Rescuing tri-heteromeric NMDA receptor function: the potential of pregnenolone-sulfate in loss-of-function GRIN2B variants.

N-methyl-D-aspartate receptors (NMDARs emerging from GRIN genes) are tetrameric receptors that form diverse channel compositions in neurons, typically consisting of two GluN1 subunits combined with two GluN2(A-D) subunits. During prenatal stages, the predominant channels are di-heteromers with two GluN1 and two GluN2B subunits due to the high abundance of GluN2B subunits. Postnatally, the expression of GluN2A subunits increases, giving rise to additional subtypes, including GluN2A-containing di-heteromers and tri-heteromers with GluN1, GluN2A, and GluN2B subunits. The latter  emerge as the major receptor subtype at mature synapses in the hippocampus. Despite extensive research on purely di-heteromeric receptors containing two identical GRIN variants, the impact of a single variant on the function of other channel forms, notably tri-heteromers, is lagging. In this study, we systematically investigated the effects of two de novo GRIN2B variants (G689C and G689S) in pure, mixed di- and tri-heteromers. Our findings reveal that incorporating a single variant in mixed di-heteromers or tri-heteromers exerts a dominant negative effect on glutamate potency, although 'mixed' channels show improved potency compared to pure variant-containing di-heteromers. We show that a single variant within a receptor complex does not impair the response of all receptor subtypes to the positive allosteric modulator pregnenolone-sulfate (PS), whereas spermine completely fails to potentiate tri-heteromers containing GluN2A and -2B-subunits. We examined PS on primary cultured hippocampal neurons transfected with the variants, and observed a positive impact over current amplitudes and synaptic activity. Together, our study supports previous observations showing that mixed di-heteromers exhibit improved glutamate potency and extend these findings towards the exploration of the effect of Loss-of-Function variants over tri-heteromers. Notably, we provide an initial and crucial demonstration of the beneficial effects of GRIN2B-relevant potentiators on tri-heteromers. Our results underscore the significance of studying how different variants affect distinct receptor subtypes, as these effects cannot be inferred solely from observations made on pure di-heteromers. Overall, this study contributes to ongoing efforts to understand the pathophysiology of GRINopathies and provides insights into potential treatment strategies.

Molecular Dynamics Simulations of the Mutated Proton-Transferring a-Subunit of E. coli FoF1-ATP Synthase.

The membrane Fo factor of ATP synthase is highly sensitive to mutations in the proton half-channel leading to the functional blocking of the entire protein. To identify functionally important amino acids for the proton transport, we performed molecular dynamic simulations on the selected mutants of the membrane part of the bacterial FoF1-ATP synthase embedded in a native lipid bilayer: there were nine different mutations of a-subunit residues (aE219, aH245, aN214, aQ252) in the inlet half-channel. The structure proved to be stable to these mutations, although some of them (aH245Y and aQ252L) resulted in minor conformational changes. aH245 and aN214 were crucial for proton transport as they directly facilitated H+ transfer. The substitutions with nonpolar amino acids disrupted the transfer chain and water molecules or neighboring polar side chains could not replace them effectively. aE219 and aQ252 appeared not to be determinative for proton translocation, since an alternative pathway involving a chain of water molecules could compensate the ability of H+ transmembrane movement when they were substituted. Thus, mutations of conserved polar residues significantly affected hydration levels, leading to drastic changes in the occupancy and capacity of the structural water molecule clusters (W1-W3), up to their complete disappearance and consequently to the proton transfer chain disruption.

DeepSub: Utilizing Deep Learning for Predicting the Number of Subunits in Homo-Oligomeric Protein Complexes.

The molecular weight (MW) of an enzyme is a critical parameter in enzyme-constrained models (ecModels). It is determined by two factors: the presence of subunits and the abundance of each subunit. Although the number of subunits (NS) can potentially be obtained from UniProt, this information is not readily available for most proteins. In this study, we addressed this gap by extracting and curating subunit information from the UniProt database to establish a robust benchmark dataset. Subsequently, we propose a novel model named DeepSub, which leverages the protein language model and Bi-directional Gated Recurrent Unit (GRU), to predict NS in homo-oligomers solely based on protein sequences. DeepSub demonstrates remarkable accuracy, achieving an accuracy rate as high as 0.967, surpassing the performance of QUEEN. To validate the effectiveness of DeepSub, we performed predictions for protein homo-oligomers that have been reported in the literature but are not documented in the UniProt database. Examples include homoserine dehydrogenase from Corynebacterium glutamicum, Matrilin-4 from Mus musculus and Homo sapiens, and the Multimerins protein family from M. musculus and H. sapiens. The predicted results align closely with the reported findings in the literature, underscoring the reliability and utility of DeepSub.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Functional Analysis of NMDAR Subunit Components in Postsynaptic Currents of Identified Cells and Synapses in Brain Slices.

Epilepsy is one of the most represented neurological diseases worldwide. However, in many cases, the precise molecular mechanisms of epileptogenesis and ictiogenesis are unknown. Because of their important role in synaptic function and neuronal excitability, NMDA receptors are implicated in various epileptogenic mechanisms. Most of these are subunit specific and require a precise analysis of the subunit composition of the NMDARs implicated. Here, we describe an express electrophysiological method to analyze the contribution of NMDAR subunits to spontaneous postsynaptic activity in identified cells in brain slices using patch clamp whole cell recordings.

Selective Cell-Surface Expression of Triheteromeric NMDA Receptors.

NMDA-type ionotropic glutamate receptors are critically involved in many brain functions and are implicated in a variety of brain disorders. Seven NMDA receptor subunits exist (GluN1, GluN2A-D, and GluN3A-B) that assemble into tetrameric receptor subtypes with distinct functional properties and physiological roles. The majority NMDA receptors are composed of two GluN1 and two GluN2 subunits, which can assemble into four diheteromeric receptors subtypes composed of GluN1 and one type of GluN2 subunit (e.g., GluN1/2A), and presumably also six triheteromeric receptor subtypes composed of GluN1 and two different GluN2 subunits (e.g., GluN1/2A/2B). Furthermore, the GluN1 subunit exists as eight splice variants (e.g., GluN1-1a and GluN1-1b isoforms), and two different GluN1 isoforms can co-assemble to also form triheteromeric NMDA receptors (e.g., GluN1-1a/1b/2A). Here, we describe a method to faithfully express triheteromeric NMDA receptors in heterologous expression systems by controlling the identity of two of the four subunits. This method overcomes the problem that co-expression of three different NMDA receptor subunits generates two distinct diheteromeric receptor subtypes as well as one triheteromeric receptor subtype, thereby confounding studies that require a homogenous population of triheteromeric NMDA receptors. The method has been applied to selectively express recombinant triheteromeric GluN1/2A/2B, GluN1/2A/2C, GluN1/2B/2D, GluN1-1a/GluN1-1b/2A, GluN1-1a/GluN1-1b/2B receptors with negligible co-expression of the respective diheteromeric receptor subtypes. This method therefore enables quantitative evaluation of functional and pharmacological properties of triheteromeric NMDA receptors, some of which are abundant NMDA receptor subtypes in the adult brain.

Characterizing Functional Contributions of Specific GluN2 Subunits to Individual Postsynaptic NMDAR Responses Using Biophysical Parameters.

The NMDAR is a heterotetramer composed of two GluN1 subunits and two GluN2 and/or GluN3 subunits, with the GluN2 subunits exhibiting significant diversity in their structure and function. Recent studies have highlighted the importance of characterizing the specific roles of each GluN2 subunit across central nervous system regions and developmental stages, as well as their unique contributions to NMDAR-mediated signaling and plasticity. Understanding the distinct functions of GluN2 subunits is critical for the development of targeted therapeutic strategies for NMDAR-related disorders. However, measuring the functional contribution of individual GluN2 subtypes in ex vivo slices is challenging. Conventionally, pharmacological or genetic approaches are used, but, in many cases, this is not possible or is restricted to population-level NMDAR responses. Here, we describe a technique for using biophysical properties of miniature synaptic NMDAR responses as a proxy to measure the functional contribution of specific GluN2-NMDAR subunits to individual synapses within a neuron.

Molecular and physiological characterization of AIP1, encoding the acetolactate synthase regulatory subunit in rice.

Flooding deprives plants of oxygen and thereby causes severe stress by interfering with energy production, leading to growth retardation. Enzymes and metabolites may help protect plants from waterlogging and hypoxic environmental conditions. Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of branched-chain amino acids (BCAAs), providing the building blocks for proteins and various secondary metabolites. Additionally, under energy-poor conditions, free BCAAs can be used as an alternative energy source by mitochondria through a catabolic enzyme chain reaction. In this study, we characterized ALS-INTERACTING PROTEIN 1 (OsAIP1), which encodes the regulatory subunit of ALS in rice (Oryza sativa). This gene was expressed in all parts of the rice plant, and its expression level was significantly higher in submerged and low-oxygen environments. Rice transformants overexpressing OsAIP1 showed a higher survival rate under hypoxic stress than did non-transgenic control plants under the same conditions. The OsAIP1-overexpressing plants accumulated increased levels of BCAAs, demonstrating that OsAIP1 is an important factor in the hypoxia resistance mechanism. These results suggest that ALS proteins are part of a defense mechanism that improves the tolerance of plants to low-oxygen environments.

CORVET-specific subunit levels determine the balance between HOPS/CORVET endosomal tethering complexes.

The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively, and HOPS also mediates the fusion of lysosomes with incoming vesicles including late endosomes and autophagosomes. These heterohexameric complexes share their four core subunits that assemble with additional two, complex-specific subunits. These features and the similar structure of the complexes could allow the formation of hybrid complexes, and the complex specific subunits may compete for binding to the core. Indeed, our biochemical analyses revealed the overlap of binding sites for HOPS-specific VPS41 and CORVET-specific VPS8 on the shared core subunit VPS18. We found that the overexpression of CORVET-specific VPS8 or Tgfbrap1 decreased the amount of core proteins VPS11 and VPS18 that are assembled with HOPS-specific subunits VPS41 or VPS39, indicating reduced amount of assembled HOPS. In line with this, we observed the elevation of both lipidated, autophagosome-associated LC3 protein and the autophagic cargo p62 in these cells, suggesting impaired autophagosome-lysosome fusion. In contrast, overexpression of HOPS-specific VPS39 or VPS41 did not affect the level of assembled CORVET or autophagy. VPS8 or Tgfbrap1 overexpression also induced Cathepsin D accumulation, suggesting that HOPS-dependent biosynthetic delivery of lysosomal hydrolases is perturbed, too. These indicate that CORVET-specific subunit levels fine-tune HOPS assembly and activity in vivo.

Modulation of proteasome subunit selectivity of syringolins.

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure-activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure-activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding ß5 and ß2 subunits.

Structural basis of Integrator-dependent RNA polymerase II termination.

The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.

How an intrinsically disordered regulatory subunit assembles a PP1:eIF2 complex.

Fatalska et al.1 use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and positions the phosphatase-substrate complex for dephosphorylation. As validation, they show that a disease mutation abolishes the interaction.

A structure-based computational model of IP3R1 incorporating Ca and IP3 regulation.

The inositol 1,4,5-triphosphate receptor (IP3R) mediates Ca release in many cell types and is pivotal to a wide range of cellular processes. High-resolution cryoelectron microscopy studies have provided new structural details of IP3R type 1 (IP3R1), showing that channel function is determined by the movement of various domains within and between each of its four subunits. Channel properties are regulated by ligands, such as Ca and IP3, which bind at specific sites and control the interactions between these domains. However, it is not known how the various ligand-binding sites on IP3R1 interact to control the opening of the channel. In this study, we present a coarse-grained model of IP3R1 that accounts for the channel architecture and the location of specific Ca- and IP3-binding sites. This computational model accounts for the domain-domain interactions within and between the four subunits that form IP3R1, and it also describes how ligand binding regulates these interactions. Using a kinetic model, we explore how two Ca-binding sites on the cytosolic side of the channel interact with the IP3-binding site to regulate the channel open probability. Our primary finding is that the bell-shaped open probability of IP3R1 provides constraints on the relative strength of these regulatory binding sites. In particular, we argue that a specific Ca-binding site, whose function has not yet been established, is very likely a channel antagonist. Additionally, we apply our model to show that domain-domain interactions between neighboring subunits exert control over channel cooperativity and dictate the nonlinear response of the channel to Ca concentration. This suggests that specific domain-domain interactions play a pivotal role in maintaining the channel's stability, and a disruption of these interactions may underlie disease states associated with Ca dysregulation.

The synergy between the PscC subunits for electron transfer to the P840 special pair in Chlorobaculum tepidum.

The primary photochemical reaction of photosynthesis in green sulfur bacteria occurs in the homodimer PscA core proteins by a special chlorophyll pair. The light induced excited state of the special pair producing P840+ is rapidly reduced by electron transfer from one of the two PscC subunits. Molecular dynamics (MD) simulations are combined with bioinformatic tools herein to provide structural and dynamic insight into the complex between the two PscA core proteins and the two PscC subunits. The microscopic dynamic model involves extensive sampling at atomic resolution and at a cumulative time-scale of 22µs and reveals well defined protein-protein interactions. The membrane complex is composed of the two PscA and the two PscC subunits and macroscopic connections are revealed within a putative electron transfer pathway from the PscC subunit to the special pair P840 located within the PscA subunits. Our results provide a structural basis for understanding the electron transport to the homodimer RC of the green sulfur bacteria. The MD based approach can provide the basis to further probe the PscA-PscC complex dynamics and observe electron transfer therein at the quantum level. Furthermore, the transmembrane helices of the different PscC subunits exert distinct dynamics in the complex.

The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

Exploring the structural assembly of rice ADP-glucose pyrophosphorylase subunits using MD simulation.

ADP-glucose pyrophosphorylase plays a pivotal role as an allosteric enzyme, essential for starch biosynthesis in plants. The higher plant AGPase comparises of a pair of large and a pair of small subunits to form a heterotetrameric complex. Growing evidence indicates that each subunit plays a distinct role in regulating the underlying mechanism of starch biosynthesis. In the rice genome, there are four large subunit genes (OsL1-L4) and three small subunit genes (OsS1, OsS2a, and OsS2b). While the structural assembly of cytosolic rice AGPase subunits (OsL2:OsS2b) has been elucidated, there is currently no such documented research available for plastidial rice AGPases (OsL1:OsS1). In this study, we employed protein modeling and MD simulation approaches to gain insights into the structural association of plastidial rice AGPase subunits. Our results demonstrate that the heterotetrameric association of OsL1:OsS1 is very similar to that of cytosolic OsL2:OsS2b and potato AGPase heterotetramer (StLS:StSS). Moreover, the yeast-two-hybrid results on OsL1:OsS1, which resemble StLS:StSS, suggest a differential protein assembly for OsL2:OsS2b. Thus, the regulatory and catalytic mechanisms for plastidial AGPases (OsL1:OsS1) could be different in rice culm and developing endosperm compared to those of OsL2:OsS2b, which are predominantly found in rice endosperm.

Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Architecture and activation of human muscle phosphorylase kinase.

The study of phosphorylase kinase (PhK)-regulated glycogen metabolism has contributed to the fundamental understanding of protein phosphorylation; however, the molecular mechanism of PhK remains poorly understood. Here we present the high-resolution cryo-electron microscopy structures of human muscle PhK. The 1.3-megadalton PhK α4ß4γ4δ4 hexadecamer consists of a tetramer of tetramer, wherein four αßγδ modules are connected by the central ß4 scaffold. The α- and ß-subunits possess glucoamylase-like domains, but exhibit no detectable enzyme activities. The α-subunit serves as a bridge between the ß-subunit and the γδ subcomplex, and facilitates the γ-subunit to adopt an autoinhibited state. Ca2+-free calmodulin (δ-subunit) binds to the γ-subunit in a compact conformation. Upon binding of Ca2+, a conformational change occurs, allowing for the de-inhibition of the γ-subunit through a spring-loaded mechanism. We also reveal an ADP-binding pocket in the ß-subunit, which plays a role in allosterically enhancing PhK activity. These results provide molecular insights of this important kinase complex.

Indirect interactions involving the PsbM or PsbT subunits and the PsbO, PsbU and PsbV proteins stabilize assembly and activity of Photosystem II in Synechocystis sp. PCC 6803.

The low-molecular-weight PsbM and PsbT proteins of Photosystem II (PS II) are both located at the monomer-monomer interface of the mature PS II dimer. Since the extrinsic proteins are associated with the final step of assembly of an active PS II monomer and, in the case of PsbO, are known to impact the stability of the PS II dimer, we have investigated the potential cooperativity between the PsbM and PsbT subunits and the PsbO, PsbU and PsbV extrinsic proteins. Blue-native polyacrylamide electrophoresis and western blotting detected stable PS II monomers in the ∆PsbM:∆PsbO and ∆PsbT:∆PsbO mutants that retained sufficient oxygen-evolving activity to support reduced photoautotrophic growth. In contrast, the ∆PsbM:∆PsbU and ∆PsbT:∆PsbU mutants assembled dimeric PS II at levels comparable to wild type and supported photoautotrophic growth at rates similar to those obtained with the corresponding ∆PsbM and ∆PsbT cells. Removal of PsbV was more detrimental than removal of PsbO. Only limited levels of dimeric PS II were observed in the ∆PsbM:∆PsbV mutant and the overall reduced level of assembled PS II in this mutant resulted in diminished rates of photoautotrophic growth and PS II activity below those obtained in the ∆PsbM:∆PsbO and ∆PsbT:∆PsbO strains. In addition, the ∆PsbT:∆PsbV mutant did not assemble active PS II centers although inactive monomers could be detected. The inability of the ∆PsbT:∆PsbV mutant to grow photoautotrophically, or to evolve oxygen, suggested a stable oxygen-evolving complex could not assemble in this mutant.

Cryo-EM structure of the Slo1 potassium channel with the auxiliary γ1 subunit suggests a mechanism for depolarization-independent activation.

Mammalian Ca2+-dependent Slo K+ channels can stably associate with auxiliary γ subunits which fundamentally alter their behavior. By a so far unknown mechanism, the four γ subunits reduce the need for voltage-dependent activation and, thereby, allow Slo to open independently of an action potential. Here, using cryo-EM, we reveal how the transmembrane helix of γ1/LRRC26 binds and presumably stabilizes the activated voltage-sensor domain of Slo1. The activation is further enhanced by an intracellular polybasic stretch which locally changes the charge gradient across the membrane. Our data provide a possible explanation for Slo1 regulation by the four γ subunits and also their different activation efficiencies. This suggests a novel activation mechanism of voltage-gated ion channels by auxiliary subunits.