Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease

Yan, Xin-Fu; Guo, Wanshou; Yuan, Yunbin

doi:10.1080/15476286.2015.1051300

Cited by 27 publications

(47 citation statements)

References 40 publications

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“…( Figure 2), in keeping with previous observations (Yan et al, 2015). The rate constant for cleavage 117 was calculated as 0.0063 ± 0.0013 min -1 at 50 °C.…”

supporting

confidence: 89%

“…The complex with an RNA fragment had two adenine, one uracil and one guanine bases in the same 252 position as the four adenine bases described here (Yan et al, 2015), there is obviously some 253 plasticity around these recognition sites. 254…”

mentioning

confidence: 66%

“…bound between adjacent dimers of Csx3 ( Figure 5A). Notably, the Csx3 structure presented here 230 crystallised in a different space group to those structures published previously (Yan et al, 2015), 231…”

Section: Csx3 Can Decrease Ca 4 -Mediated Immunity In Vivo 145mentioning

confidence: 90%

“…from Archaeoglobus fulgidus has been described as an RNA deadenylation enzyme and 73 crystallised in complex with a pseudo-symmetric RNA tetraloop (Yan et al, 2015). Subsequent 74 structural analysis revealed that the Csx3 protein is a distantly related member of the CARF family 75 of proteins (Topuzlu and Lawrence, 2016).…”

Section: Introduction 23mentioning

confidence: 99%

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Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Graham

Grüschow

Gloster

et al. 2020

Preprint

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9Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to 11 signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This 12 can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell 13once a viral infection has been defeated. Enzymes specialised for this task are known as ring 14nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR 15 associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that 16rapidly degrades cyclic tetra-adenylate (cA 4 ). The enzyme has an unusual cooperative reaction 17 mechanism involving an active site that spans the interface between two dimers, sandwiching the 18 cA 4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 19family. 20 Keywords: 21 CRISPR, Csx3, ring nuclease, cyclic tetra-adenylate, CARF, cooperative enzyme 22 103 terminal Csx3 domain fused to a C-terminal kinase/transferase domain of unknown function. B. Phosphor 104 images of native gel electrophoresis visualising cA 4 (20 nM) or RNA oligonucleotide 49-9A (50 nM) binding 105 by A. fulgidus Csx3. Csx3 binds to cA 4 with high affinity (apparent K D ~ 50 nM) and binds the RNA 49-9A 106 with significantly lower affinity (apparent K D ~ 10 µM). Images are representative of three technical 107 replicates. 108 109 Figure 1figure supplement 1. Multiple sequence alignment of Csx3 proteins showing conserved 110 residues. Csx3 proteins are shown from Archaeoglobus fulgidus; Methanosarcinia mazei; candidatus 111 Bathyarchaeota; Thermococcus celericrescens; Methylacidiphilum fumariolicum; Aquifex aeolicus; 112 Dictyoglomus turgidum; Spirulina major; Oscillatoria nigro-viridis. Absolutely conserved residues are shaded. 113 Conserved residues H60 and D69 (Afu numbering) are indicated by asterisks. 114 131 Figure 2. Csx3 is a potent ring nuclease. A. TLC analysis of the reaction products of radiolabelled cA 4 132 (200 nM) incubated with A. fulgidus Csx3 (8 µM dimer) in reaction buffer at 50 °C (time points every 5 s from 133 10-60 s then 1.5, 2, 3, 4, 5 and 10 min, n = 6 technical replicates). The cA 4 was rapidly converted into A 2 -P, 134 with a small amount of A 2 >P (linear A 2 with a cyclic 2',3' terminal phosphate). B. Denaturing polyacrylamide 135 gel electrophoresis visualising cleavage of 5'-end radiolabelled RNA 49-9A (50 nM) by Csx3 (8 µM dimer) at 136 50 °C (time points every 5 min from 5-40 min then 50, 60 and 90 min, n = 3 technical replicates). C. Plot 137 comparing single-turnover kinetics of cA 4 (blue) and RNA 49-9A (green) cleavage by AfCsx3 at 50 °C. Data 138 points are the average of three technical replicates and error bars represent the standard deviation of the 139 mean. D. Liquid-chromatography high-resolution mass spectrometry analysis of reaction products when cA 4

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“…( Figure 2), in keeping with previous observations (Yan et al, 2015). The rate constant for cleavage 117 was calculated as 0.0063 ± 0.0013 min -1 at 50 °C.…”

supporting

confidence: 89%

mentioning

confidence: 66%

Section: Csx3 Can Decrease Ca 4 -Mediated Immunity In Vivo 145mentioning

confidence: 90%

Section: Introduction 23mentioning

confidence: 99%

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Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Graham

Grüschow

Gloster

et al. 2020

Preprint

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show abstract

“…1 In this research article the authors present compelling evidence that Csx3 possesses manganese dependent RNase activity and also present crystal structures showing that the manganese and RNA binding sites (Fig. 1A) lie at opposite ends of the Csx3 homodimer.…”

mentioning

confidence: 75%

Recognition of a pseudo-symmetric RNA tetranucleotide by Csx3, a new member of the CRISPR associated Rossmann fold superfamily

Topuzlu

Lawrence

2016

RNA Biology

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The CRISPR/Cas adaptive immune system shows extreme diversity in the number of CRISPR/Cas types and subtypes, and in the multitude of CRISPR associated protein families of which they are composed. Despite this diversity, the roles of many Cas protein families are now defined with regard to spacer acquisition, crRNA biogenesis, and DNA or RNA surveillance and targeting. However, a number of unclassified CRISPRCas proteins remain. Such proteins have traditionally been designated as CRISPR subtype x (Csx). Here we revisit the structural analysis of one such protein, Csx3, and show that this homodimeric protein utilizes a Rossmann fold for the recognition of an RNA tetranucleotide. Tertiary and quaternary structural similarities of Csx3 to CRISPR/Cas proteins Csx1 and Csa3 are identified and suggest Csx3 is a new member of the CRISPR Associated Rossmann Fold (CARF) superfamily. The structure of the Csx3/RNA complex illustrates one way CARF domain proteins may recognize pseudo-symmetric polynucleotides. Yan et al. recently published "Crystal structures of CRISPRassociated Csx3 reveal a manganese-dependent deadenylation exoribonuclease" in RNA Biology. 1 In this research article the authors present compelling evidence that Csx3 possesses manganese dependent RNase activity and also present crystal structures showing that the manganese and RNA binding sites (Fig. 1A) lie at opposite ends of the Csx3 homodimer. In all, their work represents a significant advance in our understanding of Csx3, providing critical new information relevant to its potential roles in CRISPR/Cas.They also describe the fold of Csx3 (Fig. 1B) as a "ferredoxin-like" fold. At first glance, it is not surprising that Csx3 might also harbor a ferredoxin-like fold. The ferredoxin-like fold (Fig. 1C) is commonly observed in proteins involved in CRISPR-Cas, where it may also be referred to as an RRM (RNA Recognition Motif) or RAMP (Repeat Associated Mysterious Protein) domain. RAMP domains are found in the Cas5, Cas6, Cas7 and Cmr3 families and RAMP-like domains are also found in Cas2 and Cas10. 2,3 However, the ferredoxin-like fold is defined in the SCOP database 4 (and elsewhere) as a babbab or (bab) 2 fold, resulting in a 4 stranded antiparallel b sheet arranged as b 2 b 3 b 1 b 4 . The two helices, present as righthanded crossovers between the b strands, lie on the same face of the b-sheet where they run antiparallel to each other (Fig. 1C). In this context, the Csx3 structure does indeed show a b-sheet flanked on one side by 2 a-helices. But in contrast to the ferredoxin-like fold, Csx3 lacks a 4-stranded antiparallel b sheet, the helices run antiparallel to each other, and the order and connectivity between the b-strands is substantially different (Fig. 1B).The Csx3 fold can, however, be described as a bbababbb(a) structure resulting in a 6-stranded mixed b-sheet in which the b-strands run sequentially across the sheet from b1-b6 (right to left in Fig. 1B). In addition, as a result of right-handed crossover connections provided by helices a1...

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Specific recognition of cyclic oligonucleotides by Cap4 for phage infection

Chang

You

Tien

et al. 2023

International Journal of Biological Macromolecules

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Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease

Cited by 27 publications

References 40 publications

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Recognition of a pseudo-symmetric RNA tetranucleotide by Csx3, a new member of the CRISPR associated Rossmann fold superfamily

Specific recognition of cyclic oligonucleotides by Cap4 for phage infection

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