Tetrameric Protein Complex Research Articles

Physcomitrium patens flavodiiron proteins form heterotetrametric complexes

Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by using electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero tetrameric protein complex with cooperative nature. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo activity.

Comparative study of the steady-state subcellular distribution of lysosome-associated membrane glycoprotein-2 (LAMP-2) isoforms with GYXXΦ-type tyrosine-based motifs that interact differently with four adaptor protein (AP) complexes.

Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and μ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with μ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four μ-subunits (μ1, μ2, μ3A and μ4 of AP-1, AP-2, AP-3 and AP-4, respectively). The interaction with μ3A was more robust than that with other μ-subunits. LAMP-2B-CT interacted exclusively and moderately with μ3A. LAMP-2C-CT did not detectably interact with any of the four μ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3.

Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.

Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.

Abstract 2302: The pan-cancer regulator of the mutant TERT promoter and a new model of the GABP-TERT axis

Abstract Ninety percent of human tumors reactivate telomerase reverse transcriptase (TERT) to achieve cellular immortality. The route to immortality found in over 50 cancer types is mutation of the TERT promoter (TERTp) mutations. Studies in glioblastoma have shown the two most common TERTp mutations, G228A and G250A, reactivate TERT expression through de novo E26 transformation specific (ETS) site generation that, in tandem with a native TERTp ETS site, recruits the tetrameric GA-binding protein (GABP) complex formed by two GABP alpha (GABPA) subunits and two tetramer specific GABPB1 (B1) subunits, GABPB1L (B1L). While ten additional TERTp mutations have been described across cancer types, functional studies have been limited to select mutations and cancer types. We found that each de novo ETS motif generating TERTp mutation increases TERTp transcriptional activity in a GABP-dependent manner. Furthermore, our pan-cancer analysis demonstrated selective enrichment of GABPA at the mutant TERTp across 14 cancer types and further determined that this recruitment is necessary for TERT expression. Prior investigations found that CRISPR-cas9 mediated mutagenesis of the B1 ninth exon, specific to B1L, reduces TERT transcriptional activity in a TERTp-mutation dependent manner. We explored B1L targeting in the reversal of tumor cell immortality in several cancers. We found that the B1 dimer restricted isoform, GABPB1S (B1S), is consistently and significantly increased following B1L reduction, which we determined to be the consequence of a disengagement of a GABP tetramer mediated negative feedback loop acting on the B1 promoter. Interestingly, this upregulated B1S expression enables GABP dimer binding and activation of the mutant TERTp thereby compensating for B1L loss. Indeed, knockout of B1L and B1S together resulted in a near complete elimination of GABPA TERTp recruitment and a striking reduction in TERT expression, leading to telomere shortening and ultimately resulting in tumor cell death and senescence. This data identifies GABP as the master regulator of the mutant TERTp across cancer type and de novo ETS mutation type and suggests a new model of the GABP-TERT axis involving both the GABP tetramer, and the GABP dimer. Citation Format: Nicholas O. Stevers, Carter Barger, Olivia Lenzo, Chibo Hong, Katarzyna Soczek, Samuel H. Wu, Andrew M. McKinney, Abigail Suwala, Jennifer A. Doudna, Joseph F. Costello. The pan-cancer regulator of the mutant TERT promoter and a new model of the GABP-TERT axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2302.

Association of Transthyretin Val122Ile Variant With Incident Heart Failure Among Black Individuals

A genetic variant in the TTR gene (rs76992529; Val122Ile), present more commonly in individuals with African ancestry (population frequency: 3%-4%), causes misfolding of the tetrameric transthyretin protein complex that accumulates as extracellular amyloid fibrils and results in hereditary transthyretin amyloidosis. To estimate the association of the amyloidogenic Val122Ile TTR variant with the risk of heart failure and mortality in a large, geographically diverse cohort of Black individuals. Retrospective population-based cohort study of 7514 self-identified Black individuals living in the US participating in the REGARDS (Reasons for Geographic and Racial Differences in Stroke) study with genetic data available and without heart failure at baseline. The participants were enrolled at the baseline visit (2003-2007). The end of follow-up for the majority of outcomes was on December 31, 2018. All-cause mortality data were available through December 31, 2020. TTR Val122Ile (rs76992529) genotype. The primary outcome was incident heart failure (first hospitalization for heart failure or death due to heart failure). The secondary outcomes were heart failure mortality, cardiovascular mortality, and all-cause mortality. The multivariable Cox proportional hazards regression analyses were adjusted for genetic ancestry and demographic, clinical, and social factors. Among 7514 Black participants (median age, 64 years [IQR, 57-70 years]; 61% women), the population frequency of the TTR Val122Ile variant was 3.1% (232 variant carriers and 7282 noncarriers). During a median follow-up of 11.1 years (IQR, 5.9-13.5 years), incident heart failure occurred in 535 individuals (34 variant carriers and 501 noncarriers) and the incidence of heart failure was 15.64 per 1000 person-years among variant carriers vs 7.16 per 1000 person-years among noncarriers (adjusted hazard ratio [HR], 2.43 [95% CI, 1.71-3.46]; P < .001). Deaths due to heart failure occurred in 141 individuals (13 variant carriers and 128 noncarriers) and the incidence of heart failure mortality was 6.11 per 1000 person-years among variant carriers vs 1.85 per 1000 person-years among noncarriers (adjusted HR, 4.19 [95% CI, 2.33-7.54]; P < .001). Deaths due to cardiovascular causes occurred in 793 individuals (34 variant carriers and 759 noncarriers) and the incidence of cardiovascular death was 15.18 per 1000 person-years among variant carriers vs 10.61 per 1000 person-years among noncarriers (adjusted HR, 1.69 [95% CI, 1.19-2.39]; P = .003). Deaths due to any cause occurred in 2715 individuals (100 variant carriers and 2615 noncarriers) and the incidence of all-cause mortality was 41.46 per 1000 person-years among variant carriers vs 33.94 per 1000 person-years among noncarriers (adjusted HR, 1.46 [95% CI, 1.19-1.78]; P < .001). There was no significant interaction between TTR variant carrier status and sex on incident heart failure and the secondary outcomes. Among a cohort of Black individuals living in the US, being a carrier of the TTR Val122Ile variant was significantly associated with an increased risk of heart failure.

A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection

Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin γ1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.

Subunit-specific effects of poricoic acid A on NMDA receptors.

N-methyl-D-aspartate (NMDA) receptor is a tetrameric protein complex composed of glycine-linked NR1 subunits and glutamate-linked NR2 subunits. There are four NR2 subunits (A-D) that differ in development, anatomy, and function profiles. They play various roles in normal and neuropathologic conditions. Specific agonists, antagonists, and modulators of subunits for selective NMDA receptors may be precious mediational tools and potent agents for treating diseases. The objective of this study was to determine the effect of poricoic acid A on NMDA receptor known to mediate excitatory synaptic transmission factors and cause changes in synaptic strength. Inhibitory effect of poricoic acid A on NR1a combined with NR2A, NR2B, NR2C, or NR2D receptor was evaluated. Glutamate-mediated currents for each NR1a and NR2 subunits were investigated using two-electrode voltage-clamp techniques. Molecular modeling and molecular dynamics simulation studies were carried out with Autodock Tools. Poricoic acid A and NMDA receptor protein complex were examined with Ligplot and Pymol docking program. Ligplot shows binding activity at the protein and the ligand. The inhibitory effect of poricoic acid A on glutamate-induced inward current in a concentration-dependent manner that was reversible. Half inhibitory concentrations of glutamate on NR1a/NR2A, NR1a/NR2B, NR1a/NR2C, and NR1a/NR2D receptors were 9.6 ± 1.2, 5.7 ± 0.4, 46.1 ± 21.5, and 21.5 ± 8.2μM, respectively. This corresponded to the order of inhibitory effect of oocyte expressing NR1a and NR2s subunit of NR1a/NR2B > NR1a/NR2A > NR1a/NR2C > NR1a/NR2D. Taken together, these results indicate that poricoic acid A can modulate the expression of NMDA receptor. In addition, the regulation of excitatory ligand-gating ion channel by poricoic acid A may have pharmaceutical functions on excitatory synaptic transmission of neuronal system.

Recent Findings on AMPA Receptor Recycling.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) are tetrameric protein complexes that mediate most of the fast-excitatory transmission in response to the neurotransmitter glutamate in neurons. The abundance of AMPA-Rs at the surface of excitatory synapses establishes the strength of the response to glutamate. It is thus evident that neurons need to tightly regulate this feature, particularly in the context of all synaptic plasticity events, which are considered the biological correlates of higher cognitive functions such as learning and memory. AMPA-R levels at the synapse are regulated by insertion of newly synthesized receptors, lateral diffusion on the plasma membrane and endosomal cycling. The latter is likely the most important especially for synaptic plasticity. This process starts with the endocytosis of the receptor from the cell surface and is followed by either degradation, if the receptor is directed to the lysosomal compartment, or reinsertion at the cell surface through a specialized endosomal compartment called recycling endosomes. Although the basic steps of this process have been discovered, the details and participation of additional regulatory proteins are still being discovered. In this review article, we describe the most recent findings shedding light on this crucial mechanism of synaptic regulation.

Multimer recognition and secretion by the non-classical secretion pathway in Bacillus subtilis

Non-classical protein secretion in bacteria is a common phenomenon. However, the selection principle for non-classical secretion pathways remains unclear. Here, our experimental data, to our knowledge, are the first to show that folded multimeric proteins can be recognized and excreted by a non-classical secretion pathway in Bacillus subtilis. We explored the secretion pattern of a typical cytoplasmic protein D-psicose 3-epimerase from Ruminococcus sp. 5_1_39BFAA (RDPE), and showed that its non-classical secretion is not simply due to cell lysis. Analysis of truncation variants revealed that the C- and N-terminus, and two hydrophobic domains, are required for structural stability and non-classical secretion of RDPE. Alanine scanning mutagenesis of the hydrophobic segments of RDPE revealed that hydrophobic residues mediated the equilibrium between its folded and unfolded forms. Reporter mCherry and GFP fusions with RDPE regions show that its secretion requires an intact tetrameric protein complex. Using cross-linked tetramers, we show that folded tetrameric RDPE can be secreted as a single unit. Finally, we provide evidence that the non-classical secretion pathway has a strong preference for multimeric substrates, which accumulate at the poles and septum region. Altogether, these data show that a multimer recognition mechanism is likely applicable across the non-classical secretion pathway.

New High Resolution Ion Mobility Mass Spectrometer Capable of Measurements of Collision Cross Sections from 150 to 520 K.

We present a new variable temperature (VT), high resolution ion mobility (IM) drift tube coupled to a commercial mass spectrometer (MS). Ions are generated in an electrospray ion source with a sampling cone interface and two stacked ring RF guides which transfer ions into the mobility analyzer located prior to a quadrupole time-of-flight mass spectrometer. The drift cell can be operated over a pressure range of 0.5-3 Torr and a temperature range of 150-520 K with applied fields typically between 3 and 14 V cm-1. This makes the instrument suitable for rotationally averaged collision cross section (CCS) measurements at low E/N ratios where ions are near thermal equilibrium with the buffer gas. Fundamental studies of the effective ion temperatures can be performed at high E/N ratios. An RF ion trap/buncher is located at the beginning of the drift region, which modulates the continuous ion beam into spatially narrow packets. Packets of ions then drift in a linear electric field, which is 50.5 cm long, and are separated according to their mobility in an inert buffer gas. Post-drift, an ion funnel focuses the radially spread pulses of ions into the inlet of a commercial MS platform (Micromass QToF2). We present the novel features of this instrument and results from VT-IM-MS experiments on a range of model systems-IMS CCS standards (Agilent ESI Tune Mix), the monomeric protein Ubiquitin (8.6 kDa), and the tetrameric protein complex Concanavalin A (103 kDa). We evaluate the performance of the instrument by comparing ambient DTCCSHe values of model compounds with those found in the literature. Several effects of temperature on collision cross sections and resolution are observed. For small rigid molecules, changes in resolution are consistent with anticipated thermal diffusion effects. Changes in measured DTCCSHe for these rigid systems at different temperatures are attributed primarily to the effect of temperature on the long-range attractive interaction. Similar effects are seen for protein ions at low temperatures, although there is also some evidence for structural transitions. By heating the protein ions, their conformational profiles are significantly altered. Very high temperatures narrow the conformational space presented by both Ubiquitin and Concanavalin; it appears that diverse conformational families are "melted" into more homogeneous populations. Because of this conformational heterogeneity, the apparent IMS resolution obtained for proteins at ambient and reduced temperatures is an order of magnitude lower than the expected diffusion limited resolution (Rmax). This supports a hypothesis that the broad DTCCSHe features frequently observed for proteins do not correspond to interconverting conformers, but rather to high numbers of intrinsically stable structures.

193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology.

Protein-protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins.

Roles of collagen Q in MuSK antibody-positive myasthenia gravis

The low-density lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific receptor tyrosine kinase (MuSK) form a tetrameric protein complex on the postsynaptic membrane at the neuromuscular junction (NMJ). Binding of agrin to LRP4 triggers phosphorylation of MuSK. Activated MuSK drives clustering of acetylcholine receptor (AChR). Wnt ligands also directly bind to MuSK to induce AChR clustering. MuSK anchors the acetylcholinesterase (AChE)/collagen Q (ColQ) complex to the synaptic basal lamina. In addition, an extracellular proteoglycan, biglycan, binds to MuSK.Anti-MuSK autoantibodies (MuSK-IgG) are observed in 5–15% of autoimmune myasthenia gravis (MG) patients. MuSK-IgG blocks both ColQ-MuSK and LRP4-MuSK interactions. MuSK-IgG, LRP4, ColQ, and biglycan bind to the immunoglobulin-like domains 1 and 4 of MuSK. Lack of the effects of cholinesterase inhibitors in MuSK-MG patients is likely due to hindrance of ColQ-MuSK interaction by MuSK-IgG and subsequent deficiency of AChE observed in model mice, which, however, has not been proven in MuSK-MG patients. As ColQ enhances expression of membrane-bound MuSK, inhibition of ColQ-MuSK interaction by MuSK-IgG may account for lack of AChR clusters in MuSK-MG. We thus made passive transfer models using Colq+/+ and Colq−/− mice to dissect the effect of ColQ on AChR clustering in MuSK-MG. We found that MuSK-IgG-mediated suppression of LRP4-MuSK interaction, not of ColQ-MuSK interaction, caused defective AChR clustering. We also unexpectedly observed that both MuSK-IgG and ColQ suppressed agrin/LRP4/MuSK signaling in dose-dependent manners. Quantitative comparison revealed that MuSK-IgG blocked agrin-LRP4-MuSK signaling more than ColQ.We propose that attenuation of AChR clustering in MuSK-MG is due to hindrance of LRP4-MuSK interaction in the presence of agrin by MuSK-IgG.

Characterisation of serum transthyretin by electrospray ionisation-ion mobility mass spectrometry: Application to familial amyloidotic polyneuropathy type I (FAP-I)

Transthyretin (TTR) is a homotetrameric protein which is known to misfold and aggregate causing different types of amyloidosis, such as familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR (Met30)) that can be easily detected analysing the monomeric forms of the mutant protein. Meanwhile, the mechanism of protein aggregation onset, which could be triggered by structural changes on the native tetrameric protein complex, remains uncertain. We developed and described herein a new sample pretreatment based on immunoprecipitation (IP) to purify TTR from serum under non-denaturing conditions. Later, a nano-electrospray ionization-ion mobility mass spectrometry (nano-ESI-IM-MS or IM-MS) method was optimised to analyse the protein complexes in serum samples from healthy controls and FAP-I patients. IM-MS allowed separation and characterisation of tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time, which is related to ion size and charge. The tetramer-to-dimer abundance ratio was differential between healthy controls and FAP-I patients (asymptomatic, symptomatic and an iatrogenic patient originally without the mutation who received a liver transplant from an FAP-I patient), and was also indicative of the effectiveness of liver transplantation as a treatment for FAP-I.

ADAR2-dependent GluA2 editing regulates cocaine seeking.

Activation of AMPA receptors in the nucleus accumbens is necessary for the reinstatement of cocaine-seeking behavior, an animal model of drug craving and relapse. AMPA receptors are tetrameric protein complexes that consist of GluA1-GluA4 subunits, of which GluA2 imparts calcium permeability. Adenosine deaminase acting on RNA (ADAR2) is a nuclear enzyme that is essential for editing GluA2 pre-mRNA at Q/R site-607. Unedited GluA2(Q) subunits form calcium permeable AMPA receptors (CP-AMPARs), whereas edited GluA2(R) subunits form calcium impermeable channels (CIAMPARs). Emerging evidence suggests that the reinstatement of cocaine seeking is associated with increased synaptic expression of CP-AMPARs in the nucleus accumbens. However, the role of GluA2 Q/R site editing and ADAR2 in cocaine seeking is unclear. In the present study, we investigated the effects of forced cocaine abstinence on GluA2 Q/R site editing and ADAR2 expression in the nucleus accumbens. Our results demonstrate that 7 days of cocaine abstinence is associated with decreased GluA2 Q/R site editing and reduced ADAR2 expression in the accumbens shell, but not core, of cocaine-experienced rats compared to yoked saline controls. In order to examine the functional significance of ADAR2 and GluA2 Q/R site editing in cocaine seeking, we used viral-mediated gene delivery to overexpress ADAR2b in the accumbens shell. Increased ADAR2b expression in the shell attenuated cocaine priming-induced reinstatement of drug seeking and was associated with increased GluA2 Q/R site editing and surface expression of GluA2-containing AMPARs. Taken together, these findings support the novel hypothesis that an increased contribution of accumbens shell CP-AMPARs containing unedited GluA2(Q) promotes cocaine seeking. Therefore, CP-AMPARs containing unedited GluA2(Q) represent a novel target for cocaine addiction pharmacotherapies.

Expression of NMDA receptor subunit 1 in the rat retina

N-methyl-d-aspartate receptors (NMDARs) belong to the ionotropic glutamate receptors, which play key roles in neuronal communication in the retina. NMDA receptors are tetrameric protein complexes usually comprising two obligatory NMDA receptor 1 (NR1) subunits and modulatory NMDA receptor 2/3 (NR2/3) subunits. Although the expression patterns of different NMDA receptor subunits have been extensively studied, in this study we focused on NR1 protein expression in the rat retina by immunofluorescence double labeling. We show that NR1 labeling is diffusely distributed in the outer plexiform layer (OPL) and throughout the whole inner plexiform layer (IPL). The NR1-immunoreactivity (IR) was displayed in a variety of cells in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Interestingly, NR1 was expressed in both rod and cone bipolar cells identified by specific bipolar cell markers Chx10, protein kinase C (PKC) and recoverin. All the amacrine cells that we studied, including cholinergic, dopaminergic, GABAergic and glycinergic amacrine cells, were NR1-IR positive. In the ganglion cell layer, NR1-IR was expressed in all cells that were positive for the ganglion cell marker Brn3a. Our study suggests that the NR1 subunit is expressed more widely than was previously appreciated.

HnRNP C Tetramer Measures RNA Length to Classify RNA Polymerase II Transcripts for Export

Specific RNA recognition is usually achieved by specific RNA sequences and/or structures. However, we show here a mechanism by which RNA polymerase II (Pol II) transcripts are classified according to their length. The heterotetramer of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 measures the length of the transcripts like a molecular ruler, by selectively binding to the unstructured RNA regions longer than 200 to 300 nucleotides. Thus, the tetramer sorts the transcripts into two RNA categories, to be exported as either messenger RNA or uridine-rich small nuclear RNA (U snRNA), depending on whether or not they are longer than the threshold, respectively. Our findings reveal a new function of the C tetramer and highlight the biological importance of RNA recognition by the length.

Cucurbit[8]uril-mediated protein homotetramerization

A supramolecular protein tetramerization approach has been devised which enables the controlled formation of a discrete protein tetramer. The supramolecular element cucurbit[8]uril has been used as an inducer of the protein tetramerization in combination with intrinsic affinities between the proteins, which preorganize the protein in dimerized form. The combination of a dimerizing interface on the fluorescent proteins under study (dYFP, dCFP), with a genetically encoded N-terminal phenylalanine-glycine-glycine (FGG) peptide motif allows cucurbit[8]uril to selectively induce FGG-dYFP or FGG-dCFP tetramerization. The concept of cucurbit[8]uril-induced protein tetramerization was elucidated by using a combination of fluorescence anisotropy, dynamic light scattering and size exclusion chromatography experiments. The cucurbit[8]uril-induced tetrameric protein complex is formed via a “dimers of dimers” pathway, is highly stable and can be separated by size exclusion chromatography. This supramolecular induced protein tetramerization approach opens up a novel entry in generating well-defined synthetic protein assemblies.

Gas-phase protein assemblies: Unfolding landscapes and preserving native-like structures using noncovalent adducts

Mass spectrometry is a rapidly emerging technology for characterising the native structures of protein complexes. One challenge in interpreting results from mass spectrometry experiments is that the structures of protein complexes in the gas phase may differ from those in solution. As such, there is great interest in using small molecules to stabilise the structure of large proteins and their complexes in the gas-phase. Here, we investigate the stabilisation properties of trisH+, a cationic non-volatile electrospray buffer component, by experimentally characterising the unfolding and dissociation of three gas-phase tetrameric protein complexes. We find that trisH+ preferentially stabilises the compact native-like state of the complexes studied here. We put these results in context, and look beyond the water-soluble complexes studied here to discuss the mechanistic implications of this work on the stabilisation of membrane protein complexes during electrospray ionisation.

An Electrostatic Charge Partitioning Model for the Dissociation of Protein Complexes in the Gas Phase

Electrosprayed multi-protein complexes can be dissociated by collisional activation in the gas phase. Typically, these processes follow a mechanism whereby a single subunit gets ejected with a disproportionately high amount of charge relative to its mass. This asymmetric behavior suggests that the departing subunit undergoes some degree of unfolding prior to being separated from the residual complex. These structural changes occur concomitantly with charge (proton) transfer towards the subunit that is being unraveled. Charge accumulation takes place up to the point where the subunit loses physical contact with the residual complex. This work develops a simple electrostatic model for studying the relationship between conformational changes and charge enrichment during collisional activation. Folded subunits are described as spheres that carry continuum surface charge. The unfolded chain is envisioned as random coil bead string. Simulations are guided by the principle that the system will adopt the charge configuration with the lowest potential energy for any backbone conformation. A finite-difference gradient algorithm is used to determine the charge on each subunit throughout the dissociation process. Both dimeric and tetrameric protein complexes are investigated. The model reproduces the occurrence of asymmetric charge partitioning for dissociation events that are preceded by subunit unfolding. Quantitative comparisons of experimental MS/MS data with model predictions yield estimates of the structural changes that occur during collisional activation. Our findings suggest that subunit separation can occur over a wide range of scission point structures that correspond to different degrees of unfolding.

Bound anions differentially stabilize multiprotein complexes in the absence of bulk solvent.

The combination of ion mobility separation with mass spectrometry is an emergent and powerful structural biology tool, capable of simultaneously assessing the structure, topology, dynamics, and composition of large protein assemblies within complex mixtures. An integral part of the ion mobility-mass spectrometry measurement is the ionization of intact multiprotein complexes and their removal from bulk solvent. This process, during which a substantial portion of protein structure and organization is likely to be preserved, imposes a foreign environment on proteins that may cause structural rearrangements to occur. Thus, a general means must be identified to stabilize protein structures in the absence of bulk solvent. Our approach to this problem involves the protection of protein complex structure through the addition of salts in solution prior to desorption/ionization. Anionic components of the added salts bind to the complex either in solution or during the electrospray process, and those that remain bound in the gas phase tend to have high gas phase acidities. The resulting 'shell' of counterions is able to carry away excess energy from the protein complex ion upon activation and can result in significant structural stabilization of the gas-phase protein assembly overall. By using ion mobility-mass spectrometry, we observe both the dissociation and unfolding transitions for four tetrameric protein complexes bound to populations of 12 different anions using collisional activation. The data presented here quantifies, for the first time, the influence of a large range of counterions on gas-phase protein structure and allows us to rank and classify counterions as structure stabilizers in the absence of bulk solvent. Our measurements indicate that tartrate, citrate, chloride, and nitrate anions are among the strongest stabilizers of gas-phase protein structure identified in this screen. The rank order determined by our data is substantially different when compared to the known Hofmeister salt series in solution. While this is an expected outcome of our work, due to the diminished influence of anion and protein solvation by water, our data correlates well to expected anion binding in solution and highlights the fact that both hydration layer and anion-protein binding effects are critical for Hofmeister-type stabilization in solution. Finally, we present a detailed mechanism of action for counterion stabilization of proteins and their complexes in the gas-phase, which indicates that anions must bind with high affinity, but must dissociate readily from the protein in order to be an effective stabilizer. Anion-resolved data acquired for smaller protein systems allows us to classify anions into three categories based on their ability to stabilize protein and protein complex structure in the absence of bulk solvent.