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM.

We describe a spraying-plunging method for preparing cryoelectron microscopy (cryo-EM) grids with vitreous ice of controllable, highly consistent thickness using a microfluidic device. The new polydimethylsiloxane (PDMS)-based sprayer was tested with apoferritin. We demonstrate that the structure can be solved to high resolution with this method of sample preparation. Besides replacing the conventional pipetting-blotting-plunging method, one of many potential applications of the new sprayer is in time-resolved cryo-EM, as part of a PDMS-based microfluidic reaction channel to study short-lived intermediates on the timescale of 10-1,000 ms.

Cerebral vascular amyloid seeds drive amyloid ß-protein fibril assembly with a distinct anti-parallel structure.

Cerebrovascular accumulation of amyloid ß-protein (Aß), a condition known as cerebral amyloid angiopathy (CAA), is a common pathological feature of patients with Alzheimer's disease. Familial Aß mutations, such as Dutch-E22Q and Iowa-D23N, can cause severe cerebrovascular accumulation of amyloid that serves as a potent driver of vascular cognitive impairment and dementia. The distinctive features of vascular amyloid that underlie its unique pathological properties remain unknown. Here, we use transgenic mouse models producing CAA mutants (Tg-SwDI) or overproducing human wild-type Aß (Tg2576) to demonstrate that CAA-mutant vascular amyloid influences wild-type Aß deposition in brain. We also show isolated microvascular amyloid seeds from Tg-SwDI mice drive assembly of human wild-type Aß into distinct anti-parallel ß-sheet fibrils. These findings indicate that cerebrovascular amyloid can serve as an effective scaffold to promote rapid assembly and strong deposition of Aß into a unique structure that likely contributes to its distinctive pathology.

Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy.

Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.

Mechanism of Nucleated Conformational Conversion of Aß42.

Soluble oligomers and protofibrils of the Aß42 peptide are neurotoxic intermediates in the conversion of monomeric Aß42 into the amyloid fibrils associated with Alzheimer's disease. Nuclear magnetic resonance and Fourier transform infrared spectroscopy, along with single-touch atomic force microscopy, are used to establish the structural transitions involved in fibril formation. We show that under conditions favorable for the nucleated conformation conversion, the Aß42 peptide aggregates into largely unstructured low-molecular weight (MW) oligomers that are able to stack to form high-MW oligomers and to laterally associate to form protofibrils. ß-Sheet secondary structure develops during the irreversible lateral association of the oligomers. The first step in this conversion is the formation of an antiparallel ß-hairpin stabilized by intramonomer hydrogen bonding. The antiparallel ß-hairpins then associate into a cross ß-sheet structure with parallel and in-register ß-strands having intermonomer hydrogen bonding.

Capping of aß42 oligomers by small molecule inhibitors.

Aß42 peptides associate into soluble oligomers and protofibrils in the process of forming the amyloid fibrils associated with Alzheimer's disease. The oligomers have been reported to be more toxic to neurons than fibrils, and have been targeted by a wide range of small molecule and peptide inhibitors. With single touch atomic force microscopy (AFM), we show that monomeric Aß42 forms two distinct types of oligomers, low molecular weight (MW) oligomers with heights of 1-2 nm and high MW oligomers with heights of 3-5 nm. In both cases, the oligomers are disc-shaped with diameters of ~10-15 nm. The similar diameters suggest that the low MW species stack to form the high MW oligomers. The ability of Aß42 inhibitors to interact with these oligomers is probed using atomic force microscopy and NMR spectroscopy. We show that curcumin and resveratrol bind to the N-terminus (residues 5-20) of Aß42 monomers and cap the height of the oligomers that are formed at 1-2 nm. A second class of inhibitors, which includes sulindac sulfide and indomethacin, exhibit very weak interactions across the Aß42 sequence and do not block the formation of the high MW oligomers. The correlation between N-terminal interactions and capping of the height of the Aß oligomers provides insights into the mechanism of inhibition and the pathway of Aß aggregation.