Plexins are cell surface receptors that bind semaphorins and transduce signals for regulating neuronal axon guidance and other processes. Plexin signaling depends on their cytoplasmic GTPase activating protein (GAP) domain, which specifically inactivates the Ras homolog Rap through an ill-defined non-canonical catalytic mechanism. The plexin GAP is activated by semaphorin-induced dimerization, the structural basis for which remained unknown. Here we present the crystal structures of the active dimer of zebrafish PlexinC1 cytoplasmic region in the apo state and in complex with Rap. The structures show that the dimerization induces a large-scale conformational change in plexin, which opens the GAP active site to allow Rap binding. Plexin stabilizes the switch II region of Rap in an unprecedented conformation, bringing Gln63 in Rap into the active site for catalyzing GTP hydrolysis. The structures also explain the unique Rap-specificity of plexins. Mutational analyses support that these mechanisms underlie plexin activation and signaling.
A key question in neurobiology is how the brain becomes wired up. How do axons—the ‘wires’ along which neural signals flow—know in which direction to grow to reach their intended targets? A family of signalling proteins called semaphorins contribute to this process by acting as stop signals for axons that are heading in the wrong direction. The actions of semaphorins are mediated by receptors known as plexins, which are found on the membranes of axons.
Plexins contain an extracellular domain that binds semaphorin, and a large domain inside the cell that can turn semaphorin binding into cellular responses. When a semaphorin protein binds to the extracellular domain of a plexin receptor, the domain inside the cell joins with the intracellular domain of a neighbouring receptor to form a dimer. This activates the intracellular domain, which turns on its ability to inactivate a molecule called Rap. The end result is that the axon stops growing and changes direction, but the molecular mechanisms through which these events occur are not well understood.
Now, Wang, Pascoe et al. have worked out the structure of the dimers formed by the intracellular plexin domains, both alone and in complex with Rap. The structures reveal how the dimer drives a shape change of the intracellular domain to enable it to bind Rap, and show that Rap itself adopts a novel conformation upon binding to plexin. This conformational change in Rap catalyses the breakdown of a signalling molecule called GTP, which inactivates Rap and triggers an intracellular signalling cascade that causes the axon to collapse and change direction.
Lastly, Wang, Pascoe et al. have shown that the highly specific nature of these interactions depends on particular amino-acid residues in both Rap and the plexin receptor. Further work is now required to determine whether this pattern of activation represents a general mechanism for signalling by plexin receptors, and for the inhibition of Rap.
plexin; axon guidance; Rap; GAP; signal transduction; dimerization; Human; Mouse; Zebrafish
The receptor tyrosine kinase Her2, an intensely pursued drug target, differs from other members of the EGFR family in that it does not bind EGF-like ligands, relying instead on heterodimerization with other (ligand-bound) EGFR-family receptors for activation. The structural basis for Her2 heterodimerization, however, remains poorly understood. The unexpected recent finding of asymmetric ectodomain dimer structures of Drosophila EGFR (dEGFR) suggests a possible structural basis for Her2 heterodimerization, but all available structures for dimers of human EGFR family ectodomains are symmetric. Here, we report results from long-timescale molecular dynamics simulations indicating that a single ligand is necessary and sufficient to stabilize the ectodomain interface of Her2 heterodimers, which assume an asymmetric conformation similar to that of dEGFR dimers. This structural parallelism suggests a dimerization mechanism that has been conserved in the evolution of the EGFR family from Drosophila to human.
ErbB proteins are found in most multi-cellular organisms, and are involved in the regulation of a number of important cellular processes, including proliferation, migration, and differentiation. Humans have four ErbB proteins, which span the plasma membrane of cells. These proteins respond to interactions with molecules outside the cell—such as growth factors and hormones—by sending signals along the appropriate signaling pathway within the cell.
ErbB proteins have three portions: an ectodomain that extends outside the cell; a single helix that spans the membrane; and a cytoplasmic domain inside the cell. When a signaling ligand molecule outside the cell binds to the ectodomain of an ErbB protein, this protein must then combine with another ErbB protein to form a dimer before a signal can be sent within the cell. These dimers can include two copies of the same ErbB protein or two different ErbB proteins. However, one of the ErbB proteins—Her2—works in a different way. It cannot bind ligands outside the cell, and it can only send a signal within the cell if it first forms a dimer with an ErbB protein of another type, which itself must be bound to an external ligand.
The four ErbB proteins diverged from a common ancestor relatively recently, yet they are now diverse enough to play key roles in a variety of complex signaling networks. In particular, the fact that Her2 cannot bind external ligands, and that it must form a dimer with a different ErbB protein before it can send a signal, has led to suggestions that the role of Her2 is to amplify the signals from other ErbB proteins. Since high levels of Her2 are associated with aggressive forms of breast and ovarian cancer, understanding how it is activated could improve our understanding of these cancers.
Arkhipov et al. have now used computer simulations to model how Her2 forms dimers with other ErbB proteins in human cells. They based these simulations on crystal structures of human ErbB proteins and dEGFR, a growth-factor receptor found in fruit flies that closely resembles the ErbB proteins found in humans. They found that the dimers were stable as long as one protein within the dimer was bound to a ligand. Removing this ligand, however, distorted the ectodomain of the host protein, creating a gap that weakened the dimer and prevented Her2 from sending a signal within the cell. Similar results were obtained with the fruit fly dEGFR proteins. These simulations suggest that ErbB proteins form dimers and send signals through a mechanism conserved in evolution. Research in this field might help ongoing efforts to develop new treatments for human tumors characterized by high levels of Her2 expression.
Her2/ErbB2 activation; extracellular domain conformation; dimerization interface; None
Background: NSF and α-SNAP disassemble all SNARE complexes.
Results: The disassembly kinetics is conserved for different ternary and binary SNARE complexes. α-SNAP and the ternary SNARE complex form a 1:1 complex.
Conclusion: NSF uses a conserved mechanism to disassemble all SNARE complexes, starting from a 1:1 SNAP-SNARE complex interaction.
Significance: We illuminate a broad mechanism allowing NSF to support SNARE-mediated exocytosis.
Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.
ATPases; Biophysics; Biosensors; Circular Dichroism (CD); Enzyme Kinetics; Fluorescence; Membrane Fusion; Protein-Protein Interactions; SNARE Proteins; Structural Biology
T cells discriminate between self and foreign antigenic peptides, displayed on antigen presenting cell surfaces, via the TCR. While the molecular interactions between TCR and its ligands are well characterized in vitro, quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling. We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes. Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70, as a readout of TCR triggering. Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems. Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio. Thus any signal amplification must occur downstream of TCR triggering.
The immune system identifies and combats foreign objects, including pathogens, in the body. T cells are key components of the immune system, and each has a unique variant of a signalling complex known as the T cell receptor on its surface. T cells scan the surfaces of other cells in search of antigens, which are peptides (fragments of proteins) that derive from foreign pathogens such as viruses. Successful recognition of a foreign peptide leads to an immune response that, in most cases, ultimately rids the body of the pathogen. Most importantly, however, the immune system must be able to discriminate between peptides that are produced naturally in the body (‘self’ peptides) and foreign or ‘non-self’ peptides. This is challenging because self peptides may have similar structures to non-self peptides and are often much more abundant.
Many models have been proposed to explain how T cells are able to detect just a few molecules of foreign peptide. According to some hypotheses the T cell receptors get together in clusters to function cooperatively; alternatively, it has been suggested that rapid binding of a foreign peptide to multiple T cell receptors sequentially can build up a strong signal. However, none of these phenomena have been directly observed.
O'Donoghue et al. now image the interactions between T cell receptors and peptides bound to molecules called major histocompatibility complexes (MHCs), and show that T cell activation can occur when a single foreign peptide binds to a single receptor. These interactions are long-lived and ultimately result in the recruitment of ZAP70, which has an important role in the activation of T cells, to the complex formed by the T cell, the peptide and the MHC molecule. Therefore, any amplification of the activating signal transmitted by non-self peptides occurs following receptor binding, in contrast to previous models.
TCR triggering; single molecule kinetics; T cells; Mouse
This Protocol describes a single vesicle-vesicle microscopy system to study Ca2+-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and SNAP-25, and are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at zero Ca2+-concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca2+-injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-hundred millisecond timescale. Other factors, such as complexin, can be easily added. Our system is unique by monitoring both content and lipid mixing, and by starting from a metastable state of interacting vesicle pairs prior to Ca2+-injection.
Membrane biology; vesicle fusion; membrane fusion; sulforhodamine B; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate; DiIC18(5); DiD; cascade blue; SNARE; Ca2+; fluorescence microscopy; TIR; total internal reflection
α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself.
The central nervous system coordinates many different activities by sending instructions to large numbers of cells and, simultaneously, processing all the signals that are sent back to the brain. All these messages are carried by electrical pulses that travel along chains of neurons, with neurotransmitter molecules enclosed inside synaptic vesicles conveying the messages across the synapses between neurons. A protein called α-synuclein is thought to have a role in the transport of neurotransmitter molecules across synapses, but the details of its involvement are not fully understood.
Mutations in the gene that codes for α-synuclein, and also duplications and triplications of this gene, are known to lead to an increased risk of early onset Parkinson's disease, a condition where the central nervous system degenerates. Moreover, the Lewy bodies found in the neurons of patients with Parkinson's disease contain high concentrations of α-synuclein. Again, however, none of this is fully understood.
Diao et al. have shed new light on these questions by creating synthetic vesicles to mimic what happens in real synapses, and using optical microscopy to observe the behaviour of these vesicles. They found that native α-synuclein (and another set of membrane proteins) increases the availability of synthetic vesicles at the synapse by causing them to cluster together. In a second experiment, Diao et al. showed that native α-synuclein does not decrease calcium-triggered fusion between membranes, the process that releases neurotransmitter into the synaptic cleft. In contrast, it is known that pathogenic α-synuclein aggregates directly interfere with the release of the neurotransmitter molecules. Moreover, when Diao et al. used a particular mutant form of α-synuclein that is associated with Parkinson's disease, the vesicles did not form clusters. If these results are confirmed in vivo, the role played by native α-synuclein in the central nervous system, and the connection between α-synuclein and Parkinson's disease, will be much clearer.
Parkinson's disease; synaptic terminals; alpha-synuclein; Mouse
Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism.
Influenza is caused by viruses that infect birds and mammals. These viruses enter cells when two lipid bilayers—one surrounding the virus, the other enclosing the cellular compartment into which the virus has been engulfed—merge to form a single unified membrane. This process, known as membrane fusion, allows the RNA of the virus to gain access to the host cell's molecular machinery, which it commandeers to produce multiple copies of itself and to direct the assembly of new virus particles. The process of membrane fusion generally includes an intermediate hemifused state in which only the adjacent monolayers from each bilayer have merged. In addition to its role in virology, membrane fusion is critical for many other biological processes, including exocytosis, protein trafficking and the fertilization of eggs by sperm.
Efficient membrane fusion requires a catalyst, and a glycoprotein known as the influenza hemagglutinin performs this role for the influenza virus. The hemagglutinin is found on the surface of the virus, and a typical influenza virus particle can have a few hundred such molecules on its surface. When an influenza virus particle binds to the surface of a cell (as a result of these hemagglutinin molecules interacting with cellular receptor molecules), the cell engulfs the virus into an internal compartment called an endosome. Acidification of the endosome, part of the cell's normal activity, triggers a sequence of conformational changes in the hemagglutinin molecules on the surface of the virus. One part of the hemagglutinin inserts itself into the endosomal membrane, and further conformational changes draw the endosomal and viral membranes together into an intermediate, hemifused state; the process then continues until fusion of the two membranes is complete.
Previous work has suggested that an average of three hemagglutinin molecules are required to fuse the endosomal and viral membranes. Ivanovic et al. have now investigated the molecular details of this process and described the time course of conformational changes undergone by the hemagglutinin molecules from the moment the pH is lowered within the endosome until the time when hemifusion of the endosomal and viral membranes is complete. They find, among other things, that hemifusion proceeds rapidly only when three or four immediately adjacent hemagglutinin molecules have inserted into the endosomal membrane. Since membrane fusion is a very general cellular process, the findings of Ivanovic et al. are relevant to many areas of cell biology, in addition to having potential applications in virology.
influenza; enveloped viruses; membrane fusion; single molecule; virus entry; lipid bilayer; Viruses
Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation–libration–screw (TLS) model. Modeling of 20 protein datasets at 1.1–3.1 Å resolution reduced cross-validated Rfree values by 0.3–4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a ‘molten core’ likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order–disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure–dynamics–function relationships.
It has been clear since the early days of structural biology in the late 1950s that proteins and other biomolecules are continually changing shape, and that these changes have an important influence on both the structure and function of the molecules. X-ray diffraction can provide detailed information about the structure of a protein, but only limited information about how its structure fluctuates over time. Detailed information about the dynamic behaviour of proteins is essential for a proper understanding of a variety of processes, including catalysis, ligand binding and protein–protein interactions, and could also prove useful in drug design.
Currently most of the X-ray crystal structures in the Protein Data Bank are ‘snap-shots’ with limited or no information about protein dynamics. However, X-ray diffraction patterns are affected by the dynamics of the protein, and also by distortions of the crystal lattice, so three-dimensional (3D) models of proteins ought to take these phenomena into account. Molecular-dynamics (MD) computer simulations transform 3D structures into 4D ‘molecular movies’ by predicting the movement of individual atoms.
Combining MD simulations with crystallographic data has the potential to produce more realistic ensemble models of proteins in which the atomic fluctuations are represented by multiple structures within the ensemble. Moreover, in addition to improved structural information, this process—which is called ensemble refinement—can provide dynamical information about the protein. Earlier attempts to do this ran into problems because the number of model parameters needed was greater than the number of observed data points. Burnley et al. now overcome this problem by modelling local molecular vibrations with MD simulations and, at the same time, using a course-grain model to describe global disorder of longer length scales.
Ensemble refinement of high-resolution X-ray diffraction datasets for 20 different proteins from the Protein Data Bank produced a better fit to the data than single structures for all 20 proteins. Ensemble refinement also revealed that 3 of the 20 proteins had a ‘molten core’, rather than the well-ordered residues core found in most proteins: this is likely to be important in various biological functions including ligand binding, filament formation and enzymatic function. Burnley et al. also showed that a HIV enzyme underwent an order–disorder transition that is likely to influence how this enzyme works, and that similar transitions might influence the interactions between the small-molecule drug Imatinib (also known as Gleevec) and the enzymes it targets. Ensemble refinement could be applied to the majority of crystallography data currently being collected, or collected in the past, so further insights into the properties and interactions of a variety of proteins and other biomolecules can be expected.
protein; crystallography; structure; function; dynamics; None
The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.
The central nervous system relies on electrical signals travelling along neurons and through synapses at high speeds. Signals often have to pass between two neurons, or from a neuron to a muscle fiber, and the nervous system relies on a process called membrane fusion to ensure that the neurotransmitter molecules that carry the signal across the synapses are released as quickly as possible. Membrane fusion is an important process in many areas of biology, including intracellular transport and fertilization, but it occurs much faster (millisecond time scale) in the nervous system than anywhere else in the body. The reasons for this have long been a mystery, although calcium ions are known to trigger the fusion process.
The fusion of two biological membranes is similar in many regards to the way that small soap bubbles merge together to form large bubbles. Just as soap bubbles can form a variety of discernible intermediate structures when they merge, so can biological membranes. This means that it is possible to produce a so-called hemifusion intermediate in which the outer layers of the membranes have merged, but the inner layers have not, so it is not possible for anything—such as serotonin, dopamine and other neurotransmitter molecules—to transfer from one membrane to the other.
Diao et al. have used a combination of advanced optical imaging and cryogenic electron microscopy to explore membrane fusion between synthetic membranes that contained reconstituted synaptic proteins, including synaptotagmin and a family of protein receptors called SNAREs. When calcium ions were injected into the synthetic system, the basic characteristics of neurotransmitter release—such as membrane fusion on a millisecond time scale—was observed. Contrary to some theories of membrane fusion, the fastest fusion events did not begin or proceed via a discernible hemifusion intermediate state. Rather, these events proceeded from a ‘point contact’ state in which the membranes were close to each other (just 1–5 nm apart) without being fused, and were ready to undergo fast fusion once the calcium ions had been injected. And when Diao et al. introduced a protein called complexin, which is known to be important for fast neurotransmitter release in vivo, they observed more immediate fusion events and fewer events that involved a hemifusion intermediate.
With a synthetic system it is possible to perform experiments that are currently not possible with live neurons, and this has allowed Diao et al. to clarify the roles of the individual components in the process of membrane fusion, and could prove useful in efforts to develop novel therapeutic treatments to combat neurological disorders.
neurotransmitter release; synaptic vesicle fusion; SNARE; synaptotagmin; complexin; Other
In X-ray crystallography, molecular replacement and subsequent refinement is challenging at low resolution. We compared refinement methods using synchrotron diffraction data of photosystem I at 7.4 Å resolution, starting from different initial models with increasing deviations from the known high-resolution structure. Standard refinement spoiled the initial models moving them further away from the true structure and leading to high Rfree-values. In contrast, DEN-refinement improved even the most distant starting model as judged by Rfree, atomic root-mean-square differences to the true structure, significance of features not included in the initial model, and connectivity of electron density. The best protocol was DEN-refinement with initial segmented rigid-body refinement. For the most distant initial model, the fraction of atoms within 2 Å of the true structure improved from 24% to 60%. We also found a significant correlation between Rfree-values and the accuracy of the model, suggesting that Rfree is useful even at low resolution.
DEN refinement; membrane protein; low-resolution refinement; simulated annealing; free R value
A density-based procedure is described for improving a homology model that is locally accurate but differs globally. The model is deformed to match the map and refined, yielding an improved starting point for density modification and further model-building.
An approach is presented for addressing the challenge of model rebuilding after molecular replacement in cases where the placed template is very different from the structure to be determined. The approach takes advantage of the observation that a template and target structure may have local structures that can be superimposed much more closely than can their complete structures. A density-guided procedure for deformation of a properly placed template is introduced. A shift in the coordinates of each residue in the structure is calculated based on optimizing the match of model density within a 6 Å radius of the center of that residue with a prime-and-switch electron-density map. The shifts are smoothed and applied to the atoms in each residue, leading to local deformation of the template that improves the match of map and model. The model is then refined to improve the geometry and the fit of model to the structure-factor data. A new map is then calculated and the process is repeated until convergence. The procedure can extend the routine applicability of automated molecular replacement, model building and refinement to search models with over 2 Å r.m.s.d. representing 65–100% of the structure.
molecular replacement; automation; macromolecular crystallography; structure similarity; modeling; Phenix; morphing
DEN refinement and automated model building with AutoBuild were used to determine the structure of a putative succinyl-diaminopimelate desuccinylase from C. glutamicum. This difficult case of molecular-replacement phasing shows that the synergism between DEN refinement and AutoBuild outperforms standard refinement protocols.
Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.
reciprocal-space refinement; DEN refinement; real-space refinement; automated model building; succinyl-diaminopimelate desuccinylase
The deformable elastic network (DEN) method for reciprocal-space crystallographic refinement improves crystal structures, especially at resolutions lower than 3.5 Å. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements.
Deformable elastic network (DEN) restraints have proved to be a powerful tool for refining structures from low-resolution X-ray crystallographic data sets. Unfortunately, optimal refinement using DEN restraints requires extensive calculations and is often hindered by a lack of access to sufficient computational resources. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements in parallel on the Open Science Grid, the US cyberinfrastructure. Access to the grid is provided through a simple and intuitive web interface integrated into the SBGrid Science Portal. Using this portal, refinements combined with full parameter optimization that would take many thousands of hours on standard computational resources can now be completed in several hours. An example of the successful application of DEN restraints to the human Notch1 transcriptional complex using the grid resource, and summaries of all submitted refinements, are presented as justification.
deformable elastic network restraints; low-resolution refinement; DEN refinement
A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis
Crystallographic structures encompassing GPCR autoproteolytic sequences (GPS) delineate a novel conserved structural domain called GAIN, which is found in cell-adhesion GPCRs, polycystic kidney disease proteins conserved throughout evolution.
The G protein-coupled receptor (GPCR) Proteolysis Site (GPS) of cell-adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ∼40-residue GPS motif represents an integral part of a much larger ∼320-residue domain that we termed GPCR-Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell-adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five β-strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell-adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine-tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.
adhesion GPCRs; autoproteolysis; latrotoxin; polycystic kidney disease-1; synapse
Neurexins are presynaptic cell-adhesion molecules that form trans-synaptic complexes with postsynaptic neuroligins. When overexpressed in non-neuronal cells, neurexins induce formation of postsynaptic specializations in co-cultured neurons, suggesting that neurexins are synaptogenic. However, we now find that when overexpressed in neurons, neurexins do not increase synapse density, but instead selectively suppressed GABAergic synaptic transmission without decreasing GABAergic synapse numbers. This suppression was mediated by all subtypes of neurexins tested, in a cell-autonomous and neuroligin-independent manner. Strikingly, addition of recombinant neurexin to cultured neurons at sub-micromolar concentrations induced the same suppression of GABAergic synaptic transmission as neurexin overexpression. Moreover, experiments with native brain proteins and with purified recombinant proteins revealed that neurexins directly and stoichiometrically bind to GABAA-receptors, suggesting that they decrease GABAergic synaptic responses by interacting with GABAA-receptors. Our findings suggest that besides their other well-documented interactions, presynaptic neurexins directly act on postsynaptic GABAA-receptors, which may contribute to regulate the excitatory/inhibitory balance in brain.
Background: A key step in intoxication by botulinum neurotoxins is the translocation of the protease domain by the translocation domain (TD) across endosomes. The requirements for translocation remain poorly understood.
Results: A construct encompassing the TD yet devoid of the belt embodies a minimum channel-forming unit.
Conclusion: The belt is dispensable for channel formation.
Significance: The belt restricts cargo dissociation from channel during translocation.
Botulinum neurotoxin, the causative agent of the paralytic disease botulism, is an endopeptidase composed of a catalytic domain (or light chain (LC)) and a heavy chain (HC) encompassing the translocation domain (TD) and receptor-binding domain. Upon receptor-mediated endocytosis, the LC and TD are proposed to undergo conformational changes in the acidic endocytic environment resulting in the formation of an LC protein-conducting TD channel. The mechanism of channel formation and the conformational changes in the toxin upon acidification are important but less well understood aspects of botulinum neurotoxin intoxication. Here, we have identified a minimum channel-forming truncation of the TD, the “beltless” TD, that forms transmembrane channels with ion conduction properties similar to those of the full-length TD. At variance with the holotoxin and the HC, channel formation for both the TD and the beltless TD occurs independent of a transmembrane pH gradient. Furthermore, acidification in solution induces moderate secondary structure changes. The subtle nature of the conformational changes evoked by acidification on the TD suggests that, in the context of the holotoxin, larger structural rearrangements and LC unfolding occur preceding or concurrent to channel formation. This notion is consistent with the hypothesis that although each domain of the holotoxin functions individually, each domain serves as a chaperone for the others.
Membrane Proteins; Membrane Reconstitution; Neurotoxin; Patch Clamp; Protein Translocation; Botulinum Neurotoxin; Membranes; Protein Domains; Protein Translocases; Single Channels
Most current crystallographic structure refinements augment the diffraction data with a priori information consisting of bond, angle, dihedral, planarity restraints and atomic repulsion based on the Pauli exclusion principle. Yet, electrostatics and van der Waals attraction are physical forces that provide additional a priori information. Here we assess the inclusion of electrostatics for the force field used for all-atom (including hydrogen) joint neutron/X-ray refinement. Two DNA and a protein crystal structure were refined against joint neutron/X-ray diffraction data sets using force fields without electrostatics or with electrostatics. Hydrogen bond orientation/geometry favors the inclusion of electrostatics. Refinement of Z-DNA with electrostatics leads to a hypothesis for the entropic stabilization of Z-DNA that may partly explain the thermodynamics of converting the B form of DNA to its Z form. Thus, inclusion of electrostatics assists joint neutron/X-ray refinements, especially for placing and orienting hydrogen atoms.
This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators.
► Validation criteria used by the PDB for X-ray crystal structures have been reassessed ► Key scores should be presented prominently in an easily understood format ► A concise validation report should be available to referees of papers on crystal structures
Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.
single molecule fluorescence; FRET; molecular dynamics; protein-protein interactions
X-ray diffraction plays a pivotal role in understanding of biological systems by revealing atomic structures of proteins, nucleic acids, and their complexes, with much recent interest in very large assemblies like the ribosome. Since crystals of such large assemblies often diffract weakly (resolution worse than 4 Å), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, while others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex1. Determining such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution below 5 Å generally exceed the number of degrees of freedom. Here we introduce a new method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with Rfree determines the optimum deformation and influence of the homology model. For test cases at 3.5 – 5 Å resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model coordinate accuracy, the definition of secondary structure, and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the PDB, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to studying weakly diffracting crystals using X-ray micro-diffraction2 as well as data from new X-ray light sources3. Use of homology information is not restricted to X-ray crystallography and cryo-electron microscopy: as optical imaging advances to sub-nanometer resolution4,5, it can use similar tools.
X-ray crystallography; homology modeling; cross-validation; Rfree value; refinement
Synchronous neurotransmission is triggered when Ca2+ binds to synaptotagmin 1, a synaptic vesicle protein that interacts with SNAREs and membranes. We used single-molecule FRET between synaptotagmin’s two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intra-synaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive synaptotagmin 1–SNARE complex by using a multi-body docking approach with 34 FRET-derived distances as restraints. The Ca2+-binding loops point away from the SNARE complex, so they could interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca2+ bound synaptotagmin 3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs plays a role in Ca2+-triggered fusion.
membrane fusion; neurotransmitter release; protein-protein interactions; synaptic vesicle; single molecule FRET
In neurons, SNAREs, synaptotagmin, and other factors catalyze Ca2+-triggered fusion of vesicles with the plasma membrane. The molecular mechanism of this process remains an enigma, especially regarding the interaction between synaptotagmin and SNAREs. Here we characterized this interaction by single-molecule fluorescence microscopy and crystallography. The two rigid Ca2+-binding domains of synaptotagmin 3 undergo large relative motions in solution. Interaction with SNARE complex amplifies a particular state of the two domains that is further enhanced by Ca2+. This state is represented by the first SNARE-induced Ca2+-bound crystal structure of a synaptotagmin fragment containing both domains. The arrangement of the Ca2+-binding loops of this structure of synaptotagmin 3 matches that of SNARE-bound synaptotagmin 1, suggesting a conserved feature of synaptotagmins. The loops resemble the membrane-interacting loops of certain viral fusion proteins in the postfusion state, suggesting unexpected similarities between both fusion systems.
neurotransmitter release; synaptic vesicle; SNARE-induced fusion; virus-induced fusion; Ca2+ sensor; single molecule fluorescence microscopy
The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their Ki values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2′ position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.
Formation of a binary complex between syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) at the active zone is believed to precede assembly of the ternary SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex that is essential for neurotransmitter release. Despite its importance in models of synaptic neurotransmitter release, this binary complex has been difficult to characterize by bulk methods due to the prevalence of a 2:1 dead-end species. Here we used single molecule fluorescence resonance energy transfer (smFRET) to study the structure and dynamics of the 1:1 syntaxin/SNAP-25 binary complex. The binary complex is conformationally variable with FRET efficiency states often changing on the second timescale. One state corresponds to a parallel three-helix bundle configuration, while other states correspond to configurations with one of the SNAP-25 SNARE domains dissociated. All configurations of the binary complex are rapidly locked into the single three-helix bundle configuration by the addition of synaptobrevin. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex serves as a dynamic acceptor for synaptobrevin binding, and interactions with accessory proteins stabilize this acceptor. In a high protein density cellular environment the syntaxin/SNAP-25 complex is therefore expected to be in the configuration where it can rapidly interact with synaptobrevin so its formation is unlikely a limiting step for SNARE-mediated neurotransmitter release.
membrane fusion; vesicle trafficking; synaptic neurotransmission; single molecule fluorescence spectroscopy; t-SNARE
SNAREs are essential components of the machinery for Ca2+-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. While much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca2+ sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single molecule methodology. In this review we discuss first applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such the possibility of parallel and anti-parallel SNARE complexes, or vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.
FRET; membrane fusion; neurotransmission; synaptic vesicle