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Biochemical and Biophysical Research... Feb 2020The Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation...
The Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation and target RNA binding in the type III system. However, the exact functional mechanism of Csm5 has remained elusive. Here, we report the crystal structure of the apo form of the Csm5 subunit at a resolution of 2.6 Å. Structural comparison of amino acids in the complex bound to RNA exhibits notable conformational changes in the crRNA and the target RNA binding sites. Shifts in the β-hairpin motif (β5-β6), α13 helix (resides 352-383), and G-rich loop (residues 335-337) in the C-terminal domain indicate an induced movement by crRNA binding. The positively charged residues (Lys 92, Arg 95 and Lys 96) located in the β-α4 loop of the target RNA interface show high conformational flexibility, while three-helix bundles (α1-α3) of the N-domain involved in Csm2 binding exhibit a rotational shift. The altered architecture of the Csm5 subunit demonstrates remarkable versatility of the ferredoxin-like fold in the RNA binding protein and provides a structural basis for the mechanism for crRNA and target RNA binding in the type III-A Crispr-Cas system.
Topics: Apoproteins; CRISPR-Associated Proteins; CRISPR-Cas Systems; Crystallography, X-Ray; Models, Molecular; Protein Subunits
PubMed: 31836139
DOI: 10.1016/j.bbrc.2019.12.046 -
Biochemical and Biophysical Research... Dec 2020c-Myc modulator 1 (MM1), also known as PFDN5, is the fifth subunit of prefoldin. It was previously reported that MM1-based prefoldin promotes folding of actin during...
c-Myc modulator 1 (MM1), also known as PFDN5, is the fifth subunit of prefoldin. It was previously reported that MM1-based prefoldin promotes folding of actin during assembly of cytoskeleton, which plays key roles in cell migration. However, no evidence supports that MM1 affects cell migration. In the present study, we found that MM1 promotes cell migration in multiple cell lines. Further study revealed that MM1 promotes polymerization of β-actin into filamentous form and increases both density and length of filopodia. Effects of MM1 on filopodia formation and cell migration depend on its prefoldin activity. Though c-Myc is repressed by MM1, simultaneous knock-down of c-Myc fails to rescue migration inhibition induced by MM1 ablation. Taken together, we here, for the first time, report that prefoldin subunit MM1 is involved in cell migration; this involvement of MM1 in cell migration is due to its prefoldin activity to boost polymerization of β-actin during filopodia formation. Our findings may be helpful to elucidate the mechanism of cell migration and cancer metastasis.
Topics: Actins; Cell Line; Cell Movement; Humans; Molecular Chaperones; Protein Subunits; Pseudopodia
PubMed: 32981679
DOI: 10.1016/j.bbrc.2020.09.063 -
Methods in Enzymology 2021Most membrane proteins, and ion channels in particular, assemble to multimeric biological complexes. This starts with the quarternary structure and continues with the...
Most membrane proteins, and ion channels in particular, assemble to multimeric biological complexes. This starts with the quarternary structure and continues with the recruitment of auxiliary subunits and oligomerization or clustering of the complexes. While the quarternary structure is best determined by atomic-scale structures, stoichiometry of heteromers and dynamic changes in the assembly cannot necessarily be investigated with structural methods. Here, single subunit counting has proven a powerful method to study the composition of these complexes. Single subunit counting uses the irreversible photodestruction of fluorescent tags as means to directly count a labeled subunit and thereby derive the composition of the assemblies. In this chapter, we discuss single subunit counting and its limitations. We present alternative methods and provide a detailed protocol for recording and analysis of single subunit counting data.
Topics: Ion Channels; Protein Subunits
PubMed: 34099180
DOI: 10.1016/bs.mie.2021.02.017 -
Current Opinion in Structural Biology Apr 2013The eukaryotic Elongator complex was initially identified in yeast as a RNA polymerase II (Pol II) associated transcription elongation factor, although there is... (Review)
Review
The eukaryotic Elongator complex was initially identified in yeast as a RNA polymerase II (Pol II) associated transcription elongation factor, although there is accumulating evidence that its main cellular function is the specific modification of uridines at the wobble base position of tRNAs. Elongator complex is built up by six highly conserved subunits and was shown to be involved in a variety of different cellular activities. Here, we summarize structural and functional information on individual Elongator subunits or subcomplexes. On the basis of homology models of the Elp1, Elp2 and Elp3 subunits and the crystal structure of the Elp456 subcomplex, the role of each subunit in Elongator complex assembly and catalytic activity is discussed.
Topics: Carrier Proteins; Humans; Multiprotein Complexes; Peptide Chain Elongation, Translational; Peptide Elongation Factors; Protein Binding; Protein Subunits; RNA, Transfer
PubMed: 23510783
DOI: 10.1016/j.sbi.2013.02.009 -
Experimental Cell Research Nov 2014During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that apelin-13 stimulation can lead to the activation of...
During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that apelin-13 stimulation can lead to the activation of Gαi2 or Gαi3 through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi1 subunit and Gβγ subunits demonstrated little change after apelin-13 stimulation. These results demonstrated that stimulation of APJ with apelin-13 causes activation of Gαi2, Gαi3, Gαo, Gαq; among which Gαi2, Gαi3 were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.
Topics: Apelin Receptors; Cell Line; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins; Protein Subunits; Receptors, G-Protein-Coupled
PubMed: 25193074
DOI: 10.1016/j.yexcr.2014.08.035 -
Photochemistry and Photobiology 2008Membrane-inserted complexes consisting of two photochemically reactive sensory rhodopsin (SR) subunits flanking a homodimer of a transducing protein subunit (Htr) are... (Review)
Review
Membrane-inserted complexes consisting of two photochemically reactive sensory rhodopsin (SR) subunits flanking a homodimer of a transducing protein subunit (Htr) are used by halophilic archaea for sensing light gradients to modulate their swimming behavior (phototaxis). The SR-Htr complexes extend into the cytoplasm where the Htr subunits bind a his-kinase that controls a phosphorylation system that regulates the flagellar motors. This review focuses on current progress primarily on the mechanism of signal relay within the SRII-HtrII complexes from Natronomonas pharaonis and Halobacterium salinarum. The recent elucidation of a photoactive site steric trigger crucial for signal relay, advances in understanding the role of proton transfer from the chromophore to the protein in SRII activation, and the localization of signal relay to the membrane-embedded portion of the SRII-HtrII interface, are beginning to produce a clear picture of the signal transfer process. The SR-Htr complexes offer unprecedented opportunities to resolve first examples of the chemistry of signal relay between membrane proteins at the atomic level, which would provide a major contribution to the general understanding of dynamic interactions between integral membrane proteins.
Topics: Binding Sites; Euryarchaeota; Models, Molecular; Protein Conformation; Protein Subunits; Sensory Rhodopsins; Signal Transduction
PubMed: 18346091
DOI: 10.1111/j.1751-1097.2008.00314.x -
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 -
Advances in Experimental Medicine and... 2010The archaeal exosome is aprotein complex with structural similarities to the eukaryotic exosome and bacterial PNPase. Its catalytic core is formed by alternating Rrp41... (Review)
Review
The archaeal exosome is aprotein complex with structural similarities to the eukaryotic exosome and bacterial PNPase. Its catalytic core is formed by alternating Rrp41 and Rrp42 polypeptides, arranged in a hexameric ring. A flexible RNA binding cap composed of the evolutionarily conserved proteins Rrp4 and/or Cs14 is bound at the top of the ring and seems to be involved in recruitment of specific substrates and their unwinding. Additionally, the protein complex contains an archaea-specific subunit annotated as DnaG, the function of which is still unknown. The archaeal exosome degrades RNA phosphorolytically in 3' to 5' direction. In a reverse reaction, it synthesizes heteropolymeric RNA tails using nucleoside diphosphates. The functional similarity between the archaeal exosome and PNPase shows that important processes of RNA degradation and posttranscriptional modification in Archaea are similar to the processes in Bacteria and organelles.
Topics: Archaea; Archaeal Proteins; Exoribonucleases; Exosomes; Models, Molecular; Protein Conformation; Protein Subunits; RNA, Archaeal
PubMed: 21618872
DOI: No ID Found -
The Journal of Biological Chemistry Jul 2011Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based...
Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O](n) multimers within the protein cage, en route to the cavity, at sites distributed over ~50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³⁺O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A(650 nm)) and on mineral growth (Fe³⁺O-A(350 nm)), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²⁺ access/selectivity to the active sites (Glu¹³⁰), (ii) distribution of Fe²⁺ to each of the three active sites near each ion channel (Asp¹²⁷), (iii) product (diferric oxo) release into the Fe³⁺O nucleation channels (Ala²⁶), and (iv) [Fe³⁺O](n) transit through subunits (Val⁴², Thr¹⁴⁹). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²⁺ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.
Topics: Amino Acid Substitution; Animals; Anura; Biocatalysis; Catalytic Domain; Conserved Sequence; Ferritins; Iron; Minerals; Models, Molecular; Movement; Nanostructures; Oxygen; Protein Structure, Secondary; Protein Subunits
PubMed: 21592958
DOI: 10.1074/jbc.M110.205278 -
Molecular Biology of the Cell Jul 2004The Na,K-ATPase consists of an alpha- and beta-subunit. Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK) express low levels of Na,K-ATPase beta(1)-subunit....
The Na,K-ATPase consists of an alpha- and beta-subunit. Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK) express low levels of Na,K-ATPase beta(1)-subunit. Ectopic expression of Na,K-ATPase beta(1)-subunit in these cells increased the protein levels of the alpha(1)-subunit of Na,K-ATPase. This increase was not due to altered transcription of the alpha(1)-subunit gene or half-life of the alpha(1)-subunit protein because both alpha(1)-subunit mRNA levels and half-life of the alpha(1)-subunit protein were comparable in MSV-MDCK and beta(1)-subunit expressing MSV-MDCK cells. However, short pulse labeling revealed that the initial translation rate of the alpha(1)-subunit in beta(1)-subunit expressing MSV-MDCK cells was six- to sevenfold higher compared with MSV-MDCK cells. The increased translation was specific to alpha(1)-subunit because translation rates of occludin and beta-catenin, membrane and cytosolic proteins, respectively, were not altered. In vitro cotranslation/translocation experiments using rabbit reticulocyte lysate and rough microsomes revealed that the alpha(1)-subunit mRNA is more efficiently translated in the presence of beta(1)-subunit. Furthermore, sucrose density gradient analysis revealed significantly more alpha(1)-subunit transcript associated with the polysomal fraction in beta(1)-subunit expressing MSV-MDCK cells compared with MSV-MDCK cells, indicating that in mammalian cells the Na,K-ATPase beta(1)-subunit is involved in facilitating the translation of the alpha(1)-subunit mRNA in the endoplasmic reticulum.
Topics: Animals; Cell Extracts; Cell Line, Transformed; Cell Membrane; Dogs; Gene Expression Regulation, Enzymologic; Moloney murine sarcoma virus; Polyribosomes; Protein Biosynthesis; Protein Subunits; RNA, Messenger; Sodium-Potassium-Exchanging ATPase; Up-Regulation
PubMed: 15133131
DOI: 10.1091/mbc.e04-03-0222