Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli

Crystallography has advanced our understanding of G protein–coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled receptor signaling, receptor maturation, and desensitization.Neurotensin is a 13-amino-acid peptide, which plays important roles in the pathogenesis of Parkinson’s disease, schizophrenia, antinociception, and hypothermia and in lung cancer progression (1–4). It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A G protein–coupled receptors (GPCRs) (5, 6), whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol (5, 7). Activated NTR1 is rapidly phosphorylated and internalizes by a β-arrestin– and clathrin-mediated process (8), which is crucial for desensitizing the receptor (9). Several lines of evidence suggest that internalization is also linked to G protein–independent NTR1 signaling (10, 11). To improve our mechanistic understanding of NTR1 and to gain additional insight into GPCR features such as helix 8 (H8), we were interested in obtaining a structure of this receptor in a physiologically relevant state.To date, by far the most successful strategy for GPCR structure determination requires the replacement of the intracellular loop 3 by a fusion protein, as the intracellular domain is otherwise too small to provide crystal contacts. The fusion protein approach has provided a wealth of valuable structural data on GPCRs, but as it renders the crystallized constructs signaling-inactive, the most important functionality—the activation of G proteins—cannot be confirmed for these structures. This leads inevitably to a degree of uncertainty regarding the physiological relevance of intracellular structural aspects, and it also impedes the elucidation of signaling mechanisms, as functional assays and structure determination cannot be performed with the same GPCR constructs.Crystallization in the absence of fusion proteins was so far mainly possible for rhodopsin (12), the A_2A adenosine receptor (A2AR) (13), and the β₁-adrenergic receptor (14). Together, they share a high stability, which is either given naturally (rhodopsin) or it is due to stabilizing mutations. High stability appeared to be crucial for crystallographic success, as it allowed the application of harsh short-chain detergents. These tend to form small micelles, which may explain why crystal contact formation can occur under these conditions despite the small extra- and intracellular domains of class A GPCRs.Besides the stability requirement and/or the necessity of fusion proteins, structural studies of GPCRs have also been complicated by the need of eukaryotic expression systems [e.g., Spodoptera frugiperda (Sf9) insect cells], as prokaryotes exhibit generally low functional expression levels of wild-type GPCRs. However, prokaryotes such as Escherichia coli offer several advantages compared with insect cells, including quick genetic modification strategies, growth to high cell densities, fast doubling times, inexpensive media, absence of glycosylation, and robust handling. Furthermore, E. coli is well suited for producing fully isotope-labeled proteins—a crucial requirement for many NMR studies, which are limited to date.To exploit these advantages, we recently developed a directed evolution method for high functional GPCR expression levels in E. coli (15). In contrast to screening a few hundred mutants one by one, this strategy allows the simultaneous, competitive testing of >10⁸ different protein variants for highest prokaryotic expression and functionality. Briefly, diverse libraries of NTR1 variants were either obtained synthetically (16, 17) or by error-prone PCR on the wild-type sequence (15). The libraries were ligated to a plasmid encoding an inducible promoter, which was subsequently used to transform E. coli. Selection pressure for high functional expression levels was applied by incubating the induced cells with fluorescently labeled neurotensin, which allowed enrichment of the best expressing cells by fluorescence-activated cell sorting (FACS). The outlined procedure was performed in cycles, leading to a gradual adaptation of the NTR1 population toward high functional expression levels, and additionally, it gave rise to an increase in thermostability for certain variants.In a second technology, termed CHESS (cellular high-throughput encapsulation, solubilization and screening), we adapted this concept to directly evolve NTR1 variants for high thermostability in short-chain detergent micelles—a property that is not only beneficial for structural studies but also for in vitro drug screening (18). The crucial development of CHESS was to surround, simultaneously, every E. coli cell by a semipermeable polysaccharide capsule. This allows us to solubilize the receptor mutants with harsh short-chain detergents, each mutant inside its own encapsulated cell, all at once and in the same test tube. Both the solubilized receptors and their encoding plasmids are maintained within the same capsules. Long-term incubation under these conditions followed by labeling of the encapsulated solubilized receptors with fluorescent neurotensin and rounds of FACS enrichment ensured a strong selection pressure and a gradual adaption of the NTR1 population toward high stability in harsh short-chain detergents (18).In this work, we present the crystal structures of three evolved NTR1 variants, which were either obtained by evolving high functional expression levels in E. coli or by directed evolution for stability in detergent micelles. In contrast to the majority of crystallized GPCRs, our NTR1 variants are devoid of bulky modifications at the cytoplasmic face and can thus remain signaling-active, which allows us to gain unique insights into the structure–function relationship of NTR1.     

Self-assembled lipid and membrane protein polyhedral nanoparticles

We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry. Subsequent single-particle electron cryomicroscopy yielded a reconstruction at ∼1-nm resolution, revealing a conformation closely resembling the nonconducting state. The generality of this approach has been addressed by the successful preparation of MPPNs for two unrelated proteins, the mechanosensitive channel of large conductance and the connexon Cx26, using a recently devised microfluidics-based free interface diffusion system. MPPNs provide not only a starting point for the structural analysis of membrane proteins in a phospholipid environment, but their closed surfaces should facilitate studies in the presence of physiological transmembrane gradients, in addition to potential applications as drug delivery carriers or as templates for inorganic nanoparticle formation.The functions of many membrane proteins are intimately coupled to the generation, utilization, and/or sensing of transmembrane gradients (1). Despite advances in the structure determination of membrane proteins (2), the high-resolution structural analysis of membrane proteins in a biological membrane is uncommon and in the presence of a functionally relevant gradient remains an as-yet unrealized experimental challenge. This stems from the fact that the primary 2D- and 3D ordered specimens used in structural studies of membrane proteins by X-ray crystallography and electron microscopy lack closed membrane surfaces, thus making it impossible to establish physiologically relevant transmembrane gradients.As an alternative, we have been developing methodologies for the self-assembly of lipids and membrane proteins into closed polyhedral structures that can potentially support transmembrane gradients for structural and functional studies. The possibility of generating polyhedral arrangements of membrane proteins in proteoliposomes was motivated by the existence of polyhedral capsids of membrane-enveloped viruses (3, 4), the ability of surfactant mixtures to self-assemble into polyhedral structures (5, 6), and the formation of proteoliposomes from native membranes containing bacteriorhodopsin (7, 8) and light-harvesting complex II (LHCII) (9). Significantly, the high-resolution structure of LHCII was determined from crystals of icosahedral proteoliposomes composed of protein subunits in chloroplast lipids (10). Whereas detergent solubilized membrane proteins and lipid mixtures can self-assemble to form 2D-ordered crystalline sheets or helical tubes favorable for structure determination by electron microscopy (11–14), simple polyhedral ordered assemblies have only been described to form from select native membranes (7–9). To expand the repertoire of membrane protein structural methods, we have prepared membrane protein polyhedral nanoparticles (MPPNs) of the bacterial mechanosensitive channel of small conductance (MscS) (15, 16) from detergent solubilized protein and phospholipids, and demonstrated that they are amenable to structural analysis using electron microscopy.Conditions for generating MPPNs were anticipated to resemble those for other types of 2D-ordered bilayer arrangements of membrane proteins, particularly 2D crystals, in that membrane protein is mixed with a particular phospholipid at a defined ratio, followed by dialysis to remove the solubilizing detergent (17). The main distinction is that because MPPNs are polyhedral, conditions are sought that will stabilize highly curved surfaces of polyhedra rather than the planar (flat) specimens desired for 2D crystals. We used the Escherichia coli MscS as a model system. MscS is an intrinsically stretch-activated channel identified by Booth and coworkers (15) that confers resistance to osmotic downshock in E. coli. MscS forms a heptameric channel with 21 transmembrane helices (3 from each subunit) and a large cytoplasmic domain with overall dimensions of ∼8 × ∼12 nm parallel and perpendicular to the membrane plane; structures have been reported in both nonconducting (16, 18) and open-state conformations (19). Different phospholipids were added to purified the E. coli MscS solubilized in the detergent Fos-Choline 14 and the system was allowed to reach equilibrium by dialysis at different temperatures. To gain insight into the biophysical parameters that govern MPPN formation, we investigated the role of lipid head group, alkyl chain length, pH, and protein construct. Table S1 shows the observed influence of these various factors on our ability to form uniform MPPNs (as opposed to disordered aggregates or polydisperse proteoliposomes). The optimal conditions for MPPN formation used 1,2-dimyristoyl-sn-glycero-3-phosphocholine [added to ∼1:0.1 (wt/wt) protein:phospholipid] at pH 7 with the His-tagged MscS that is anticipated to be positively charged under these conditions. The biophysical properties of the protein are important as the best results were achieved using a His-tagged construct and the presence of a FLAG tag at the C terminus of MscS interfered with MPPN formation, even though the tag is ∼10 nm from the membrane-spanning region of MscS.To monitor MPPN formation, dynamic light scattering (DLS) was used. Under optimal conditions, we observed (Fig. 1A) the complete transition of solubilized MscS particles with a narrow distribution centered around a mean radii of 4.5 nm to MPPNs with a narrow distribution centered around a mean radii of 20 nm. We further characterized these particles using negative-stain electron microscopy. Fig. 1B is a field view negative-stain electron micrograph of a solution of detergent-solubilized MscS and lipid before initiation of the self-assembly process. Fig. 1C is a field view negative-stain electron micrograph of the same sample after the self-assembly process. We observed the incorporation of MscS into highly uniform polyhedra with mean radii of ∼20 nm (90%) and ∼17 nm (10%) by negative-stain electron microscopy. To gain more insight into the biophysical properties of these particles, we performed protein and phosphorus analysis on multiple samples to determine the lipid:protein ratio (Fig. S1). The observed lipid:protein ratio of the MscS MPPNs was 11 ± 1 (mole lipid:mole protein subunit) and consistent with a single layer of lipids forming a bilayer surrounding each protein. This ratio is comparable to the observed lipid to protein ratio found in 2D crystals of membrane proteins such as bacteriorhodopsin (lipid:protein ratio of 10; refs. 20 and 21) and aquaporin (lipid:protein ratio of 9; ref. 22).Open in a separate windowFig. 1.Preparation of MscS MPPNs. (A) DLS analysis of particles before dialysis and after completion of dialysis when MPPNs are formed. The observed radius of MscS alone was 4.5 nm and the particle radius at the end of dialysis was observed to be 20 nm. In both cases 99% of the scattering mass was observed in the distributions centered at 4.5 nm and 20 nm, respectively. (B) Negative-stain electron microscopy analysis of MscS before dialysis. Individual MscS proteins can be observed as small doughnut-shaped particles. (C) Negative-stain electron microscopy analysis of MPPNs following dialysis of the sample in B. MPPNs can be clearly observed and appear as uniform assemblies of individual MscS molecules. (Scale bars, 100 nm.)To further elucidate the structural nature of these particles and to unambiguously determine the symmetry, we performed electron cryotomography with image reconstruction using IMOD (23) combined with Particle Estimation for Electron Tomography (PEET) program (ref. 24 and SI Materials and Methods). In principle, electron tomography provides a complete 3D map of the particles and would allow us to unambiguously determine the MPPN symmetry. However, the alignment process was highly biased by the missing wedge phenomenon (24) due to poor signal:noise and resulted in an incomplete map (Fig. 2A). To overcome this alignment bias, we assigned random initial orientation values to all particles and constrained possible angular shifts to less than 30° to achieve a more uniform distribution of orientations (Fig. S2). This strategy resulted in a much improved density map (Fig. 2B) that revealed individual molecules with a size and shape that are in good agreement to the known molecular structure of MscS (Fig. 2C and Fig. S3). Building on the analysis of Haselwandter and Phillips (25), a systematic analysis was conducted (Table S2) of the symmetry relationships between MscSs in MPPNs that identified the arrangement corresponding to the snub cuboctahedron (dextro), an Archimedean solid. The snub cuboctahedron has cubic (octahedral) symmetry which, as recognized by Crick and Watson (26), provides an efficient way to pack identical particles in a closed, convex shell. In this particular arrangement, 24 MscS molecules are related by the 432-point group symmetry axes that pass through the faces, but not the vertices, of the snub cuboctahedron. Because the MscS molecules are positioned on the vertices of this chiral polyhedron, they occupy general positions that permit the ordered packing of the heptamers of a biomacromolecule (or indeed any type of particle). This is an important observation as it means that the individual MscS molecules with sevenfold symmetry are capable of packing into a symmetric assembly that is amenable to averaging. Whereas 24 objects can be arranged with identical environments in a snub cuboctahedron, certain integer multiples of this number can also be accommodated using the principles of quasiequivalence (27, 28) to form larger closed shells.Open in a separate windowFig. 2.Cryotomography of MscS MPPNs. (A) The PEET isosurface derived from 162 individual particles selected from eight single-tilt tomograms. The strong bias due to the missing wedge is observed along the lower part of the surface, but individual MscS heptamers are still discernible in the image. (B) The corresponding PEET isosurface, following introduction of randomized starting Euler angles to minimize missing-wedge bias. (C) The use of randomized starting Euler angles results in a much-improved map with apparent octahedral (432) symmetry that could be fit with 24 molecules of the MscS crystal structure. Isosurface renderings of the volume averages were generated using Chimera (31).Using the symmetry derived by electron cryotomography, we proceeded to collect high-resolution single-particle electron cryomicroscopy data. Samples prepared identically for cryotomography were imaged under low-dose conditions and a total of 4,564 particles were processed using the Electron Micrograph Analysis 2 (EMAN2) software package (SI Materials and Methods) (29). The final map had a resolution of 9 Å by Fourier shell correlation (Fig. S4) and allowed us to model the inner and outer helices of the transmembrane pore (Fig. 3). The arrangement of the helices more closely resembles the nonconducting conformation (16, 18) than the open-state structure (19), although some differences in the positioning of the outer helices relative to the nonconducting structure are indicated in sections 2 and 3 of Fig. 3. These results demonstrate that membrane proteins are capable of assembling into MPPNs that are amenable to high-resolution structure analysis by single-particle electron cryomicroscopy. Higher resolution data will be required, however, to detail the precise conformational differences between MscS in the phospholipid environment of MPPNs compared with those in the detergent-solubilized state used in the X-ray crystal structure analyses.Open in a separate windowFig. 3.Single-particle image analysis reconstructed from 4,564 particles processed with EMAN2 and subsequently the density surrounding a single MscS heptamer was extracted and sevenfold averaged as described in SI Materials and Methods. (Left) A cross-section through the electron density revealing the translocation pathway and cytoplasmic vestibule, and showing the overall fit of the closed structure of MscS (red ribbons) fit to the map (cyan). (Right) Stereoviews of cross-sections in the density map normal to the sevenfold axis at sections 1, 2, and 3. The closed-structure coordinates (red ribbons) of MscS were fit to the map using rigid body refinement in Chimera (31) showing the position of the transmembrane helices.In these promising initial studies we used traditional dialysis methods to screen conditions for MPPN formation. These methods are time consuming and require substantial quantities of a sample. To more efficiently screen conditions for MPPN formation with a variety of membrane proteins, we designed and fabricated a free interface diffusion microfluidic device (30) (Fig. 4A and Fig. S5) This device greatly simplifies the screening process and minimizes the amount of sample required for determining suitable conditions for MPPN formation. Using this device, we were able to produce MPPNs from MscS but more importantly from several other proteins that had previously failed to produce MPPNs using traditional dialysis. Fig. 4 B and C shows the results of using this device for the mechanosensitive channel of large conductance (MscL) and the connexon Cx26, respectively, where polyhedra were only observed in the presence of the target protein. Intriguingly, several different particles sizes could be observed for both MscL and Cx26 and we hypothesize that the variable-sized polyhedra may correspond to different packing arrangements similar to triangulation numbers observed in viral polyhedral assemblies. This microfluidic device will provide rapid screening of conditions for the formation of MPPNs and it is hoped will expedite membrane protein structural analysis in native lipid environments.Open in a separate windowFig. 4.Preparation of MPPNs using a microfluidics-based free interface diffusion system. (A) Schematic illustration of the device used for lipid–protein nanoparticle formation. From left to right, molecules in the center flow diffuse into the outer flow by the concentration gradient, with small molecules (larger diffusion coefficient) moving more quickly than larger molecules. Specifically, monomer detergents are removed through interfacial diffusion, whereas larger membrane proteins remain in the center flow, forming nanoparticles. Both the ratio of input:buffer and the flow rate influence particle formation. (B). Negative-stain electron microscopy images of MPPNs of MscL and (C) Cx26 formed using the microfluidic device from A. (Scale bar, 100 nm.) Insets show 2.5× magnification of a select region of interest.The self-assembly of membrane proteins into polyhedral nanoparticles demonstrates a potentially powerful method for studying the structure and function of membrane proteins in a lipid environment. MPPNs represent a novel form of lipid–protein assemblies which lie between single particles and large crystalline sheets or tubes. We have demonstrated that conditions favorable for MPPN formation can be identified and have elucidated the structure, symmetry, and potential application to membrane protein structure analysis. In addition we have designed and fabricated microfluidic devices for high-throughput screening of conditions for MPPN formation. MPPNs may allow a variety of perturbations to be achieved such as pH, voltage, osmotic, concentration gradients, etc. that cannot be achieved with other membrane protein assemblies and will potentially allow us to activate various types of gated channels and receptors so that active conformational states can be structurally investigated. The potential of such materials for targeted drug delivery with precisely controlled release mechanisms offers an intriguing avenue for future biomedical applications.     

Unitary step of gliding machinery in Mycoplasma mobile

Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0–4.5 μm⋅s⁻¹ with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.The fastest of the Mycoplasma species is Mycoplasma mobile (M. mobile); they glide with a speed of 2.0–4.5 μm⋅s⁻¹ (1, 2). Under an optimal-growth condition, cultivated single M. mobile cells are flask-shaped (Fig. 1A) and glide smoothly across a substrate covered with surface-immobilized sialylated oligosaccharides (3) in the direction of protrusion at a constant speed (Movie S1). Genomic sequencing and analysis have revealed that the mechanism must differ from other forms of motor protein systems and bacterial motility, because M. mobile lacks genes encoding conventional motor proteins in eukaryotes, such as myosin, kinesin, and dynein, in addition to lacking other motility structures in bacteria, such as flagella and pili (4). So far, three proteins have been identified as a part of the gliding machinery (Fig. 1B, Bottom): Gli123 (5), Gli521 (6), and Gli349 (7). The machinery units localize around the cell neck, and their number has been estimated to be ∼450 (2, 5, 8). Gli349 extends out from the cell membrane and shows a rod structure, ∼100 nm in total, with two flexible hinges when isolated (9). Notably, the machinery is driven by hydrolysis of ATP to ADP and inorganic phosphate, caused by an unknown ATPase (10). Because of the large size and characteristic structure of Gli349, and a series of studies with mutants and inhibitory antibodies (2, 11), it has been hypothesized that Gli349 works as a “leg” by binding to and releasing from a substrate covered with randomly arranged sialylated oligosaccharides (2) consuming the chemical energy of ATP. In addition, the pivoting movement of an elongated cell suggests that there are units working not simultaneously but rather independently to propel the cell forward (12). To test this hypothesis and identify conformational changes of a key part of the gliding machinery, we here designed an assay to detect the movement of M. mobile by high precision colocalization microscopy. In the presence of an excess number of binding targets in the solution, which decreased the number of active legs, stepwise displacement was shown for the first time, to our knowledge, to occur in gliding bacteria.Open in a separate windowFig. 1.Nanometer-scale tracking of Mycoplasma gliding. (A) A dark-field image of M. mobile. The image was captured with center-stop optics to maintain the high numerical aperture of the objective, which enabled a high spatial resolution (35). (Scale bar: 1 μm.) (B, Upper) Illustration of the fluorescent ghost. The gliding machinery was distributed around the neck portion, but only the active machinery bound to the glass is shown for simplicity. (Bottom) A construction model of the gliding machinery comprising three proteins: Gli123, Gli521, and Gli349. See the review by Miyata (2) for more detail. (C) A fluorescent image of the labeled ghost was acquired with a time resolution of 2 ms. (Scale bar: 1 μm; pixel size: 240 nm.) (D) The intensity profile of C. The XY area is 5 × 5 μm. (E) Gaussian fitting to D. Nanometer-scale tracking is achieved by positioning the peak of the 2D Gaussian function fitting to the intensity profile of the ghost. (F, Left) The speed of gliding ghosts at different [ATP]s in the solution (n = 129). The cyan curve shows a fit with Michaelis–Menten kinetics; Vmaxspeed and K_m are 2.6 µm⋅s⁻¹ and 61 µM, respectively. The dotted cyan curve shows a fit with the kinetics including the Hill coefficient; Vmaxspeed, [ATP₅₀] and n are 2.2 µm⋅s⁻¹, 43 µM, and 2.4, respectively. (Right) The speed of living cells with no ATP in the solution (2.1 ± 0.1 µm⋅s⁻¹; n = 22). (G) Effect of SL on the gliding velocity of the ghost at saturated [ATP]s, 0.3–1.0 mM (n = 50).     

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Molecular chaperones are an essential part of the machinery that avoids protein aggregation and misfolding in vivo. However, understanding the molecular basis of how chaperones prevent such undesirable interactions requires the conformational changes within substrate proteins to be probed during chaperone action. Here we use single-molecule fluorescence spectroscopy to investigate how the DnaJ–DnaK chaperone system alters the conformational distribution of the denatured substrate protein rhodanese. We find that in a first step the ATP-independent binding of DnaJ to denatured rhodanese results in a compact denatured ensemble of the substrate protein. The following ATP-dependent binding of multiple DnaK molecules, however, leads to a surprisingly large expansion of denatured rhodanese. Molecular simulations indicate that hard-core repulsion between the multiple DnaK molecules provides the underlying mechanism for disrupting even strong interactions within the substrate protein and preparing it for processing by downstream chaperone systems.Maintaining protein homeostasis in vivo requires a tight regulation of protein folding to prevent misfolding and aggregation. Molecular chaperones have evolved as an essential part of the cellular machinery that facilitates such processes in the complex and crowded environment of a living cell (1, 2). To assist protein folding, many chaperones proceed through complex conformational cycles in an ATP-dependent manner (3–5). For several chaperone systems, these cycles have been investigated in great detail by experiment and simulation (6–8). A remarkable example are the heat shock protein (Hsp) 70 chaperones, which are essential in prokaryotes and eukaryotes and are involved in co-translational folding, refolding of misfolded and aggregated proteins, protein translocation, and protein degradation (9). The Hsp70 chaperone DnaK from Escherichia coli together with its co-chaperone DnaJ and the nucleotide exchange factor GrpE form an ATP-driven catalytic reaction cycle (7) (Fig. 1A). Many denatured or misfolded substrate proteins are first captured by DnaJ and subsequently transferred to the DnaK–ATP complex, with DnaK in an open conformation. Substrate and DnaJ synergistically trigger DnaK’s ATPase activity, which leads to locking of the substrate in the DnaK–ADP complex, with DnaK in the closed conformation. Driven by the following GrpE-catalyzed ADP–ATP exchange, the DnaK–substrate complex dissociates (10). Since this ATP-driven cycle can even solubilize protein aggregates (11, 12), substantial forces must be transduced to the substrate protein (13–15). However, as for other chaperone systems (16), surprisingly little is known about how these forces and the resulting constraints of the underlying free energy surfaces affect the conformations of the denatured or misfolded substrate proteins. To better understand this important link between chaperone action and function, we probed the conformation of a substrate protein along the different stages of the chaperone cycle of DnaK with single-molecule Förster resonance energy transfer (smFRET), correlation spectroscopy, and microfluidic mixing.Open in a separate windowFig. 1.DnaK expands the denatured substrate protein. (A) Illustration of the DnaK–ATPase cycle. (B) Surface representation of rhodanese (PDB ID code 1RHS) with the subdomains indicated in different gray levels and the label positions of fluorescent dyes for single-molecule FRET measurements shown schematically. (C) FRET efficiency histograms of native rhodanese (gray) and denatured rhodanese under native conditions transiently populated in the microfluidic mixer (colored, measured 125 ms after dilution of rhodanese into native conditions). (D) FRET efficiency histograms of DnaJ–rhodanese complexes (0.5 µM DnaJ). (E) FRET efficiency histograms of DnaK–rhodanese complexes (0.5 µM DnaJ, 10 µM DnaK, and 1 mM ATP; DnaK and DnaJ were added simultaneously to rhodanese). Black lines indicate the DnaK–rhodanese complex population resulting from a fit that takes into account the residual population of refolded and DnaJ-bound rhodanese. The vertical lines in C–E indicate the positions of the FRET efficiency peaks of the native population of the respective rhodanese variants. The small populations at zero transfer efficiency in D (note the axis scaling and the small amplitudes of this population compared with E) originate from incomplete elimination of molecules with inactive acceptor fluorophores by pulsed interleaved excitation.     

Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection

Adenovirus cement proteins play crucial roles in virion assembly, disassembly, cell entry, and infection. Based on a refined crystal structure of the adenovirus virion at 3.8-Å resolution, we have determined the structures of all of the cement proteins (IIIa, VI, VIII, and IX) and their organization in two distinct layers. We have significantly revised the recent cryoelectron microscopy models for proteins IIIa and IX and show that both are located on the capsid exterior. Together, the cement proteins exclusively stabilize the hexon shell, thus rendering penton vertices the weakest links of the adenovirus capsid. We describe, for the first time to our knowledge, the structure of protein VI, a key membrane-lytic molecule, and unveil its associations with VIII and core protein V, which together glue peripentonal hexons beneath the vertex region and connect them to the rest of the capsid on the interior. Following virion maturation, the cleaved N-terminal propeptide of VI is observed, reaching deep into the peripentonal hexon cavity, detached from the membrane-lytic domain, so that the latter can be released. Our results thus provide the molecular basis for the requirement of maturation cleavage of protein VI. This process is essential for untethering and release of the membrane-lytic region, which is known to mediate endosome rupture and delivery of partially disassembled virions into the host cell cytoplasm.Human adenoviruses (HAdVs) are large (∼150 nm in diameter, 150-MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause respiratory, ocular, and enteric diseases (1). Although these diseases are self-limiting in immunocompetent individuals, they cause significant morbidity in AIDS, cancer, and organ transplant patients with compromised immune systems (2–4). Because of their broad cell tropism and ease of genome manipulation, replication-deficient or conditionally replicating HAdVs are also being evaluated in the clinic as potential vaccine and gene therapy vectors (5).The capsid shell of an adenovirus (Ad) comprises multiple copies of three major capsid proteins (MCPs; hexon, penton base, and fiber) and four minor/cement proteins (IIIa, VI, VIII, and IX) that are organized with pseudo-T = 25 icosahedral symmetry (Fig. 1 A and B). In addition, six other proteins (V, VII, μ, IVa2, terminal protein, and adenovirus protease) are encapsidated along with the 36-kb dsDNA genome inside the capsid (Fig. 1A). The crystal structures of all three MCPs are known, and so is their organization in the capsid from prior X-ray crystallography (6–8) and cryoelectron microscopy (cryo-EM) analyses (9, 10). Recently, high-resolution structures of recombinant HAdV5 vectors have been determined using cryo-EM (11) and X-ray methods (12) that revealed the structures and organization of some of the cement proteins. Both studies agree closely on the organization of the MCPs and confirm the earlier cryo-EM observations (9, 10, 13), but neither provided significant information on the structure and location of protein VI, which serves key roles in the virus life cycle. Of note, however, the two studies differ significantly in their assignments of the cement proteins IIIa and IX. Recent cryo-EM studies reported that only protein IX molecules form “triskelion” as well as “four-helix bundle” (4-HLXB) structures and mediate the network of interactions between hexon subunits on the capsid exterior (11, 14, 15). They also suggested that the densities ascribed to α-helices beneath the vertex region belong to protein IIIa. However, based on our X-ray crystallographic data and considering the principles of quasi-equivalence (16), we earlier suggested that although the IX molecules form triskelion structures, it is rather unlikely that the C termini of IX would form 4-HLXB structures (12). Instead, we proposed that this 4-HLXB is most likely derived from a subdomain of IIIa (12).Open in a separate windowFig. 1.Structure and organization of human adenovirus. (A) A schematic illustration of the organization of capsid and core proteins in human adenovirus. The locations of various proteins are represented by different-colored symbols and the corresponding names are shown (Right). The indicated locations of the core proteins are approximate. Shown in blue-colored lettering are the proteins whose structures have been identified in this study. (B) Overall organization of hexon and penton base subunits exhibiting pseudo-T = 25 icosahedral symmetry. Structurally unique hexons (1–4) are color-coded in light blue, pink, green, and khaki, respectively. Penton vertices are shown in magenta. Outer cement proteins IIIa and IX are shown in purple and blue, respectively. Fiber molecules associated with the penton base are disordered. The outline of the triangular icosahedral facet is shown as a gray triangle, whereas the border of the GON hexons is indicated by yellow-colored rope. (C) An exterior view of the triangular icosahedral facet that comprises 12 hexons along with penton base vertices shown in magenta. Color representations are the same as in B. (D) An interior view of the facet in C, with three minor proteins, V (green), VI (red), and VIII (orange). It is noteworthy that a copy of V, VI, and VIII forms a ternary complex beneath the vertices, whereas VIII (orange) molecules are arranged as staples along the border (yellow-colored rope) of the GON hexons.Here we report a revised interpretation, a paradigm shift, of the structures and locations of all of the cement proteins based on the refined crystal structure of Ad5F35 (HAdV5 vector encoding the type 35 fiber) that includes detailed models for the ordered regions of all four cement proteins (IIIa, VI, VIII, and IX). Additionally, we identified a segment of core protein V, which associates closely with protein VI. The 4-HLXB structure on the capsid exterior is a subdomain of IIIa (amino acids 101–355) that mediates interactions between group-of-nine (GON) (17) hexons (Fig. 1 C and D). The backbone path of each IX molecule is reversed from what was assigned by the cryo-EM studies (11, 14). Proteins V, VI, and VIII form a ternary complex that stabilizes the adjacent peripentonal hexons (PPHs) underneath each of the 12 vertex regions (Fig. 1D). This complex was incorrectly assigned to protein IIIa in cryo-EM studies (11, 14). Following virion maturation, the cleaved propeptide(s) of VI (pVIn; amino acids 1–33) is observed in the inner cavities of the PPHs, in agreement with recent interpretations from hydrogen–deuterium exchange mass spectrometry studies (18).     

A cellular solution to an information-processing problem

Signaling receptors on the cell surface are mobile and have evolved to efficiently sense and process mechanical or chemical information. We pose the problem of identifying the optimal strategy for placing a collection of distributed and mobile sensors to faithfully estimate a signal that varies in space and time. The optimal strategy has to balance two opposing objectives: the need to locally assemble sensors to reduce estimation noise and the need to spread them to reduce spatial error. This results in a phase transition in the space of strategies as a function of sensor density and efficiency. We show that these optimal strategies have been arrived at multiple times in diverse cell biology contexts, including the stationary lattice architecture of receptors on the bacterial cell surface and the active clustering of cell-surface signaling receptors in metazoan cells.The molecular characteristics of signaling receptors and their spatiotemporal organization have evolved to optimize different facets of information processing at the cell surface. A canonical information-processing problem involves designing strategies for a collection of distributed, noisy, mobile sensors to faithfully estimate a signal or function that varies in space and time (1). This problem appears naturally in many contexts, biological and nonbiological: (i) chemoattractant protein sensors on the bacteria cell surface (2, 3); (ii) galectin-glycoprotein assemblies designed for effective immune response on the surface of metazoan cells (4, 5); (iii) ligand-activated signaling protein receptors on the surface of eukaryotic cells (6–10); (iv) coclustering of integrin receptors to faithfully read and discriminate the rigidity and chemistry of a substrate (11); (v) clustering of e-cadherin receptors for effective adherence at cell–cell junctions (12); and even (vi) radio frequency (RF) sensor networks monitoring the environment or mobile targets (13). In the signal-processing community, this problem is known as data fusion or more generally information fusion (14, 15); however typical applications do not consider mobile sensors.In this paper we show how biology has, on multiple occasions, arrived at a solution to this optimization problem. The optimal solution needs to balance two opposing objectives, the need to locally assemble sensors to reduce estimation noise and the need to spread them out for broader spatial coverage. We show that in the space of strategies, this leads to a phase transition as a function of sensor density, sensor characteristics, and function properties. At very low sensor density, the optimal design corresponds to freely diffusing sensors. For sensor density above a threshold, there are two different optimal solutions as a function of a dimensionless parameter constructed from the sensor advection velocity and the correlation length and time of the incident signal. One optimal solution is that the sensors are static and located on a regular lattice grid. This is the strategy used in bacteria, such as Escherichia coli, to organize their chemoattractant receptors in a regular lattice array (3, 16), and in metazoan cells, where galectin-glycoproteins are organized in a lattice on the cell surface to effect an optimal immune response (4, 5). To realize this strategy, the cell needs to provide a rigid cortical scaffold that holds the receptors in place. Another optimal solution is to make the receptors mobile in such a way that a fraction of them form multiparticle nanoclusters, which then break up and reform randomly, the rest being uniformly distributed. Recent studies on the steady-state distribution of several cell-surface proteins reveal a stereotypical distribution of a fixed fraction of monomers and dynamic nanoclusters (6–9), and our information theoretic perspective could provide a general explanation for this. To realize this dynamic strategy, the cell surface needed to be relieved of the constraints imposed by the rigid scaffold and to be more regulatable. This strategy change needed the innovation of motor proteins and dynamic actin filaments, a regulated actomyosin machinery fueled by ATP, and a coupling of components of the cell surface to this cortical dynamic actin (17).     

Functional map of arrestin-1 at single amino acid resolution

Arrestins function as adapter proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin–GPCR complexes. The presented functional map quantitatively connects critical interactions in the polar core and along the C tail of arrestin. A series of amino acids (Phe375, Phe377, Phe380, and Arg382) anchor the C tail in a position that blocks binding of the receptor. Interaction of phosphates in the rhodopsin C terminus with Arg29 controls a C-tail exchange mechanism in which the C tail of arrestin is released and exposes several charged amino acids (Lys14, Lys15, Arg18, Lys20, Lys110, and Lys300) for binding of the phosphorylated receptor C terminus. In addition to this arrestin phosphosensor, our data reveal several patches of amino acids in the finger (Gln69 and Asp73–Met75) and the lariat loops (L249–S252 and Y254) that can act as direct binding interfaces. A stretch of amino acids at the edge of the C domain (Trp194–Ser199, Gly337–Gly340, Thr343, and Thr345) could act as membrane anchor, binding interface for a second rhodopsin, or rearrange closer to the central loops upon complex formation. We discuss these interfaces in the context of experimentally guided docking between the crystal structures of arrestin and light-activated rhodopsin.The human genome encodes more than 800 G protein-coupled receptors (GPCRs), which mediate signaling between cells and provide an important link to our environment as the principal receptors for taste, smell, and vision. The visual system with its photoreceptor rhodopsin is an excellent system to understand GPCR signaling, as detailed information exists on the structures and dynamic interactions of the protein constituents (1). G protein-mediated signaling by light-activated rhodopsin is terminated by a process that begins with the phosphorylation of rhodopsin’s C terminus by the rhodopsin kinase GRK1. The phosphorylated, light-activated rhodopsin binds then to arrestin-1, which stops signaling by occluding the G protein-binding site. Further cloning efforts yielded two ubiquitously expressed nonvisual arrestins (arrestin-2 and arrestin-3 or β-arrestin-1 and β-arrestin-2) and the cone-specific arrestin-4. It seems clear today that most GPCRs share a common mechanism of signal termination involving receptor phosphorylation and the binding of arrestins. Arrestin-bound receptors may be internalized and degraded, internalized and recycled, and/or initiate G protein-independent signaling (2).In recent years there has been tremendous progress in the structure determination of active GPCR states including those of light-activated rhodopsin (3–5) and the first GPCR-G protein complex (6). Detailed structural information on the inactive state of arrestin-1 has been available for some time (7, 8). These inactive structures have recently been complemented with structures of a preactivated state of the arrestin-1 splice variant p44 (9) and of arrestin-2 bound to a receptor phosphopeptide (10). Analysis of these 3D structures provides many clues of how arrestins function. However, structures alone do not tell the whole story, as they contain little information about which subset of residues stabilize a particular conformation or contribute to receptor binding. The approximate binding surface was established by peptide (11) and antibody (12) competition experiments. Series of targeted mutagenesis studies based on the cell-free expression of radiolabeled arrestin (13) were used to probe the function of specific arrestin regions. However, these targeted studies are difficult to compare quantitatively as they have been gathered in over 20 y of work. Here we present an unbiased and complete scan of the arrestin-1 sequence to compare the relative impact of each amino acid on binding to light-activated, phosphorylated rhodopsin. These data provide a functional dimension to the available crystal structures and will guide our molecular understanding of GPCR–arrestin interactions.     

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5–10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular condition that afflicts as many as 1 of 350 males and 420 females over the age of 18 (1). In ALS, degeneration of upper and lower motor neurons causes progressive muscle paralysis and spasticity, affecting mobility, speech, swallowing, and respiration (2). Half of affected individuals die within 3 y, and less than 20% survive for more than 5 y (3); 90–95% of ALS cases are sporadic (SALS) in which some apparently facilitating gene mutations, such as repeat expansions in the gene that encodes ataxin-2 (4), have been identified. The remaining 5–10% of ALS cases are familial (FALS) and predominantly associated with Mendelian-inherited mutations in the genes encoding Cu/Zn superoxide dismutase (SOD1), TAR-DNA–binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLS), C9ORF72, and other genes (reviewed in ref. 3).Despite the profusion of functionally diverse genes implicated in FALS and SALS, clinical and pathological similarities between all forms of ALS suggest the existence of a common pathogenic pathway that could be united by a single gene/protein (5). One of the mechanisms by which a mutant or wild-type (WT) protein can dominate pathogenesis of phenotypically diverse diseases is by propagated protein misfolding, such as that underpinning the prion diseases, which has been increasingly implicated in other neurodegenerative and systemic disorders (6, 7). A role for propagated protein misfolding in ALS is supported by the prion-like spatiotemporal progression of disease through the neuroaxis (8, 9). However, given the disparity in protein inclusion pathology between subtypes of ALS, a single unifying prion-like protein that could explain such a progression remains obscure.Whereas it is generally accepted SOD1 is not found in large perikaryal cytoplasmic inclusions outside of SOD1 FALS cases, misfolded SOD1 has been increasingly identified in SALS and non-SOD1 FALS (5, 10, 11). Indeed, we have reported that misfolded human wild-type SOD1 (HuWtSOD1) can be detected by spinal cord immunohistochemistry (IHC) in FALS secondary to FUS mutation, and in SALS patients with cytosolic WT TDP-43 accumulation (11). Moreover, in cell models, overexpression of WTTDP-43, or expression of mutant FUS or TDP-43, is associated with HuWtSOD1 misfolding (11). Collectively, these data are consistent with SOD1 being a molecular common denominator for all types of ALS. Furthermore, prion-like activity has been described for the cell-to-cell transmission of misfolding of mutant SOD1 (12), and we have reported that mutant SOD1 can confer its misfold on HuWtSOD1 (13). However, mutant SOD1 cannot explain propagation in SALS.To test if HuWtSOD1 participates in cell-to-cell transmission of protein misfolding, we make use of previously developed mouse mAb probes for misfolded/oxidized SOD1, recognizing either full-length human mutant or WT SOD1, generated against regions that are antibody-inaccessible in natively folded SOD1 (13–15). Misfolded SOD1 mAbs used in this work are 10E11C11 and 3H1, directed against an unstructured electrostatic loop [disease-specific epitope-2 (DSE2)], and 10C12, directed against a C-terminal dimer interface peptide in which the cysteine at position 146 is substituted by a cysteic acid residue to mimic oxidation of this residue (DSE1a) (13). The use of such antibody probes have enabled us to unambiguously determine the role of misfolded mutant G127X in the induced misfolding of HuWtSOD1, which upon misfolding acquires a marked increase in sensitivity to protease digestion, consistent with global loosening of structure (13). The finding that misfolded endogenous HuWtSOD1 was observed long after transfected G127X-SOD1 was degraded suggested that HuWtSOD1, once misfolded, is capable of triggering an intracellular propagated misfolding reaction (13). We now report for the first time that misfolded HuWtSOD1 can transit cell to cell both via exosomes, and release of protein aggregates and subsequent uptake in neuronal cells. In addition, misfolded HuWtSOD1 can sustain intercellular propagated misfolding in vitro and is detectable in the spinal cord of all ALS patients tested, regardless of the genetic etiology of the disease. Collectively, these data indicate that HuWtSOD1 is competent to participate in propagated misfolding, suggesting a common pathogenic mechanism linking FALS and SALS.     

甲状旁腺素对人甲状腺髓样癌细胞增殖和凋亡的影响

目的探讨甲状旁腺素对人甲状腺髓样癌细胞体外增殖抑制及凋亡作用。方法体外培养甲状腺髓样癌细胞株,经甲状旁腺素和甲状旁腺素受体单抗干预处理后,倒置相差显微镜下观察细胞生长状况,流式细胞仪检测细胞凋亡。结果倒置相差显微镜下细胞变化明显,各浓度的甲状旁腺素和甲状旁腺素受体单抗均分别能有效地抑制甲状腺髓样癌细胞增殖、诱导细胞凋亡,凋亡作用呈时间和浓度依赖。当甲状旁腺素浓度为2．0μmol／L、甲状旁腺素受体单抗浓度为1．0μmol／L时,对细胞凋亡作用显著（P〈0．05）,凋亡率分别为13．24％及20．78％。结论甲状旁腺素对人甲状腺髓样癌细胞增殖有抑制作用并能诱导其凋亡。