Scaffold proteins contain multiple binding modules that allow for co-localization of proteins that lack a direct interaction. Evolution resulted in different combinations of binding modules that rewired existing signal transduction pathways. The synaptic Membrane-Associated Guanylate Kinase (MAGuK) scaffold proteins post-synaptic density protein 93 (PSD-93) and post-synaptic density protein 95 (PSD-95) share the same organization of highly conserved binding domains connected by divergent linkers, which generates opposing functions in synaptic plasticity. Both are targets for tyrosine phosphorylation by Src kinase, but the extent and impacts of phosphorylation are poorly understood. Here, we characterized the in vitro phosphorylation of PSD-93 and PSD-95. Both were targeted at multiple sites that were similarly distributed, but PSD-93 was a more robust substrate. While Src kinase has been linked to synaptic plasticity, the direct effects on MAGuK function are not known. We found that phosphorylation differentially affected the affinity of PSD-93 and PSD-95 for postsynaptic client proteins. Phosphorylation also elicited opposite responses in recruitment of key synaptic clients by MAGuKs into supercomplexes, which we equate with scaffolding activity. Surprisingly, phosphorylation affected biomolecular condensation in PSD-93 but not PSD-95, which suggests that the affinity of protein interactions is disconnected from phase separation. Phosphorylation had minimal impacts on the supertertiary structure as assessed by small angle x-ray scattering although single molecule fluorescence resonance energy transfer revealed altered dynamics leaving the origin of functional effects unclear. PSD-95 appears optimized to work with small protein numbers while PSD-93 modulates condensation at higher protein concentrations. This difference suggests action at different stages of synapse maturation, which could be linked to synaptic plasticity.

Pore forming proteins are a diverse collection of polypeptides, which share little structural or amino acid sequence homology and span several mechanistic classes. Their commonality lies in their ability to create transmembrane pores in biological membranes, which places some pore forming proteins among nature's most toxic substances. Such membrane pores can dissipate chemical and electrical gradients, release cellular contents, and even deliver toxic cargo. Detecting pore forming activity commonly relies on dye release assays, which measure a change in brightness as quenched dyes are diluted. Single molecule detection provides the ultimate sensitivity, but measuring relative brightness is challenging due to intensity variation across the population. An ideal sensor could allow interrogation of the entire population of liposomes after pore formation without requiring foreknowledge of the initial intensity. To achieve this we have developed a FRET biosensor approach using ligand-responsive oligonucleotides, which are encapsulated within liposomes that sustain chemical gradients. We show that dissipation of transmembrane gradients can be measured with single liposome resolution using TIRF microscopy, which allows detection of pore forming proteins regardless of mechanistic class. Our encapsulated oligonucleotide biosensors could detect the presence of Botulinum neurotoxin down to picomolar concentrations without the need for protein-specific immunoreagents and highlighted the role of proteolytic activation in pore formation by the toxin. Adapting this approach to additional oligonucleotide sensors would provide a general platform to detect transmembrane solute movement and dissect the underlying transport mechanisms.

Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique for studying the structural dynamics of protein molecules or detecting interactions between protein molecules in real time. Due to the high sensitivity in spatial and temporal resolution, smFRET can decipher sub-populations within heterogeneous native state conformations, which are generally lost in traditional measurements due to ensemble averaging. In addition, the single-molecule reconstitution allows protein molecules to be observed for an extensive period of time and can recapitulate the geometry of the cellular environment to retain biological function. Here we provide a detailed method of using smFRET to monitor the conformational dynamics of syntaxin-3b from the ribbon synapses during assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.

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.

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 P DZ3, S H3, and G uanylate 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.

Multidomain proteins perform a multitude of biological functions facilitated by the diverse interactions of their constituent domains. Often, flexible linker regions connect these domains, allowing them to adopt a series of supertertiary structures. However, it is increasingly evident that linkers act as more than spacers; instead, they can modulate protein-protein interactions as participants at binding interfaces or by effectively limiting the supertertiary conformations. Previously, we showed that the two N-terminal PDZ domains of PSD-95 (PDZ12 tandem), connected by a short and flexible linker, adopt at least two distinct conformations characterized by weak interactions.

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.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ionotropic channels with high calcium permeability that play important roles in synaptic plasticity. The extracellular and transmembrane domains maintain the communication between pre- and post-synapse through ligand-induced changes in ion permeability, which have been extensively explored down to the atomic level. In contrast, structural and functional understanding of the intracellular C-terminal domain (CTD) remains still elusive. The long CTDs of GluN2 subunits recruit other proteins as part of the signaling pathways downstream of NMDARs, which have been linked to condensate formation through PSD-95 and SynGAP.

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.

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.

PSD-95, a member of the membrane-associated guanylate kinase (MAGUK) family, is a scaffold protein responsible for the clustering of ionotropic glutamate receptors and ion channels on the postsynaptic membrane of the excitatory synapse. The interdomain organization within the supertertiary structure of PSD-95 reveals two independent supramodules, PDZ1-PDZ2 tandem and PDZ3-SH3-GuK (PSG) core, which are conserved within the synaptic MAGUK homologs. Uncovering the inter-domain interactions and dynamic associations within each supramodule is crucial for elucidating the functional role of PSD-95 in molecular scaffolding and signal transduction. Here, we study the supertertiary structure of the PSG core of PSD-95 by combining replica exchange molecular dynamics simulations with single-molecular FRET (smFRET), Multiparameter Fluorescence Detection (MFD), and disulfide mapping gel assays. Computationally derived inter-residue distances were consistent with smFRET measurements for a large set of 44 inter-dye labeling sites with a Pearson correlation coefficient of ∼0.71. Free energy analysis of DMD simulations revealed three distinct conformational states, where the lowest energy basin had PDZ3 binding to the α-helix of SH3 along its canonical CRIPT binding interface. PDZ3 was also observed to bind GuK in simulations without competing with the MAP1a binding site in the GuK. The third state corresponded to an extended state with PDZ3 dissociated from SH3-GuK. These identified conformational states not only agreed with MFD measurements, but were also confirmed by disulfide mapping experiments where double-cysteine mutants were introduced according to computationally derived supertertiary structures of PSG. Our results underscore the predictive power of the hybrid approach combining DMD simulations with multi-scale FRET experiments and offered structural and dynamic insights to the role of PSG supramodule in PSD-95 scaffolding and signal transduction.

Postsynaptic Density Protein 95 (PSD95) is a MAGUK-family scaffolding protein found in the excitatory postsynaptic density (PSD). PSD95 plays key roles in regulating synaptic organization and strength through interactions with neuroreceptors at the postsynaptic membrane. Consisting of 5 subdomains (3 PDZ domains, and SH3 domain, and a catalytically-inactive guanylate kinase (GuK) domain) connected by flexible polypeptide linkers, PSD95 further adopts a supertertiary structure in which the first two PDZ domains and the latter three domains partition into two supramodules (PDZ1-2 tandem and PSG supramodule). However, while the structures of the subdomains of PSD95 are well-studied, the underlying mechanisms governing PSD95 function and regulation are not entirely understood. A key characteristic of PSD95 is its structural dynamics. Dynamic intramolecular interactions between the five subdomains provide an attractive explanation for a degree of self-regulation of PSD95 that could extend to other PDZ-containing MAGUKs. In the present work we report a study of full-length PSD95, utilizing an integrative approach combining single-molecule FRET and multiparameter fluorescence detection (MFD) with discrete molecular dynamics (DMD) simulations and using biochemical methods for cross-validation. We analyzed 16 FRET variants to probe dynamics between pairwise combinations of PSD95 subdomains within and between the supramodules. Several key conclusions were drawn from this study: i) we corroborate previous results for the PDZ1-2 tandem identifying fast dynamics between two distinct conformations, now using full-length PSD95, ii) we characterize distinct dynamics within the supramodules and spanning the full PSD95, and iii) we identify three distinct, dynamically interacting structural conformations in the PSG supramodule via DMD and FRET screening. Our findings provide insights into how the interplay between structure and dynamics give rise to biomolecular function and regulation for PSD proteins.

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. X-ray crystal structures of the full-length TcdB exotoxin of bacterial pathogen Clostridium difficile reveal pH-dependent conformational changes that allow translocation of the toxin from endosomes into the cytosol.

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.

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.

A variety of de novo missense mutations associated with neurological disorders occur in ionotropic glutamate receptors (iGluRs) subunits. These disease mutations often appear in motifs critical to the process of glutamate-induced channel opening or gating. Numerous missense mutations are found in the NMDA receptor M4 segments, a transmembrane segment peripheral to the pore domain in eukaryotic iGluRs. Subsets of these missense mutations affect receptor gating but very dramatic effects, including in one instance halting gating, occurred at a conserved glycine, positioned at the extracellular end of M4. Even alanine substitutions at this glycine, most notably in GluN1, severely restricted the stability of the open state. Molecular dynamics simulations suggest that this glycine in GluN1 permits unique backbone interactions as well as the extreme extracellular end of M4 to unravel in the open state. Surprisingly, in an AMPAR structure, reorientation of the extreme extracellular end of M4 centered on this conserved glycine occurs, but in contrast to NMDARs, in a closed state. Consistent with these structural differences, an alanine substitution at this glycine in AMPARs destabilizes a closed state, in direct contrast to that for NMDARs. Hence, structural features unique to glycine stabilize opposing conformations in iGluR subtypes. In NMDARs, missense mutations at this conserved glycine dramatically alter their function at synapses. These results have strong implications for how such disorders are caused at the ion channel level and highlight how structural features in NMDAR and AMPAR have evolved to permit them to carry out different functional roles at synapses.