Fuzzy supertertiary interactions within PSD-95 enable ligand binding.

The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by PDZ3, SH3, and Guanylate Kinase domains, regulate PSD-95 activity. Here we combined discrete molecular dynamics and single molecule Förster resonance energy transfer (FRET) to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand.

Conformational change of Syntaxin-3b in regulating SNARE complex assembly in the ribbon synapses.

Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigated the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

Structure and conformational dynamics of Clostridioides difficile toxin A.

Clostridioides difficile toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with C. difficile infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843-T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formation of TcdA. A rationally designed TcdA mutation that trapped the CROPs in the closed conformation showed drastically reduced cytotoxicity. Taken together, these studies shed new lights into the conformational dynamics of TcdA and its roles in TcdA intoxication.

Different Forms of Disorder in NMDA-Sensitive Glutamate Receptor Cytoplasmic Domains Are Associated with Differences in Condensate Formation.

The N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor (NMDAR) helps assemble downstream signaling pathways through protein interactions within the postsynaptic density (PSD), which are mediated by its intracellular C-terminal domain (CTD). The most abundant NMDAR subunits in the brain are GluN2A and GluN2B, which are associated with a developmental switch in NMDAR composition. Previously, we used single molecule fluorescence resonance energy transfer (smFRET) to show that the GluN2B CTD contained an intrinsically disordered region with slow, hop-like conformational dynamics. The CTD from GluN2B also undergoes liquid-liquid phase separation (LLPS) with synaptic proteins. Here, we extend these observations to the GluN2A CTD. Sequence analysis showed that both subunits contain a form of intrinsic disorder classified as weak polyampholytes. However, only GluN2B contained matched patterning of arginine and aromatic residues, which are linked to LLPS. To examine the conformational distribution, we used discrete molecular dynamics (DMD), which revealed that GluN2A favors extended disordered states containing secondary structures while GluN2B favors disordered globular states. In contrast to GluN2B, smFRET measurements found that GluN2A lacked slow conformational dynamics. Thus, simulation and experiments found differences in the form of disorder. To understand how this affects protein interactions, we compared the ability of these two NMDAR isoforms to undergo LLPS. We found that GluN2B readily formed condensates with PSD-95 and SynGAP, while GluN2A failed to support LLPS and instead showed a propensity for colloidal aggregation. That GluN2A fails to support this same condensate formation suggests a developmental switch in LLPS propensity.

Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer.

Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.

Structure of the full-length Clostridium difficile toxin B.

Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB-antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.

Spontaneous Switching among Conformational Ensembles in Intrinsically Disordered Proteins.

The common conception of intrinsically disordered proteins (IDPs) is that they stochastically sample all possible configurations driven by thermal fluctuations. This is certainly true for many IDPs, which behave as swollen random coils that can be described using polymer models developed for homopolymers. However, the variability in interaction energy between different amino acid sequences provides the possibility that some configurations may be strongly preferred while others are forbidden. In compact globular IDPs, core hydration and packing density can vary between segments of the polypeptide chain leading to complex conformational dynamics. Here, we describe a growing number of proteins that appear intrinsically disordered by biochemical and bioinformatic characterization but switch between restricted regions of conformational space. In some cases, spontaneous switching between conformational ensembles was directly observed, but few methods can identify when an IDP is acting as a restricted chain. Such switching between disparate corners of conformational space could bias ligand binding and regulate the volume of IDPs acting as structural or entropic elements. Thus, mapping the accessible energy landscape and capturing dynamics across a wide range of timescales are essential to recognize when an IDP is acting as such a switch.

A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1.

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic ß-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Identifying weak interdomain interactions that stabilize the supertertiary structure of the N-terminal tandem PDZ domains of PSD-95.

Previous studies of the N-terminal PDZ tandem from PSD-95 produced divergent models and failed to identify interdomain contacts stabilizing the structure. We used ensemble and single-molecule FRET along with replica-exchange molecular dynamics to fully characterize the energy landscape. Simulations and experiments identified two conformations: an open-like conformation with a small contact interface stabilized by salt bridges, and a closed-like conformation with a larger contact interface stabilized by surface-exposed hydrophobic residues. Both interfaces were confirmed experimentally. Proximity of interdomain contacts to the binding pockets may explain the observed coupling between conformation and binding. The low-energy barrier between conformations allows submillisecond dynamics, which were time-averaged in previous NMR and FRET studies. Moreover, the small contact interfaces were likely overridden by lattice contacts as crystal structures were rarely sampled in simulations. Our hybrid approach can identify transient interdomain interactions, which are abundant in multidomain proteins yet often obscured by dynamic averaging.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

Divergent roles of a peripheral transmembrane segment in AMPA and NMDA receptors.

Ionotropic glutamate receptors (iGluRs), including AMPA receptor (AMPAR) and NMDA receptor (NMDAR) subtypes, are ligand-gated ion channels that mediate signaling at the majority of excitatory synapses in the nervous system. The iGluR pore domain is structurally and evolutionarily related to an inverted two-transmembrane K+ channel. Peripheral to the pore domain in eukaryotic iGluRs is an additional transmembrane helix, the M4 segment, which interacts with the pore domain of a neighboring subunit. In AMPARs, the integrity of the alignment of a specific face of M4 with the adjacent pore domain is essential for receptor oligomerization. In contrast to AMPARs, NMDARs are obligate heterotetramers composed of two GluN1 and typically two GluN2 subunits. Here, to address the function of the M4 segments in NMDARs, we carry out a tryptophan scan of M4 in GluN1 and GluN2A subunits. Unlike AMPARs, the M4 segments in NMDAR subunits makes only a limited contribution to their biogenesis. However, the M4 segments in both NMDAR subunits are critical for receptor activation, with mutations at some positions, most notably at the extreme extracellular end, completely halting the gating process. Furthermore, although the AMPAR M4 makes a minimal contribution to receptor desensitization, the NMDAR M4 segments have robust and subunit-specific effects on desensitization. These findings reveal that the functional roles of the M4 segments in AMPARs and NMDARs have diverged in the course of their evolution and that the M4 segments in NMDARs may act as a transduction pathway for receptor modulation at synapses.

The insulin and IGF1 receptor kinase domains are functional dimers in the activated state.

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

Reconstitution of multivalent PDZ domain binding to the scaffold protein PSD-95 reveals ternary-complex specificity of combinatorial inhibition.

Multidomain scaffold proteins serve as hubs in the signal transduction network. By physically colocalizing sequential steps in a transduction pathway, scaffolds catalyze and direct incoming signals. Much is known about binary interactions with individual domains, but it is unknown whether "scaffolding activity" is predictable from pairwise affinities. Here, we characterized multivalent binding to PSD-95, a scaffold protein containing three PDZ domains connected in series by disordered linkers. We used single molecule fluorescence to watch soluble PSD-95 recruit diffusing proteins to a surface-attached receptor cytoplasmic domain. Different ternary complexes showed unique concentration dependence for scaffolding despite similar pairwise affinity. The concentration dependence of scaffolding activity was not predictable based on binary interactions. PSD-95 did not stabilize specific complexes, but rather increased the frequency of transient binding events. Our results suggest that PSD-95 maintains a loosely connected pleomorphic ensemble rather than forming a stereospecific complex containing all components.

Modulating the intrinsic disorder in the cytoplasmic domain alters the biological activity of the N-methyl-D-aspartate-sensitive glutamate receptor.

The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating.

Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved.

Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from single-molecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.

Immobilization of proteins for single-molecule fluorescence resonance energy transfer measurements of conformation and dynamics.

Fluorescence resonance energy transfer provides information about protein structure and dynamics. Single-molecule analysis can capture the information normally lost through ensemble averaging of heterogeneous and dynamic samples. Immobilization of single molecules, under conditions that retain their biological activity, allows for extended observation of the same molecule for tens of seconds. This can capture slow conformational transitions or protein binding and unbinding cycles. Using an open geometry for immobilization allows for direct observation of the response to changing solution conditions or adding ligands. Here we provide detailed methods for immobilization and observation of fluorescently labeled single proteins using total internal reflection microscopy that are widely applicable to the study of intrinsically disordered proteins.

Effect of Src kinase phosphorylation on disordered C-terminal domain of N-methyl-D-aspartic acid (NMDA) receptor subunit GluN2B protein.

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.

Domain orientation in the N-Terminal PDZ tandem from PSD-95 is maintained in the full-length protein.

Tandem PDZ domains have been suggested to form structurally independent supramodules. However, dissimilarity between crystallography and NMR models emphasize their malleable conformation. Studies in full-length scaffold proteins are needed to examine the effect of tertiary interactions within their native context. Using single-molecule fluorescence to characterize the N-terminal PDZ tandem in PSD-95, we provide the first direct evidence that PDZ tandems can be structurally independent within a full-length scaffold protein. Molecular refinement using our data converged on a single structure with an antiparallel alignment of the ligand-binding sites. Devoid of interaction partners, single-molecule conditions captured PSD-95 in its unbound, ground state. Interactions between PDZ domains could not be detected while fluctuation correlation spectroscopy showed that other conformations are dynamically sampled. We conclude that ultra-weak interactions stabilize the conformation providing a "low-relief" energy landscape that allows the domain orientation to be flipped by environmental interactions.