supplementary information structure and function of the ...€¦ · 1 supplementary information...

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Supplementary Information

Structure and function of the bacterial decapping enzyme NudC

Katharina Höfer1,4, Sisi Li2,4, Florian Abele1, Jens Frindert1, Jasmin Schlotthauer1, Julia

Grawenhoff1, Jiamu Du3, Dinshaw J. Patel2*, Andres Jäschke1*

1 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, 69120

Heidelberg, Germany

2 Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065,

USA

3 Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences,

Chinese Academy of Sciences, Shanghai 201602, China

4 These authors contributed equally to this work.

* Correspondence to [email protected] and [email protected]

Nature Chemical Biology: doi:10.1038/nchembio.2132

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Supplementary Results

Supplementary Figures

Supplementary Figure 1 | The electron density of the bound ligand in the two complexes.

Sigma-A weighted 2Fo-Fc map of NAD (a) and NMN (b) at 1 sigma level.


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Supplementary Figure 2 | Mutagenesis of NudC. (a) Y188-Y188’ interaction at the dimer

interface. (b) Size exclusion chromatography of NudC, NudC Y188A and NudC Y188Q

mutants.


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Supplementary Figure 3 | NudC-RNA interactions. (a) Decapping kinetics of full-length

E. coli NXD-RNAI depending on the nature of the 5’-terminal nucleotide. Conditions as in

Fig. 3a. Full denaturing PAGE gels are shown in Supplementary Fig. 8c. (b) Cleft above

the adenosine ribose moiety.


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Supplementary Figure 4 | Kinetic characterization of NudC and NudE. (a)

Representative gels for the experiment shown in Fig. 4c. Full denaturing PAGE gels are

shown in Supplementary Fig. 8d. (b) pH-dependent decapping of E. coli NAD-RNAI by

NudC. Data points represent mean ± standard deviation (s.d.), n=2. Assay as in Fig. 3a. Full

denaturing PAGE gels are shown in Supplementary Fig. 8e. (c) Representative gels for the

experiment shown in Fig. 4d. Full denaturing PAGE gels are shown in Supplementary Fig.

8f. (d) Hydrolysis of NAD and NADH (5 mM) by NudC. Data points represent mean ±

standard deviation (s.d.), n=3. (e) Hydrolysis of 32P-NAD by NudE, separation by TLC. (f)

Comparison of NAD-RNAI processing by Nudix enzymes NudC and NudE. Assay as in Fig.

3a. Full denaturing PAGE gels are shown in Supplementary Fig. 8g.


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Supplementary Figure 5 | NudC as an RNA-binding protein. (a) Size distribution of NudC-

bound RNAs with and without DNase I or RNase A/T1 treatment in comparison to total RNA

from the same strain under the same conditions (left gel). Size distribution of RNAs bound to

wild-type NudC in comparison to catalytically inactive E178Q mutant (right gel). 10%

denaturing PAGE, SYBR gold staining. Experiment was carried out with independent

cultures in duplicate. (b) Abundance of NudC-bound RNAs from E. coli strain BL21 (DE3)

versus total RNA isolated from the same strain under the same conditions (top panel) and

abundance of RNAs bound to wild-type NudC versus RNAs bound to NudC E178Q mutant

(bottom panel). NGS analysis (reads per million (RPM)), (c) Hydrolysis of 32P-NAD by highly

pure, RNA-free NudC (left) and by crude RNA-containing NudC (right). TLC analysis.


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Supplementary Figure 6 | Analysis of purity and dimerization of NudC and single

mutants. (a) Analysis of purified NudC mutants by SDS PAGE and (b) by size exclusion

chromatography.


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Supplementary Figure 7 | CD Spectra of NudC and single mutants.


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Supplementary Figure 8 | Full denaturing PAGE gels. (a) Decapping of full-length E. coli

NAD-RNAI by NudC and single mutants. (b) Decapping of 5 nM NAD-RNAI (asterisk) in

presence of 5 mM NAD+ or NADH. Full denaturing PAGE gels. (c) Decapping kinetics of full-

length E. coli NXD-RNAI (asterisk) depending on the nature of the 5’-terminal nucleotide. (d)

Analysis of the role of secondary structure on NudC decapping. Corresponding RNA bands

are marked with asterisk. (e) pH-dependent decapping of E. coli NAD-RNAI (asterisk) by. (f)

Decapping of 5 nM NAD-RNAI (asterisk) in presence of NAD and NADH (5 mM) by NudC.

(g) Comparison of NAD-RNAI (asterisk) processing by Nudix enzymes NudC and NudE.


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Supplementary Tables

Supplementary Table 1. Data collection and refinement statistics (molecular

replacement).

NudC + NMN NudC + NAD

Data collection Space group C2 P212121

Cell dimensions

a, b, c (Å) 104.9, 62.2, 86.9 51.9, 99.4, 113.7

() 90, 95.0, 90 90, 90, 90

Resolution (Å) 50.0-2.7 (2.81-2.70)*

50.0-2.6 (2.69-2.60)

Rsym or Rmerge 0.126 (0.789) 0.111 (0.529)

I / I 14.2 (2.2) 23.5 (3.5)

Completeness (%) 99.2 (98.7) 99.9 (99.9) Redundancy 4.0 (4.1) 7.0 (7.2) Refinement Resolution (Å) 50.0-2.7 (2.81-2.70) 50.0-2.6 (2.69-

2.60) No. reflections 15,382 18,662 Rwork / Rfree 19.6 / 23.5 20.0 / 24.8 No. atoms 4,287 4,407 Protein 4,130 4,181 Ligand/ion 44/2 88/2 Water 111 136 B-factors 49.9 49.4 Protein 50.0 49.3 Ligand/ion 88.7/78.0 59.7/53.2 Water 42.6 44.5 R.m.s. deviations Bond lengths (Å) 0.014 0.011

Bond angles () 1.484 1.061

One crystal was used for each structure. *Highest-resolution shell is shown in parentheses


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Supplementary Table 2. Primer used in this study.

Primer Sequence (5’-3’)

T7-RNAI fwd A

TAATACGACTCACTATTACAGTATTTGGTATCTGC

T7-RNAI fwd G TAATACGACTCACTATAGCAGTATTTGGTATCTGC

T7-RNAI fwd C TAATACGACTCACTATACCAGTATTTGGTATCTGC

T7-RNAI fwd U TAATACGACTCACTATATCAGTATTTGGTATCTGC

RNAI rev TCAGCAGAGCGCAGATACCAAATACTGTAATAGTGAGTCGTATTA

T7-RNAI 5’-ds TAATACGACTCACTATTAGGTATCTGCGCTCTGCT

T7-5S rRNA TAATACGACTCACTATTAGTGGCCTGGC

Rev 5S rRNA ATGCCTGGCAGTTCCCTACTCTC

T7 5’-blunt-end TAATACGACTCACTATTAGACTTCGGTCT

Rev 5’-blunt-end AGACCGAAGTCTAATAGTGAGTCGTATTA

T7 1nt-5’-overhang TAATACGACTCACTATTAAGACTTCGGTCT

Rev 1nt-5’-overhang AGACCGAAGTCTTAATAGTGAGTCGTATTA

T7 2nt-5’-overhang TAATACGACTCACTATTACAGACTTCGGTCT

Rev 2nt-5’-overhang AGACCGAAGTCTGTAATAGTGAGTCGTATTA

T7 3nt-5’-overhang TAATACGACTCACTATTACAAGACTTCGGTCT

Rev 3nt-5’-overhang AGACCGAAGTCTTGTAATAGTGAGTCGTATTA

T7 4nt-5’-overhang TAATACGACTCACTATTACAGAGACTTCGGTCT

Rev 4nt-5’-overhang AGACCGAAGTCTCTGTAATAGTGAGTCGTATTA

T7 +1nt 3’-overhang TAATACGACTCACTATTAGACTTCGGTCTA

Rev +1nt 3’-overhang TAGACCGAAGTCTAATAGTGAGTCGTATTA

T7-linear TAATACGACTCACTATTAGACTTCG

Rev-linear CGAAGTCTAATAGTGAGTCGTATTA

NudC fwd CAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAA

TGGATCGTATAATTGAAAAATTAGATCACGGC

NudC rev GTGCTCGAGTGCGGCCGCGCTGCCGCGCGGCACCAGCTCATACTCT

GCCCGACACATCGCCACCGU

Fwd NudC E178Q CACTTCGACGAATCCGGCAAGTACTGTATGG

Rev NudC E178Q GGCGAAACCCTCGAGCAGGCAGTCGCGCGGGAAGTGATGGAACAGA

GCGGAATTAAAGTTAAAAACTTGCG

Fwd NudC W194A TCTTTAATGACCGCGTTTATGGCG

Rev NudC W194A CTGAGGAAACGGCGCCGGCTGAGAAGTC

Fwd NudC P236A GCGCCGTCTGATAGAAGATACGG

Rev NudC P236A GCTACGGTGCCGGGCGCCGGGAGTAACG

Fwd NudC F160A GTACTTGCCGGAGCGGTCGAAGTGGGCGAAACC

Rev NudC F160A TGTATGGACACCGTTACGATGGCGG

Fwd NudC Y124A GAGCGTTACGCCCCGCAAATCGC

Rev NudC Y124A ACGGCAATGGCTGCACAGCATCG


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Fwd NudC E219Q CGACCCGAAACAGTTGCTCGAGGCGAAC

Rev NudC E219Q ATCACGATGTCGCCGCTGTCATATTCC

Fwd NudC E174Q CAGTCGCGCGGCAGGTGATGGAAGAGAG

Rev NudC E174Q CCTGCTCGAGGGTTTCGCCCACTTC

Fwd NudC R69A GAT ATG GGG TCG GTA GCC CAG GTC ATT GAT CTC

Rev NudC R69A GTG ACG CCG CTG CTG TTG TAC TAA CCA

Fwd NudC Y188Q CCGTGGCCGTTTCCTCAGTCTTTA

Rev NudC Y188Q CTGAGAAGTCACCTGACGCAAGTTTTTAAC

Fwd NudC Y188A GTTAAAAACTTGCGTGCGGTGACTTCTCAGCC

Rev NudC Y188A TTTAATTCCGCTCTCTTCCATCACTTCCCGC

Fwd NudE GTATGCCCATGGCTAGCAAATCATTACAAAAACCCACCATTCTG

Rev NudE GTGGTGGTGGTGGTGGTGCTCGAGCTGAAAATACAGGTTTTCTCGCC

CCTGCCCTTTCAACCATTCGCGC


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