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Science (New York, N.Y.) Apr 2019Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA...
Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1-GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo-electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.
Topics: Animals; Brain; Cryoelectron Microscopy; Ion Channel Gating; Protein Conformation; Protein Multimerization; Protein Subunits; Rats; Receptors, AMPA; Single Molecule Imaging
PubMed: 30975770
DOI: 10.1126/science.aaw8250 -
Biochemistry Jan 2022Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found in nearly all living organisms. Class I RNRs are composed...
Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found in nearly all living organisms. Class I RNRs are composed of two proteins, a large α-subunit (R1) and a smaller β-subunit (R2) that exist as homodimers, that combine to form an active heterotetramer. is a hyperthermophilic bacterium with an unusual RNR encoding a 346-residue intein in the DNA sequence encoding its R2 subunit. We present the first structures of the R1 and R2 (AaR1 and AaR2, respectively) proteins as well as the biophysical and biochemical characterization of active and inactive RNR. While the active oligomeric state and activity regulation of RNR are similar to those of other characterized RNRs, the X-ray crystal structures also reveal distinct features and adaptations. Specifically, AaR1 contains a β-hairpin hook structure at the dimer interface, which has an interesting π-stacking interaction absent in other members of the NrdAh subclass, and its ATP cone houses two ATP molecules. We determined structures of two AaR2 proteins: one purified from a construct lacking the intein (AaR2) and a second purified from a construct including the intein sequence (AaR2_genomic). These structures in the context of metal content analysis and activity data indicate that AaR2_genomic displays much higher iron occupancy and activity compared to AaR2, suggesting that the intein is important for facilitating complete iron incorporation, particularly in the Fe2 site of the mature R2 protein, which may be important for the survival of in low-oxygen environments.
Topics: Allosteric Regulation; Aquifex; Bacterial Proteins; Crystallography, X-Ray; Models, Molecular; Protein Conformation; Protein Multimerization; Protein Subunits; Ribonucleotide Reductases
PubMed: 34941255
DOI: 10.1021/acs.biochem.1c00503 -
Biochemistry Feb 2009Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step...
Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits, alpha, beta, and gamma, that in Thauera aromatica strain T1 are encoded by the tutD, tutG, and tutF genes, respectively. The large alpha-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small beta- and gamma-subunits has remained unclear. We have overexpressed all three subunits of benzylsuccinate synthase in Escherichia coli, both individually and in combination. Coexpression of the gamma-subunit (but not the beta-subunit) is essential for efficient expression of the alpha-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an alpha(2)beta(2)gamma(2) hexamer by nickel affinity chromatography through a "His(6)" affinity tag engineered onto the C-terminus of the alpha-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the beta-subunit and the other with the gamma-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly, a mutant gamma-subunit lacking the [4Fe-4S] cluster is unable to stabilize the alpha-subunit when the proteins are coexpressed.
Topics: Biocatalysis; Carbon-Carbon Lyases; Electron Spin Resonance Spectroscopy; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genes, Bacterial; Iron; Iron Chelating Agents; Iron-Sulfur Proteins; Models, Molecular; Mutation; Oxidation-Reduction; Protein Subunits; Spectrophotometry, Ultraviolet; Thauera
PubMed: 19159265
DOI: 10.1021/bi801766g -
PloS One 2019At some point early in the vertebrate lineage, two whole genome duplication events (1R, 2R) took place that allowed for the diversification and...
At some point early in the vertebrate lineage, two whole genome duplication events (1R, 2R) took place that allowed for the diversification and sub-/neo-functionalization of the glycoprotein hormones (GpHs). All jawed vertebrates possess the GpHs luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH), each of which are heterodimers with a common alpha subunit and unique beta subunits. In 2002, a novel glycoprotein hormone named thyrostimulin was described to have unique GpA2 and GpB5 subunits that were homologous to the vertebrate alpha and beta subunits. The presence of GpA2 and GpB5 in representative protostomes and deuterostomes indicates their ancestry in the GpH family. There are several reports of GpH subunit evolution, but none have included GpA2 and GpB5 for species in each major vertebrate class. Thus, we addressed the ancestry of two paralogous GpB5 subunits (GpB5a and GpB5b) that were previously only recognized in two teleost species. Our search for orthologous GpB5a and GpB5b sequences in representative vertebrates and phylogenetic analysis, in addition to the currently published evolutionary scenarios of the GpH family, supports that GpB5a and GpB5b are paralogs that arose from the first vertebrate whole genome duplication event (1R). Syntenic analysis supports lineage specific losses of GpB5a in chondrichthyes, basal actinopterygians, and tetrapods, and retention in coelacanth and teleosts. Additionally, we were unable to identify GpA2 transcripts from tilapia mRNA, suggesting that this species does not produce heterodimeric thyrostimulin. While the conserved or even species-specific functional role of thyrostimulin or its individual subunits are still unknown in vertebrates, the analyses presented here provide context for future studies on the functional divergence of the GpH family.
Topics: Amino Acid Sequence; Animals; Evolution, Molecular; Glycoproteins; Humans; Phylogeny; Protein Multimerization; Protein Subunits; Sequence Homology, Amino Acid; Species Specificity; Synteny; Vertebrates
PubMed: 31536580
DOI: 10.1371/journal.pone.0222808 -
PloS One 2011Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, k(off),...
Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, k(off), provide structural and functional information on protein complexes. However, because all existing methods for measuring k(off) require high-quality purification and specific modifications of protein complexes, dissociation kinetics has only been studied for a small set of model complexes. Here, we propose a new method, called Metabolically-labeled Affinity-tagged Subunit Exchange (MASE), to measure k(off) using metabolic stable isotope labeling, affinity purification and mass spectrometry. MASE is based on a subunit exchange process between an unlabeled affinity-tagged variant and a metabolically-labeled untagged variant of a complex. The subunit exchange process was modeled theoretically for a heterodimeric complex. The results showed that k(off) determines, and hence can be estimated from, the observed rate of subunit exchange. This study provided the theoretical foundation for future experiments that can validate and apply the MASE method.
Topics: Kinetics; Models, Biological; Multiprotein Complexes; Protein Subunits; Research Design; Staining and Labeling
PubMed: 22194924
DOI: 10.1371/journal.pone.0028827 -
The Journal of Neuroscience : the... Jun 2004Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA(A) receptor alpha1 subunit mutation (alpha1A322D). GABA(A) receptor...
The juvenile myoclonic epilepsy GABA(A) receptor alpha1 subunit mutation A322D produces asymmetrical, subunit position-dependent reduction of heterozygous receptor currents and alpha1 subunit protein expression.
Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA(A) receptor alpha1 subunit mutation (alpha1A322D). GABA(A) receptor alphabetagamma subunits are arranged around the pore in a beta-alpha-beta-alpha-gamma sequence (counterclockwise from the synaptic cleft). Therefore, each alpha1 subunit has different adjacent subunits, and heterozygous expression of alpha1(A322D), beta, and gamma subunits could produce receptors with four different subunit arrangements: beta-alpha1-beta-alpha1-gamma (wild type); beta-alpha1(A322D)-beta-alpha1-gamma (Het(betaalphabeta)); beta-alpha1-beta-alpha1(A322D)-gamma (Het(betaalphagamma));beta-alpha1(A322D)-beta-alpha1(A322D)-gamma (homozygous). Expression of a 1:1 mixture of wild-type andalpha1(A322D) subunits with beta2S and gamma2S subunits (heterozygous transfection) produced smaller currents than wild type and much larger currents than homozygous mutant transfections. Western blot and biotinylation assays demonstrated that the amount of total and surface alpha1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. alpha1(A322D) mutations were then made in covalently tethered triplet (gamma2S-beta2S-alpha1) and tandem (beta2S-alpha1) concatamers to target selectively alpha1(A322D) to each of the asymmetric alpha1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Het(betaalphabeta) or Het(betaalphagamma) receptors. Het(betaalphabeta) currents were smaller than wild type and much larger than Het(betaalphagamma) and homozygous currents. Furthermore, Het(betaalphabeta) transfections contained less beta-alpha concatamer than wild type but more than both Het(betaalphagamma) and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous alpha1(A322D)beta2Sgamma2S receptors depended on the position of the mutant alpha1 subunit, and GABA(A) receptor currents in heterozygous individuals likely result primarily from wild-type and Het(betaalphabeta) receptors with little contribution from Het(betaalphagamma) and homozygous receptors.
Topics: Cell Line; Heterozygote; Humans; Kinetics; Mutation; Myoclonic Epilepsy, Juvenile; Patch-Clamp Techniques; Protein Subunits; Receptors, GABA-A; gamma-Aminobutyric Acid
PubMed: 15201329
DOI: 10.1523/JNEUROSCI.1301-04.2004 -
FASEB Journal : Official Publication of... Mar 2011The cardiac voltage-gated Ca(2+) channel, Ca(v)1.2, mediates excitation-contraction coupling in the heart. The molecular composition of the channel includes the...
The cardiac voltage-gated Ca(2+) channel, Ca(v)1.2, mediates excitation-contraction coupling in the heart. The molecular composition of the channel includes the pore-forming α1 subunit and auxiliary α2/δ-1 and β subunits. Ca(2+) channel γ subunits, of which there are 8 isoforms, consist of 4 transmembrane domains with intracellular N- and C-terminal ends. The γ1 subunit was initially detected in the skeletal muscle Ca(v)1.1 channel complex, modulating current amplitude and activation and inactivation properties. The γ1 subunit also shifts the steady-state inactivation to more negative membrane potentials, accelerates current inactivation, and increases peak currents, when coexpressed with the cardiac α1c subunit in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. The γ1 subunit is not expressed, however, in cardiac muscle. We sought to determine whether γ subunits that are expressed in cardiac tissue physically associate with and modulate Ca(v)1.2 function. We now demonstrate that γ4, γ6, γ7, and γ8 subunits physically interact with the Ca(v)1.2 complex. The γ subunits differentially modulate Ca(2+) channel function when coexpressed with the β1b and α2/δ-1 subunits in HEK cells, altering both activation and inactivation properties. The effects of γ on Ca(v)1.2 function are dependent on the subtype of β subunit. Our results identify new members of the cardiac Ca(v)1.2 macromolecular complex and identify a mechanism by which to increase the functional diversity of Ca(v)1.2 channels.
Topics: Animals; Calcium Channels, L-Type; HEK293 Cells; Heart; Humans; Isomerism; Macromolecular Substances; Membrane Potentials; Mice; Models, Chemical; Mutagenesis, Site-Directed; Oocytes; Patch-Clamp Techniques; Protein Subunits; Rats; Xenopus
PubMed: 21127204
DOI: 10.1096/fj.10-172353 -
Physiological Reviews Oct 2004The glycine receptor chloride channel (GlyR) is a member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. Functional receptors of this family... (Review)
Review
The glycine receptor chloride channel (GlyR) is a member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. Functional receptors of this family comprise five subunits and are important targets for neuroactive drugs. The GlyR is best known for mediating inhibitory neurotransmission in the spinal cord and brain stem, although recent evidence suggests it may also have other physiological roles, including excitatory neurotransmission in embryonic neurons. To date, four alpha-subunits (alpha1 to alpha4) and one beta-subunit have been identified. The differential expression of subunits underlies a diversity in GlyR pharmacology. A developmental switch from alpha2 to alpha1beta is completed by around postnatal day 20 in the rat. The beta-subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin. The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information has recently emerged concerning GlyR molecular structure and function. Most of the information has been obtained from homomeric alpha1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritable mutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of muscle relaxant and peripheral analgesic drugs.
Topics: Animals; Central Nervous System; Chloride Channels; Humans; Molecular Structure; Neurons; Protein Subunits; Receptors, Glycine
PubMed: 15383648
DOI: 10.1152/physrev.00042.2003 -
Biochimica Et Biophysica Acta. Gene... Jul 2020In mammalian cells, the SET1/MLL complexes are the main writers of the H3K4 methyl mark that is associated with active gene expression. The activities of these complexes... (Review)
Review
In mammalian cells, the SET1/MLL complexes are the main writers of the H3K4 methyl mark that is associated with active gene expression. The activities of these complexes are critically dependent on the association of the catalytic subunit with their shared core subunits, WDR5, RBBP5, ASH2L, and DPY30, collectively referred as WRAD. In addition, some of these core subunits can bind to proteins other than the SET1/MLL complex components. This review starts with discussion of the molecular activities of these core subunits, with an emphasis on DPY30 in organizing the assembly of the SET1/MLL complexes with other associated factors. This review then focuses on the roles of the core subunits in stem cells and development, as well as in diseased cell states, mainly cancer, and ends with discussion on dissecting the responsible activities of the core subunits and how we may target them for potential disease treatment. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.
Topics: Animals; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Histone-Lysine N-Methyltransferase; Humans; Myeloid-Lymphoid Leukemia Protein; Protein Multimerization; Protein Subunits
PubMed: 32302696
DOI: 10.1016/j.bbagrm.2020.194560 -
Biomolecules May 2019The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is...
The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.
Topics: Gene Knockdown Techniques; HEK293 Cells; Humans; Proteasome Endopeptidase Complex; Protein Subunits; RNA Interference; RNA, Small Interfering
PubMed: 31159305
DOI: 10.3390/biom9060213