Structure of Csx1-cOA4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas

Abstract Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5′-C-C-3′. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.

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Probing the Cavity of the Slow Inactivated Conformation of Shaker Potassium Channels

The Journal of General Physiology ◽

10.1085/jgp.200709758 ◽

2007 ◽

Vol 129 (5) ◽

pp. 403-418 ◽

Cited By ~ 29

Author(s):

Gyorgy Panyi ◽

Carol Deutsch

Keyword(s):

Potassium Channels ◽

Binding Site ◽

Conformational Change ◽

Marked Decrease ◽

Selectivity Filter ◽

Slow Inactivation ◽

Voltage Gated ◽

Activation Gate ◽

Shaker Potassium Channels

Slow inactivation involves a local rearrangement of the outer mouth of voltage-gated potassium channels, but nothing is known regarding rearrangements in the cavity between the activation gate and the selectivity filter. We now report that the cavity undergoes a conformational change in the slow-inactivated state. This change is manifest as altered accessibility of residues facing the aqueous cavity and as a marked decrease in the affinity of tetraethylammonium for its internal binding site. These findings have implications for global alterations of the channel during slow inactivation and putative coupling between activation and slow-inactivation gates.

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Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

Biochemical Journal ◽

10.1042/bj2850419 ◽

1992 ◽

Vol 285 (2) ◽

pp. 419-425 ◽

Cited By ~ 34

Author(s):

U Christensen ◽

L Mølgaard

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Conformational Change ◽

Conformational Changes ◽

Binding Sites ◽

High Specificity ◽

Stopped Flow ◽

Protein Fluorescence ◽

Lysine Residues ◽

Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

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Insights into the binding mode of MEK type-III inhibitors. A step towards discovering and designing allosteric kinase inhibitors across the human kinome

10.1101/076711 ◽

2016 ◽

Cited By ~ 1

Author(s):

Zheng Zhao ◽

Lei Xie ◽

Philip E. Bourne

Keyword(s):

Molecular Dynamics ◽

Binding Site ◽

Protein Kinases ◽

Drug Targets ◽

Kinase Inhibitors ◽

Binding Mode ◽

Dynamics Simulation ◽

Allosteric Inhibitors ◽

Type Iii ◽

Human Kinome

AbstractProtein kinases are critical drug targets for treating a large variety of human diseases. Type-I and type-II kinase inhibitors frequently exhibit off-target toxicity or lead to mutation acquired resistance. Two highly specific allosteric type-III MEK-targeted drugs, Trametinib and Cobimetinib, offer a new approach. Thus, understanding the binding mechanism of existing type-III kinase inhibitors will provide insights for designing new type-III kinase inhibitors. In this work we have systematically studied the binding mode of MEK-targeted type-III inhibitors using structural systems pharmacology and molecular dynamics simulation. Our studies provide detailed sequence, structure, interaction-fingerprint, pharmacophore and binding-site information on the binding characteristics of MEK type-III kinase inhibitors. We propose that the helix-folding activation loop is a hallmark allosteric binding site for type-III inhibitors. Subsequently we screened and predicted allosteric binding sites across the human kinome, suggesting other kinases as potential targets suitable for type-III inhibitors. Our findings will provide new insights into the design of potent and selective kinases inhibitors.Author SummaryHuman protein kinases represent a large protein family relevant to many diseases, especially cancers, and have become important drug targets. However, developing the desired selective kinase-targeted inhibitors remain challenging. Kinase allosteric inhibitors provide that opportunity, but, to date, few have been designed other than MEK inhibitors. To more efficiently develop kinase allosteric inhibitors, we systematically studied the binding mode of the MEK type-III allosteric kinase inhibitors using structural system pharmacology and molecular dynamics approaches. New insights into the binding mode and mechanism of type-III inhibitors were revealed that may facilitate the design of new prospective type-III kinase inhibitors.

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The Distal ExsA-Binding Site in Pseudomonas aeruginosa Type III Secretion System Promoters Is the Primary Determinant for Promoter-Specific Properties

Journal of Bacteriology ◽

10.1128/jb.00106-12 ◽

2012 ◽

Vol 194 (10) ◽

pp. 2564-2572 ◽

Cited By ~ 6

Author(s):

E. D. Brutinel ◽

J. M. King ◽

A. E. Marsden ◽

T. L. Yahr

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Site ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Type Iii ◽

Primary Determinant

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Csx3 is a cyclic oligonucleotide phosphodiesterase associated with type III CRISPR–Cas that degrades the second messenger cA4

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014099 ◽

2020 ◽

Vol 295 (44) ◽

pp. 14963-14972

Author(s):

Sharidan Brown ◽

Colin C. Gauvin ◽

Alexander A. Charbonneau ◽

Nathaniel Burman ◽

C. Martin Lawrence

Keyword(s):

Second Messengers ◽

Second Messenger ◽

Nuclease Activity ◽

Type I ◽

Type Iii ◽

Cell Death Pathway ◽

Cyclase Domain ◽

Protospacer Adjacent Motif ◽

Core Elements ◽

Mechanistic Basis

Cas10 is the signature gene for type III CRISPR–Cas surveillance complexes. Unlike type I and type II systems, type III systems do not require a protospacer adjacent motif and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate second messengers. These second messengers are recognized by ancillary Cas proteins harboring CRISPR-associated Rossmann fold (CARF) domains and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn2+-dependent deadenylation exoribonuclease. However, its specific role in CRISPR–Cas defense remains to be determined. Here we show that Csx3 is strongly associated with type III systems and that Csx3 binds cyclic tetra-adenylate (cA4) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA4 signal. In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity is discussed. Overall, the work suggests that Csx3 functions within CRISPR–Cas as a counterbalance to Cas10 to regulate the duration and amplitude of the cA4 signal, providing an off ramp from the programmed cell death pathway in cells that successfully cure viral infection.

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A pH-dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system

FEBS Journal ◽

10.1111/j.1742-4658.2005.04697.x ◽

2005 ◽

Vol 272 (11) ◽

pp. 2773-2783 ◽

Cited By ~ 5

Author(s):

Tomoaki Kato ◽

Daizo Hamada ◽

Takashi Fukui ◽

Makoto Hayashi ◽

Takeshi Honda ◽

...

Keyword(s):

Escherichia Coli ◽

Conformational Change ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Escherichia Coli O157 ◽

Type Iii ◽

Ph Dependent

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Conformation-dependent inactivation of human betaine-homocysteine S-methyltransferase by hydrogen peroxide in vitro

Biochemical Journal ◽

10.1042/bj20050356 ◽

2005 ◽

Vol 392 (3) ◽

pp. 443-448 ◽

Cited By ~ 13

Author(s):

Catherine M. Miller ◽

Sandra S. Szegedi ◽

Timothy A. Garrow

Keyword(s):

Hydrogen Peroxide ◽

Binding Site ◽

Conformational Change ◽

Binding Sites ◽

Quaternary Structure ◽

Substrate Binding ◽

Tryptophan Fluorescence ◽

H2o2 Treatment ◽

Oxidative Inactivation

Betaine-homocysteine S-methyltransferase (BHMT) transfers a methyl group from betaine to Hcy to form DMG (dimethylglycine) and Met. The reaction is ordered Bi Bi; Hcy is the first substrate to bind and Met is the last product off. Using intrinsic tryptophan fluorescence [Castro, Gratson, Evans, Jiracek, Collinsova, Ludwig and Garrow (2004) Biochemistry 43, 5341–5351], it was shown that BHMT exists in three steady-state conformations: enzyme alone, enzyme plus occupancy at the first substrate-binding site (Hcy or Met), or enzyme plus occupancy at both substrate-binding sites (Hcy plus betaine, or Hcy plus DMG). Betaine or DMG alone do not bind to the enzyme, indicating that the conformational change associated with Hcy binding creates the betaine-binding site. CBHcy [S-(δ-carboxybutyl)-D,L-homocysteine] is a bisubstrate analogue that causes BHMT to adopt the same conformation as the ternary complexes. We report that BHMT is susceptible to conformation-dependent oxidative inactivation. Two oxidants, MMTS (methyl methanethiosulphonate) and hydrogen peroxide (H2O2), cause a loss of the enzyme's catalytic Zn (Zn2+ ion) and a correlative loss of activity. Addition of 2-mercaptoethanol and exogenous Zn after MMTS treatment restores activity, but oxidation due to H2O2 is irreversible. CD and glutaraldehyde cross-linking indicate that H2O2 treatment causes small perturbations in secondary structure but no change in quaternary structure. Oxidation is attenuated when both binding sites are occupied by CBHcy, but Met alone has no effect. Partial digestion of ligand-free BHMT with trypsin produces two large peptides, excising a seven-residue peptide within loop L2. CBHcy but not Met binding slows down proteolysis by trypsin. These findings suggest that L2 is involved in the conformational change associated with occupancy at the betaine-binding site and that this conformational change and/or occupancy at both ligand-binding sites protect the enzyme from oxidative inactivation.

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