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The Journal of Physiology Jan 2015NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell... (Review)
Review
NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell survival. Several features distinguish NMDARs from other iGluRs and underlie the crucial roles NMDARs play in nervous system physiology. NMDARs display slow deactivation kinetics, are highly Ca(2+) permeable, and require depolarization to relieve channel block by external Mg(2+) ions, thereby making them effective coincidence detectors. These properties and others differ among NMDAR subtypes, which are defined by the subunits that compose the receptor. NMDARs, which are heterotetrameric, commonly are composed of two GluN1 subunits and two GluN2 subunits, of which there are four types, GluN2A-D. 'Diheteromeric' NMDARs contain two identical GluN2 subunits. Gating and ligand-binding properties (e.g. deactivation kinetics) and channel properties (e.g. channel block by Mg(2+)) depend strongly on the GluN2 subunit contained in diheteromeric NMDARs. Recent work shows that two distinct regions of GluN2 subunits control most diheteromeric NMDAR subtype-dependent properties: the N-terminal domain is responsible for most subtype dependence of gating and ligand-binding properties; a single residue difference between GluN2 subunits at a site termed the GluN2 S/L site is responsible for most subtype dependence of channel properties. Thus, two structurally and functionally distinct regions underlie the majority of subtype dependence of NMDAR properties. This topical review highlights recent studies of recombinant diheteromeric NMDARs that uncovered the involvement of the N-terminal domain and of the GluN2 S/L site in the subtype dependence of NMDAR properties.
Topics: Protein Subunits; Receptors, N-Methyl-D-Aspartate
PubMed: 25556790
DOI: 10.1113/jphysiol.2014.273763 -
The Journal of Physiology Jan 2015The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding... (Review)
Review
The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.
Topics: Animals; Humans; Ion Channel Gating; Pharmaceutical Preparations; Protein Structure, Tertiary; Protein Subunits; Receptors, Ionotropic Glutamate
PubMed: 25556789
DOI: 10.1113/jphysiol.2014.273409 -
The Journal of Biological Chemistry Sep 2023Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits...
Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous β-subunits. To better reveal β-subunit functions, we engineered a human cell line devoid of endogenous Na β-subunits and their immediate phylogenetic relatives. This new cell line, β-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the β-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac Na α-subunit, Na1.5, was highly sensitive to β-subunit modulation and revealed that each β-subunit and even MPZ imparted unique gating properties. Furthermore, combining β1 and β2 with Na1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of β-subunits to regulate Na1.5 activity and can be used to improve the characterization of other α/β Na complexes.
Topics: Humans; Action Potentials; Cell Line; Intracellular Signaling Peptides and Proteins; NAV1.5 Voltage-Gated Sodium Channel; Phosphoproteins; Protein Subunits; Voltage-Gated Sodium Channel beta Subunits; Mutation
PubMed: 37544648
DOI: 10.1016/j.jbc.2023.105132 -
Methods in Molecular Biology (Clifton,... 2020Saccharomyces cerevisiae is a useful eukaryotic expression system for mitochondrial membrane proteins due to its ease of growth and ability to provide a native membrane...
Saccharomyces cerevisiae is a useful eukaryotic expression system for mitochondrial membrane proteins due to its ease of growth and ability to provide a native membrane environment. The development of the pBEVY vector system has further increased the potential of S. cerevisiae as an expression system by creating a method for expressing multiple proteins simultaneously. This vector system is amenable to the expression and purification of multi-subunit protein complexes. Here we describe the cloning, yeast transformation, and co-expression of multi-subunit outer mitochondrial membrane complexes using the pBEVY vector system.
Topics: Cell Fractionation; Cloning, Molecular; Gene Expression Regulation, Fungal; Genetic Vectors; Membrane Proteins; Mitochondrial Membranes; Mitochondrial Proteins; Organisms, Genetically Modified; Protein Multimerization; Protein Processing, Post-Translational; Protein Subunits; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transformation, Genetic
PubMed: 32112311
DOI: 10.1007/978-1-0716-0373-4_1 -
Proteomics Aug 2015Although the number of protein-encoding genes in the human genome is only about 20 000 not far from the amount found in the nematode worm genome, the number of proteins... (Review)
Review
Although the number of protein-encoding genes in the human genome is only about 20 000 not far from the amount found in the nematode worm genome, the number of proteins that are translated from these sequences is larger by several orders of magnitude. A number of mechanisms have evolved to enable this diversity. For example, genes can be alternatively spliced to create multiple transcripts; they may also be translated from different alternative initiation sites. After translation, hundreds of chemical modifications can be introduced in proteins, altering their chemical properties, folding, stability, and activity. The complexity is then further enhanced by the various combinations that are generated from the assembly of different subunit variants into protein complexes. This, in turn, confers structural and functional flexibility, and endows the cell with the ability to adapt to various environmental conditions. Therefore, exposing the variability of protein complexes is an important step toward understanding their biological functions. Revealing this enormous diversity, however, is not a simple task. In this review, we will focus on the array of MS-based strategies that are capable of performing this mission. We will also discuss the challenges that lie ahead, and the future directions toward which the field might be heading.
Topics: Computational Biology; Mass Spectrometry; Models, Molecular; Protein Conformation; Protein Processing, Post-Translational; Protein Subunits
PubMed: 25727951
DOI: 10.1002/pmic.201400517 -
International Journal of Biological... Jun 2021Protein fusion using a linker plays an important role for protein evolution. However, designing suitable linkers for protein evolution is yet challenging and...
Protein fusion using a linker plays an important role for protein evolution. However, designing suitable linkers for protein evolution is yet challenging and under-explored. To further clarify the regular pattern of suitable type of linker for fusion proteins, one nitrile hydratase (NHase) was used as a target protein and subunit fusion strategy was carried out to improve its efficient catalysis. Subunit-fused variants with three different types of linkers were constructed and characterized. All variants exhibited higher stability than that of the wild type. The longer the linker was, the higher stability NHase showed, however, too long linker affected NHase activity and expression. Among the three types of linkers, the α-helical linker seemed more suitable for NHase than flexible or rigid linkers. Though it is not clear how the linkers affecting the activity, structure analysis indicated that the stability improvement is dependent on the additional salt bridge, H-bond, and the subunit interface area increasing due to the linker insertion, among which the additional salt bridge and interface area were more important factors. The results described here may be useful for redesigning other enzymes through subunit fusion.
Topics: Biocatalysis; Catalytic Domain; Enzyme Stability; Hydro-Lyases; Kinetics; Molecular Dynamics Simulation; Protein Subunits; Recombinant Proteins; Temperature
PubMed: 33753198
DOI: 10.1016/j.ijbiomac.2021.03.103 -
PloS One 2023Most proteins form complexes consisting of two or more subunits, where complex assembly can proceed via two competing pathways: co-translational assembly of a mature and...
Most proteins form complexes consisting of two or more subunits, where complex assembly can proceed via two competing pathways: co-translational assembly of a mature and a nascent subunit, and post-translational assembly by two mature protein subunits. Assembly pathway dominance, i.e., which of the two pathways is predominant under which conditions, is poorly understood. Here, we introduce a reaction-diffusion system that describes protein complex formation via post- and co-translational assembly and use it to analyze the dominance of both pathways. Special features of this new system are (i) spatially inhomogeneous sources of reacting species, (ii) a combination of diffusing and immobile species, and (iii) an asymmetric binding competition between the species. We study assembly pathway dominance for the spatially homogeneous system and find that the ratio of production rates of the two protein subunits determines the long-term pathway dominance. This result is independent of the binding rate constants for post- and co-translational assembly and implies that a system with an initial post-translational assembly dominance can eventually exhibit co-translational assembly dominance and vice versa. For exactly balanced production of both subunits, the assembly pathway dominance is determined by the steady state concentration of the subunit that can bind both nascent and mature partners. The introduced system of equations can be applied to describe general dynamics of assembly processes involving both diffusing and immobile components.
Topics: Protein Subunits; Protein Biosynthesis
PubMed: 36827413
DOI: 10.1371/journal.pone.0281964 -
Current Opinion in Structural Biology Dec 2011Eukaryotic transcriptional coactivators are multi-subunit complexes that both modify chromatin and recognize histone modifications. Until recently, structural... (Review)
Review
Eukaryotic transcriptional coactivators are multi-subunit complexes that both modify chromatin and recognize histone modifications. Until recently, structural information on these large complexes has been limited to isolated enzymatic domains or chromatin-binding motifs. This review summarizes recent structural studies of the SAGA coactivator complex that have greatly advanced our understanding of the interplay between its different subunits. The structure of the four-protein SAGA deubiquitinating module has provided a first glimpse of the larger organization of a coactivator complex, and illustrates how interdependent subunits interact with each other to form an active and functional enzyme complex. In addition, structures of the histone binding domains of ATXN7 and Sgf29 shed light on the interactions with chromatin that help recruit the SAGA complex.
Topics: Animals; Binding Sites; Chromatin; Humans; Protein Subunits; Structure-Activity Relationship; Trans-Activators; Transcription, Genetic
PubMed: 22014650
DOI: 10.1016/j.sbi.2011.09.004 -
Proceedings of the National Academy of... Jul 2022Function follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. For design of binding to...
Function follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. For design of binding to symmetric ligands, protein homo-oligomers with matching symmetry are advantageous as each protein subunit can make identical interactions with the ligand. Here, we describe a general approach to designing hyperstable C2 symmetric proteins with pockets of diverse size and shape. We first designed repeat proteins that sample a continuum of curvatures but have low helical rise, then docked these into C2 symmetric homodimers to generate an extensive range of C2 symmetric cavities. We used this approach to design thousands of C2 symmetric homodimers, and characterized 101 of them experimentally. Of these, the geometry of 31 were confirmed by small angle X-ray scattering and 2 were shown by crystallographic analyses to be in close agreement with the computational design models. These scaffolds provide a rich set of starting points for binding a wide range of C2 symmetric compounds.
Topics: Ligands; Models, Molecular; Protein Binding; Protein Subunits
PubMed: 35862457
DOI: 10.1073/pnas.2113400119 -
Trends in Biochemical Sciences Mar 2008Protein phosphatase 2A (PP2A), a major phospho-serine/threonine phosphatase, is conserved throughout eukaryotes. It dephosphorylates a plethora of cellular proteins,... (Review)
Review
Protein phosphatase 2A (PP2A), a major phospho-serine/threonine phosphatase, is conserved throughout eukaryotes. It dephosphorylates a plethora of cellular proteins, including kinases and other signaling molecules involved in cell division, gene regulation, protein synthesis and cytoskeleton organization. PP2A enzymes typically exist as heterotrimers comprising catalytic C-, structural A- and regulatory B-type subunits. The B-type subunits function as targeting and substrate-specificity factors; hence, holoenzyme assembly with the appropriate B-type subunit is crucial for PP2A specificity and regulation. Recently, several biochemical and structural determinants have been described that affect PP2A holoenzyme assembly. Moreover, the effects of specific post-translational modifications of the C-terminal tail of the catalytic subunit indicate that a 'code' might regulate dynamic exchange of regulatory B-type subunits, thus affecting the specificity of PP2A.
Topics: Animals; Catalysis; Holoenzymes; Humans; Models, Biological; Protein Conformation; Protein Phosphatase 2; Protein Structure, Secondary; Protein Subunits; Substrate Specificity
PubMed: 18291659
DOI: 10.1016/j.tibs.2007.12.004