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Current Opinion in Structural Biology Oct 2016With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments... (Review)
Review
With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments in crystallography, novel interfaces with other biophysical methods are emerging. Here we consider how mass spectrometry can inform these techniques by providing unambiguous definition of subunit stoichiometry. Moreover recent developments that increase mass spectral resolution enable molecular details to be ascribed to unassigned density within high-resolution maps of membrane and soluble protein complexes. Importantly we also show how developments in mass spectrometry can define optimal solution conditions to guide downstream structure determination, particularly of challenging biomolecules that refuse to crystallise.
Topics: Biology; Crystallography; DNA; Humans; Mass Spectrometry; Protein Subunits; RNA
PubMed: 27721169
DOI: 10.1016/j.sbi.2016.09.008 -
The Biochemical Journal Nov 2018Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are vital eukaryotic signaling elements that convey information from ligand-regulated G protein-coupled... (Review)
Review
Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are vital eukaryotic signaling elements that convey information from ligand-regulated G protein-coupled receptors (GPCRs) to cellular effectors. Heterotrimeric G protein-based signaling pathways are fundamental to human health [ (2007) , 994-1005] and are the target of >30% of pharmaceuticals in clinical use [ (2013) , 1676-1694; (2017) , 829-842]. This review focuses on phosphorylation of G protein subunits as a regulatory mechanism in mammals, budding yeast, and plants. This is a re-emerging field, as evidence for phosphoregulation of mammalian G protein subunits from biochemical studies in the early 1990s can now be complemented with contemporary phosphoproteomics and genetic approaches applied to a diversity of model systems. In addition, new evidence implicates a family of plant kinases, the receptor-like kinases, which are monophyletic with the interleukin-1 receptor-associated kinase/Pelle kinases of metazoans, as possible GPCRs that signal via subunit phosphorylation. We describe early and modern observations on G protein subunit phosphorylation and its functional consequences in these three classes of organisms, and suggest future research directions.
Topics: Animals; Heterotrimeric GTP-Binding Proteins; Humans; Mammals; Phosphorylation; Plants; Protein Binding; Protein Subunits; Saccharomyces cerevisiae; Signal Transduction
PubMed: 30413679
DOI: 10.1042/BCJ20160819 -
Biomolecules Nov 2020The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research,... (Review)
Review
The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, mutants lacking (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in . We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants.
Topics: Bacterial Proteins; DNA-Directed RNA Polymerases; Mutant Proteins; Protein Subunits; Structure-Activity Relationship; Transcription Factors
PubMed: 33238579
DOI: 10.3390/biom10111588 -
Current Opinion in Structural Biology Feb 2019Ionotropic glutamate receptors in vertebrates are composed of three major subtypes - AMPA, kainate, and NMDA receptors - and mediate the majority of fast excitatory... (Review)
Review
Ionotropic glutamate receptors in vertebrates are composed of three major subtypes - AMPA, kainate, and NMDA receptors - and mediate the majority of fast excitatory neurotransmission at chemical synapses of the central nervous system. Among the three major families, native AMPA receptors function as complexes with a variety of auxiliary subunits, which in turn modulate receptor trafficking, gating, pharmacology, and permeation. Despite the long history of structure-mechanism studies using soluble receptor domains or intact yet isolated receptors, structures of AMPA receptor-auxiliary subunit complexes have not been available until recent breakthroughs in single-particle cryo-electron microscopy. Single particle cryo-EM studies have, in turn, provided new insights into the structure and organization of AMPA receptor - auxiliary protein complexes and into the molecular mechanisms of AMPA receptor activation and desensitization.
Topics: Animals; Protein Subunits; Receptors, AMPA
PubMed: 30825796
DOI: 10.1016/j.sbi.2019.01.011 -
International Journal of Molecular... Sep 2021Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5' end of precursor tRNAs (pre-tRNAs). In all organisms, the... (Review)
Review
Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5' end of precursor tRNAs (pre-tRNAs). In all organisms, the cleavage activity of a single phosphodiester bond adjacent to the first nucleotide of the acceptor stem is indispensable for cell viability and lies within an essential catalytic RNA subunit. Although RNase P is a ribozyme, its kinetic efficiency in vivo, as well as its structural variability and complexity throughout evolution, requires the presence of one protein subunit in bacteria to several protein partners in archaea and eukaryotes. Moreover, the existence of protein-only RNase P (PRORP) enzymes in several organisms and organelles suggests a more complex evolutionary timeline than previously thought. Recent detailed structures of bacterial, archaeal, human and mitochondrial RNase P complexes suggest that, although apparently dissimilar enzymes, they all recognize pre-tRNAs through conserved interactions. Interestingly, individual protein subunits of the human nuclear and mitochondrial holoenzymes have additional functions and contribute to a dynamic network of elaborate interactions and cellular processes. Herein, we summarize the role of each RNase P subunit with a focus on the human nuclear RNP and its putative role in flawless gene expression in light of recent structural studies.
Topics: Animals; Catalytic Domain; Humans; Kinetics; Protein Subunits; RNA Precursors; RNA, Catalytic; Ribonuclease P
PubMed: 34638646
DOI: 10.3390/ijms221910307 -
Cell Biology International Jun 2018The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium... (Review)
Review
The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium uniporter (MCU), a long-sought channel present in most eukaryotic organisms. Since then, research efforts to elucidate the role of MCU and its regulatory elements in different biological models have multiplied. MCU is the pore-forming subunit of a multimeric complex (the MCU complex or MCUC) and its predicted structure in trypanosomes is simpler than in mammalian cells, lacking two of its subunits and probably possessing other unidentified components. MCU protein has been characterized in Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African and American trypanosomiasis, respectively. Contrary to its mammalian homolog, TbMCU was found to be essential for cell growth and survival, while its paralog MCUb is an essential protein in T. cruzi. These findings could be further exploited for chemotherapeutic purposes. The emergence of new molecular tools for the genetic manipulation of trypanosomatids has been determinant for the functional characterization of the MCUC components in these organisms. However, further research has to be done to determine the role of each component in intracellular calcium signaling and cell bioenergetics. In this mini-review we summarize the original results on mitochondrial calcium uptake in trypanosomes, how did they contribute to the molecular identification of the MCU, and the functional characterization of the MCUC subunits that has so far been studied in these peculiar eukaryotes.
Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Mitochondria; Protein Subunits; Protozoan Proteins; Trypanosoma
PubMed: 29286188
DOI: 10.1002/cbin.10928 -
The FEBS Journal Aug 2013AMP-activated protein kinase (AMPK) is a sensor of energy status composed of a catalytic subunit (AMPKα), a scaffolding subunit (AMPKβ) and a regulatory subunit... (Review)
Review
AMP-activated protein kinase (AMPK) is a sensor of energy status composed of a catalytic subunit (AMPKα), a scaffolding subunit (AMPKβ) and a regulatory subunit involved in nucleotide binding (AMPKγ). Activation of AMPK results in enhancement of catabolic processes and downregulation of anabolic pathways with the aim to equilibrate the energy status of the cell. The study of the regulation of the activity of the AMPK complex has been traditionally focused on modifications of AMPKα and AMPKγ subunits by post-translational changes (i.e. phosphorylation of the catalytic subunit) and allosteric activation by AMP. In this review, we summarize recent reports that indicate that AMPKβ subunits are also critical players in AMPK function, because they can regulate the phosphorylation status and activity of the AMPK complex. AMPKβ1- and AMPKβ2-containing complexes differ in their capacity to be activated by specific drugs (i.e. A769622, salicylate) and also by the ability to undergo post-translational modifications. This selective behavior opens the possibility to design specific drugs that activate AMPK complexes containing specific β-isoforms.
Topics: AMP-Activated Protein Kinases; Animals; Enzyme Activation; Humans; Isoenzymes; Protein Interaction Domains and Motifs; Protein Processing, Post-Translational; Protein Subunits
PubMed: 23721051
DOI: 10.1111/febs.12364 -
Trends in Pharmacological Sciences Dec 2011N-Methyl-D-aspartate (NMDA) receptors are tetrameric ion channels containing two of four possible GluN2 subunits. These receptors have been implicated for decades in... (Review)
Review
N-Methyl-D-aspartate (NMDA) receptors are tetrameric ion channels containing two of four possible GluN2 subunits. These receptors have been implicated for decades in neurological diseases such as stroke, traumatic brain injury, dementia and schizophrenia. The GluN2 subunits substantially contribute to functional diversity of NMDA receptors and are distinctly expressed during development and among brain regions. Thus, subunit-selective antagonists and modulators that differentially target the GluN2 subunit might provide an opportunity to pharmacologically modify the function of select groups of neurons for therapeutic gain. A flurry of clinical, functional and chemical studies have together reinvigorated efforts to identify subunit-selective modulators of NMDA receptor function, resulting in a handful of new compounds that appear to act at novel sites. Here, we review the properties of new emerging classes of subunit-selective NMDA receptor modulators, which we predict will mark the beginning of a productive period of progress for NMDA receptor pharmacology.
Topics: Allosteric Regulation; Animals; Binding Sites; Humans; Ligands; Membrane Transport Modulators; Protein Conformation; Protein Isoforms; Protein Subunits; Receptors, N-Methyl-D-Aspartate
PubMed: 21996280
DOI: 10.1016/j.tips.2011.08.003 -
Bioorganic Chemistry Mar 2023The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for...
The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for selective labeling of biomolecules. In this work, we exploit the slow reaction between a non-conjugated terminal alkene and a tetrazine, and apply this reaction to achieving a proximity-enhanced protein crosslinking. In one protein subunit, a terminal alkene-containing amino acid was site-specifically incorporated in response to an amber nonsense codon. In another protein subunit, a tetrazine moiety was introduced through the attachment to a cysteine residue. Fast protein crosslinking was achieved due to a large increase in effective molarity of the two reactants that were brought to close proximity by the two interacting protein subunits. Such a proximity-enhanced protein crosslinking is useful for the study of protein-protein interactions.
Topics: Alkenes; Protein Subunits; Heterocyclic Compounds; Amino Acids; Cycloaddition Reaction
PubMed: 36642019
DOI: 10.1016/j.bioorg.2023.106359 -
Cell Structure and Function 2016The Saccharomyces cerevisiae autophagy-initiation complex, Atg1 kinase complex, consists of Atg1, Atg13, Atg17, Atg29, and Atg31, while the corresponding complex in most... (Review)
Review
The Saccharomyces cerevisiae autophagy-initiation complex, Atg1 kinase complex, consists of Atg1, Atg13, Atg17, Atg29, and Atg31, while the corresponding complex in most other eukaryotes, including mammals, is composed of ULK1 (or ULK2), Atg13, FIP200 (also known as RB1CC1), and Atg101. ULKs are homologs of Atg1, and FIP200 is partially homologous to Atg17. However, the sequence of Atg101 is not similar to that of Atg29 or Atg31. Although Atg101 is essential for autophagy and widely conserved in eukaryotes, its precise function and structure have remained largely unknown. Now, structural and cell biological analysis of Atg101 together with its binding partner Atg13 reveal that Atg101 is required for stabilization of "uncapped" Atg13 in most eukaryotes and also for recruitment of downstream Atg proteins through the newly identified WF motif. By contrast, S. cerevisiae has stable "capped" Atg13, which does not require Atg101 for its stabilization. Possible roles for other binding partners such as Atg29, Atg31, and Atg28 in different organisms are also discussed.
Topics: Animals; Autophagy; Humans; Protein Kinases; Protein Subunits; Yeasts
PubMed: 26754330
DOI: 10.1247/csf.15013