Báo cáo khoa học: Solution structure of the catalytic domain of RICH protein from goldfish pot

RICH consists of a highly acidic N-terminal domain, a catalytic domain with 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase CNPase activity and a C-terminal isoprenylation site.. Here, we r

from goldfish

Guennadi Kozlov, Alexey Y Denisov, Ekaterina Pomerantseva, Michel Gravel, Peter E Braun and Kalle Gehring

Department of Biochemistry, McGill University, Montreal, Quebec, Canada

Axonal injuries in the mammalian central nervous

sys-tem do not cause any significant regeneration response

due to inhibitory signaling suppressing axon outgrowth

and low trophic response [1–5] In contrast, the axons

of teleost fish regenerate upon nerve injury and have

been used as a model system to study nerve

regener-ation in the central nervous system [6] A better

under-standing of molecular processes leading to axonal

regeneration in teleost fish could find important

appli-cations for treatment of human central nervous system

injuries

Previous studies have identified numerous axonal

growth-associated proteins, which are induced during

nerve regeneration in teleost fish [6–9]

Regeneration-induced CNPase homologs (RICH) proteins are axonal

growth-associated proteins that were originally discov-ered in the studies of regenerating optical nerve in goldfish and were termed p68⁄ 70 based on their appar-ent molecular weight [10] RICH proteins are induced

in the retinal ganglion cells during axonal regrowth upon the optic nerve crush, and also expressed in the germinal neuroepithelium of retina, which generates new neurons throughout the lifespan of the fish [11] The cloning of the RICH proteins from goldfish (gRICH68 and gRICH70) and zebrafish (zRICH) revealed significant homology with mammalian brain 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterases (CNPases) [12–14] CNPases hydrolyze 2¢,3¢-cyclic nucleotides

in vitro and are abundant in oligodendrocytes and Schwann cells [15] Recent studies on CNPase-null

Keywords

CNPase; 2H phosphoesterase superfamily;

NMR solution structure; RICH protein; tRNA

splicing

Correspondence

K Gehring, Department of Biochemistry,

McGill University, 3655 Promenade Sir

William Osler, Montreal, Quebec, Canada

H3G 1Y6

Fax: +1 514 3987384

Tel: +1 514 3987287

E-mail: kalle.gehring@mcgill.ca

Website: http://www.mcgill.ca/biochemistry/

department/faculty/gehring/

(Received 6 December 2006, revised 16

January 2007, accepted 17 January 2007)

doi:10.1111/j.1742-4658.2007.05707.x

Regeneration-induced CNPase homolog (RICH) is an axonal growth-associated protein, which is induced in teleost fish upon optical nerve injury RICH consists of a highly acidic N-terminal domain, a catalytic domain with 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase (CNPase) activity and a C-terminal isoprenylation site In vitro RICH and mammalian brain CNPase specifically catalyze the hydrolysis of 2¢,3¢-cyclic nucleotides to produce 2¢-nucleotides, but the physiologically relevant in vivo substrate remains unknown Here, we report the NMR structure of the catalytic domain of goldfish RICH and describe its binding to CNPase inhibitors The structure consists of a twisted nine-stranded antiparallel b-sheet sur-rounded by a-helices on both sides Despite significant local differences mostly arising from a seven-residue insert in the RICH sequence, the active site region is highly similar to that of human CNPase Likewise, refinement

of the catalytic domain of rat CNPase using residual dipolar couplings gave improved agreement with the published crystal structure NMR titra-tions of RICH with inhibitors point to a similar catalytic mechanism for RICH and CNPase The results suggest a functional importance for the evo-lutionarily conserved phosphodiesterase activity and hint of a link with pre-tRNA splicing

Abbreviations

CNPase, 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase; RICH, regeneration-induced CNPase homolog.

mutant mice revealed that the absence of CNPase

cau-ses axonal swelling and neuronal degeneration pointing

to a role for CNPase in the maintenance of the

mye-lin–axonal interface [16] While the enzymatic activity

of CNPase has been well characterized, its

physiologi-cal substrate remains a mystery [17]

Structurally, RICH protein consists of three regions:

a glutamate- and aspartate-rich N-terminal domain, a

catalytic phosphodiesterase domain, and a C-terminal

isoprenylation site (Fig 1) Recent studies showed that

the catalytic domain is fully sufficient for the in vitro

activity of RICH [18], as previously shown for

mamma-lian CNPase [19,20] The catalytic domains of RICH

and CNPase share a pair of conserved sequence motifs

H-X-(T⁄ S)-X (Fig 1B) with three other groups of

enzymes: fungal⁄ plant RNA ligases, bacterial RNA

ligases and fungal⁄ plant cyclic phosphodiesterases

Together, the catalytic domains of these proteins form a

superfamily of so-called 2H enzymes, which occur in

evolutionary kingdoms ranging from bacteria to

mammals [21] The RNA ligases are involved in tRNA

splicing In bacteria and archaea, they join tRNA

half-molecules containing 2¢,3¢-cyclic phosphate and

5¢-hydroxyl termini Plant and yeast cyclic phosphodi-esterases (CPD or CPDase) hydrolyze ADP-ribose 1¢,2¢-cyclic phosphate to yield ADP-ribose 1¢-phosphate (at least one of these latter enzymes also hydrolyzes nucleoside 2¢,3¢-cyclic phosphates) CPDases are also thought to play a role in the tRNA splicing pathways NMR titrations with CNPase inhibitors and mutagen-esis studies of rat CNPase [20] in combination with the high-resolution crystal structure [22] of human CNPase catalytic domain have been used to propose a cata-lytic mechanism involving the catacata-lytic H-X-(T⁄ S)-X motifs

Here, we report the structure of the catalytic domain from goldfish RICH determined by NMR We show that its structure and its active site are highly similar

to that of the mammalian CNPase NMR titrations with CNPase inhibitors identified the residues in RICH involved in inhibitor binding and suggest the proteins use a similar catalytic mechanism These findings underline the importance of the evolutionarily con-served phosphodiesterase activity of 2H proteins and suggest that a not yet understood link exists between RNA metabolism and axon growth and maintenance

RICH H-T H-T

plant tRNA ligase H-T H-T

GIPGxAKS

T4 Pnk H-T H-T

GCPGSGKS

mammalian CNPase H-T H-T

GLPGSGKS

fish CNPase H-T H-T

GLPGSGKS

yeast tRNA ligase H-T H-T

GCGKT

CNPase domain acidic domain

A

B

isoprenylation site

Fig 1 (A) Domain organization of RICH and

the related 2H proteins Domains are

repre-sented by rectangles with functional motifs

added Cyclic phosphodiesterase domains

are shown in purple RICH and CNPase

contain a C-terminal isoprenylation motif

shown in red, domains with experimentally

confirmed polynucleotide kinase and

aden-ylyltransferase activity are in cyan and

green, respectively The negatively charged

low-complexity N-terminal domain of RICH

is in magenta (B) Sequence alignment of

catalytic domains of goldfish RICH

(gRICH68), human and rat CNPase (hCNP

and rCNP, respectively) and a homologous

protein from puffer fish (Gi:47207595) The

secondary structure elements refer to the

solution structure of goldfish RICH The

con-served catalytic H-X-(T ⁄ S)-X motifs are

shown in bold.

Structure of the RICH catalytic domain

We determined the structure of the 24 kDa catalytic

fragment of goldfish RICH protein (Fig 2) The

previ-ously determined resonance assignments [23] were used

to assign NOEs from 15N- and 13C-edited 3D NOESY

experiments (Fig S1) The 10 structures with the low-est energy and least number of rlow-estraint violations were chosen to represent the final ensemble (Fig 2A) The structural statistics are shown in Table 1

The folded domain extends from Leu175 to Phe386 and presents its N- and C-termini together close in space This would position the N-terminal domain of full-length RICH protein relatively close to its

Fig 2 Structure of goldfish RICH catalytic domain (A) Stereo view of the backbone superposition of 10 lowest energy structures of the RICH catalytic domain The superposition was carried out using regions Pro174–Glu208 and Leu233–Phe386 (B) Ribbon representation of the RICH catalytic domain Secondary structure elements and the N- and C-termini are labeled (C) Backbone overlay of catalytic domains of goldfish RICH (in cyan) and human CNPase (in purple) showing overall similarity of the structures The lowest-energy structure from the RICH NMR ensemble is used for the overlay (D) The surface of the RICH catalytic domain shows several negatively charged patches of res-idues The catalytic site itself is not charged Positive charges are shown in blue, negative charges are in red.

C-terminal isoprenylation site This feature could be

responsible for the smaller degree of association with

the plasma membrane observed for RICH compared

with CNPase [24] as the negative charge of the

N-ter-minal domain of RICH should lead to electrostatic

repulsion with the membrane

The catalytic domain of RICH is composed of a

highly twisted antiparallel b-sheet consisting of nine

b-strands (b1–b9) (Fig 2B) Both sides of the b-sheet

are covered with a-helices The twisted nature of the

b-sheet creates two extended grooves on the opposite

sides of the protein, which are occupied by the longest

helices a1 and a9 A number of short helical fragments

group together in the vicinity of the N-terminus This

helical patch is the most basic part of the molecule

and a potential interaction surface for the preceding

acidic N-terminal domain

Structural comparison with CNP

A structural similarity search using the Dali database

[25] showed that the best hit (Z¼ 21.0) was the

cata-lytic domain of CNPase (PDB code 1WOJ) with an rmsd of 2.8 A˚ over 197 residues (Fig 2C) The struc-tural similarity to 2¢-5¢ RNA ligase (PDB code 1VDX)

is much weaker with an rmsd of 5.3 A˚ over 123 resi-dues

As noticed previously [22], the NMR structure [20]

of the rat CNPase catalytic domain contained an erro-neously positioned helix This was caused by the sparse number of NMR constraints in this part of the mole-cule To address this, we measured residual dipolar couplings for the catalytic domain of rat CNPase using the C12E5⁄ hexanol liquid crystalline medium (data not shown) Analysis of these residual dipolar couplings added invaluable information about this region of the rat CNPase structure and allowed us to identify several misassigned NOE constraints The structure was recal-culated with the addition of residual dipolar coupling constraints and deposited to the RCSB database under the accession code 2ILX (supplementary Table S1) The corrected solution structure is in a good agree-ment with the crystal structure of human CNPase cata-lytic domain (rmsd of 2.3 A˚ over 205 residues)

Sequence alignment of the catalytic domain of RICH and related proteins (Fig 1B) identifies the big-gest difference between RICH and CNPase as the seven-residue insert in the helical region between b4 and b5 strands of RICH This insert results in addi-tional a-helical structure in RICH comprising helices a6 through a8 and causes this to be the most structur-ally dissimilar region when comparing the two pro-teins The functional significance of this difference is unclear but, of note, the recently identified, CNP-related protein from the puffer fish (gi:47207595) also contains a long 34-residue insert, on this side of the molecule, between helices a4 and a5 (Fig 1B)

The catalytic domains of RICH and CNPase differ significantly in their surface charges This changes the overall highly positive charge of the CNPase catalytic domain to a surface dominated by negatively charged patches in RICH (Fig 2D) Interestingly, the region around the catalytic H-X-(T⁄ S)-X motifs is relatively neutral in both RICH and CNP Thus, it is likely that the overall charge difference between RICH and CNPase

is more related to protein–partner interactions and less related to their catalytic activity on physiological substrate(s)

We measured heteronuclear NOEs for the RICH cat-alytic domain to identify mobile regions of the structure Besides the unstructured N-terminus, the most flexible part of the protein fragment is the internal loop immedi-ately following the helix a2 (Fig 3) This is highly remi-niscent of CNP, where the corresponding region in the primary sequence, Gly208 to Lys214, produced the most

Table 1 Structural statistics for RICH protein

Restraints for structure calculations

Final energies (kcalÆmol)1)

rmsd from idealized geometry

rmsd for experimental restraints a

Coordinate rmsd from average structure (A ˚ ) b

Ramachandran analysis (%)

Residues in most favored regions 84.5 ± 1.8

Residues in additional allowed regions 12.3 ± 1.6

Residues in generously allowed regions 3.0 ± 2.6

a Calculated structures had 3–8 dihedral angle violations >2 and

three distance violations >0.2 A ˚ b For residues 174 : 208 and

233 : 386.

intense peaks in the15N–1H heteronuclear

single-quan-tum correlation spectroscopy spectrum indicative of

backbone flexibility [20] This region is far from the

nuc-leotide binding site (vide infra) and unlikely to play a

role in catalysis

Binding to CNPase inhibitors

To obtain more information about the active site of

RICH, we titrated 15N-labeled catalytic domain of

RICH with orthophosphate and the CNPase inhibitor,

adenosine-3¢-monophosphate (3¢-AMP) The titrations

were followed by 1H–15N heteronuclear

single-quan-tum correlation spectra and the shifts of amide signals

as a function of ligand addition recorded These

sig-nals act as a fingerprint to identify amino acid residues

affected by binding and to measure the binding affinity (Fig 4)

Titration of the catalytic domain of RICH with 3¢-AMP resulted in chemical shift changes of roughly 20 backbone amides, indicating binding to the protein The biggest chemical shift changes were observed for Thr322 (0.67 p.p.m.), Thr236 (0.60 p.p.m.), Asp241 (0.36 p.p.m.), Val332 (0.35 p.p.m.), Gly335 (0.27 p.p.m.), Phe239 (0.27 p.p.m.) and Ala319 (0.27 p.p.m.) (Fig 5A) Thr236 and Thr322 are part of the H-X-(T⁄ S)-X motifs, which are essential for the catalytic activity of the related CNPase

Fig 3 Identification of the mobile regions in the RICH catalytic

domain (A) Plot of heteronuclear NOEs identifies the a2–a3 loop

(Gly215–Val221) as the most mobile place in the RICH catalytic

fragment Secondary structure and the two catalytic motifs (*) are

shown (B) Representation of flexibility in the solution structure of

the RICH catalytic domain The width of the sausage is reversely

proportional to the heteronuclear NOE values The figure was

gen-erated with MOLMOL [46].

Fig 4 NMR titration of the RICH catalytic domain with 3¢-AMP (A) Overlay of six heteronuclear single-quantum correlation spectra

of the 15 N-labeled RICH catalytic domain at different concentrations

of 3¢-AMP The color changes from cyan (unliganded RICH) to dark blue (9.3 : 1 ratio of 3¢-AMP to RICH) The most shifted amides are labeled (B) Determination of the dissociation constant of 4.6 ± 0.3 m M for 3¢-AMP binding from the amide chemical shift changes of Gly335.

[19,20] Mapping of the chemical shift changes on the

RICH catalytic domain structure (Fig 5B) shows that all

the changes are closely grouped in space, thereby

unam-biguously identifying this region as the catalytic site of the

protein (Fig 6) A similar pattern of chemical shift changes

was observed for the rat CNPase catalytic domain [20],

which confirms the structural and catalytic relatedness of

the two proteins

The binding of orthophosphate results in chemical

shift changes very similar to those observed upon

bind-ing of 3¢-AMP (Fig 5C) As previously shown for

CNPase [20,22], phosphate binds in the active site

Fewer residues are affected by phosphate binding,

which reflects the smaller size of the phosphate group

leading to a more local effect (Fig 5D) Comparison

of the 3¢-AMP and orthophosphate titrations allowed

us to identify the residues of RICH affected by binding

of the adenine group (Fig 5E) Located in the loop

between strand b2 and helix a2, Phe239 and Asp241

appear to be in a proximity of adenine base in the

RICH⁄ 3¢-AMP complex (Fig 5F)

A

0.1 0.3 0.5 0.7

Residue Number

T236

F239 D241

T322

A319 V332 G335

Residue Number 0.1

0.3

0.2

F239 D241

Residue Number 0.1

0.3 0.5 0.7

T236

T322

V332 G335

T236 T322 V332

T236 T322 V332

F239 D241

F E

D C

B

Fig 5 Chemical shift perturbation plot of

the 15 N-labeled catalytic domain of RICH

upon titration with 3¢-AMP (A) and

ortho-phosphate (C) and mapping of the chemical

shift changes upon binding of 3¢-AMP (B)

and orthophosphate (D) on the RICH

cata-lytic domain structure The color

representa-tion is white for no change to red for the

maximum change (E) The difference

between chemical shift changes from

titra-tion with 3¢-AMP and orthophosphate

identi-fies orientation of 3¢-AMP when bound to

the catalytic domain of RICH (F) Mapping

of the chemical shift changes due to the

adenine group of 3¢-AMP on the RICH

structure.

Fig 6 Catalytic site of RICH Histidines and threonines from the catalytic motifs and other residues affected by inhibitor binding are shown as sticks and labeled.

The NMR titration experiments also allowed us to

estimate binding affinities Under conditions of weak

binding and fast exchange, the shifts of the signals in

heteronuclear single-quantum correlation spectra can

be used to measure the amount of inhibitor bound

These shifts can be fitted using a simple equation,

assuming Kd [protein], to estimate the dissociation

constant (Kd) The resulting values were 4.6 ± 0.3 and

12 ± 2 mm for 3¢-AMP and orthophosphate,

respect-ively, binding to RICH (Fig 4B and Fig S2) In

com-parison, orthophosphate binding to CNPase shows an

identical binding affinity (Kd of 12 ± 2 mm), while

3¢-AMP binds CNPase with a much better Kd of

0.57 ± 0.04 mm [20] The physiological significance

of the relatively poor affinity of catalytic domain of

RICH for 3¢-AMP is unclear, since 3¢-AMP is not a

substrate of CNPase activity

Discussion

The role of 2H proteins in myelination and nerve

growth remains mysterious RICH shows highest

struc-tural similarity with CNPase in its catalytic domain

and catalytic site, which suggests that the conserved

2¢,3¢-cyclic phosphodiesterase activity is important for

the in vivo function of both proteins The

best-charac-terized members of the 2H protein superfamily are

involved in RNA-processing pathways, specifically

tRNA splicing and ligation This leads to speculation

about possible physiological substrate(s) for RICH

and CNP

One of the mechanisms of tRNA splicing involves

endonuclease cleavage of an intron-containing tRNA

at two exon–intron borders, yielding 2¢,3¢-cyclic

phos-phates and 5¢-OH termini Following cleavage, three

reactions are required to put the ends of fragmented

tRNA together: first, the 2¢,3¢-cyclic phosphate is

hydrolyzed by a cyclic phosphodiesterase; secondly,

the 5¢-OH terminus is phosphorylated by an

NTP-dependent polynucleotide kinase; and thirdly, the

modified ends are joined by an ATP-dependent RNA

ligase [26–31] RNA ligases in plants and fungi

con-sist of a single polypeptide chain with three domains

Despite the low sequence similarity, the domain

organization of plant and fungal ligases is very

sim-ilar: an N-terminal adenylyltransferase⁄ ligase domain,

followed by a polynucleotide kinase domain and a

C-terminal cyclic phosphodiesterase domain (Fig 1)

In multicellular animals, all three domains are still

essential, but are not necessarily in the same

polypeptide [31]

The C-terminal domains in all these ligases contain

two H-X-(T⁄ S)-X motifs, which identify them as 2H

proteins The central GTP-dependent polynucleotide kinase domain of yeast⁄ plant tRNA ligases contains

an NTP-binding P-loop consensus sequence of GxxGxGKS that is critical for function The sequence

of the putative P-loop in the N-terminal domain of CNPase (37GLPGSGK44S) is strikingly similar to that

of plant tRNA ligases (GIPGSAKS for Arabidopsis thaliana) (Fig 1; [32]), which reinforces the connection between CNPase and tRNA maturation While CNPase is missing a ligase domain, this activity could

be performed by another protein In T4 bacteriophage, tRNA ligation is carried out by two different enzymes The bifunctional enzyme T4 Pnk, which contains a P-loop (GCPGSGKS) almost identical to CNP, pre-pares the 3¢ and 5¢ ends of the cleaved tRNA, and T4 Rnl1 ligase reconnects the ends [33–35] This advocates the hypothesis that CNPase is a functional homolog

of T4 Pnk and participates in tRNA splicing and maturation Intriguingly, the fish homolog of CNPase (gi:47207595) contains an additional N-terminal domain, which could potentially possess an adenylyl-transferase activity (Fig 1) While showing higher sequence homology to the fish CNPase (56% identity versus 47% identity to human or rat CNPase), RICH appears to lack both the adenylyltransferase and kin-ase domains; little is known about the function of its acidic N-terminal segment

The cellular localization of CNPase does not contra-dict its involvement in pre-tRNA splicing Recent stud-ies revealed that the yeast tRNA splicing endonuclease mainly localizes on mitochondria and this localization

is important for its function [36] Interestingly, one CNPase isoform (CNP2) is specifically targeted to mitochondria [37] More intriguingly, RICH and CNPase may be involved in other RNA splicing events XBP1 mRNA, in humans, and HAC1 mRNA,

in yeast, undergo cytoplasmic splicing as part of the unfolded protein response that regulates the endoplas-mic reticulum volume and protein composition [38] While no 2¢,3¢-cyclic phosphate intermediates have been identified in these reactions or in regulation by micro RNAs, it is not impossible that the

evolutionari-ly ancient phosphoesterase activity of 2H proteins is involved in regulating membrane biogenesis in oligo-dendrocytes or neurons via RNA

In conclusion, RICH proteins have been less char-acterized than mammalian CNPases, but the strong structural similarity of these proteins suggests a sim-ilar function The structure of RICH and nucleotide binding studies presented here represent another step towards understanding of CNP⁄ RICH function and suggest new avenues to study these still enigmatic proteins

Experimental procedures

Protein expression and purification

The catalytic domain of goldfish gRICH68 protein (residues

172–389) was subcloned into pET15b (Novagen, Inc.,

Madison, WI, USA) and expressed in the Escherichia coli

expression host BL21 (DE3) (Stratagene, La Jolla, CA,

USA) as a His-tagged fusion protein The protein was

puri-fied by immobilized metal affinity chromatography on

Ni2+-loaded chelating sepharose column (Amersham

Phar-macia Biotech, Piscataway, NJ, USA) Isotopically labeled

RICH was prepared from cells grown on minimal M9

media containing15N ammonium chloride and13C glucose

(Cambridge Isotopes Laboratory, Andover, MA, USA)

The N-terminal His-tag was cleaved from RICH by

over-night dialysis with thrombin (Amersham Pharmacia

Bio-tech, Piscataway, NJ, USA) at 1 unit per mg fusion protein

at room temperature Benzamidine sepharose and Ni2+

-loa-ded chelating sepharose were used to remove thrombin and

the His-tag peptide from RICH The resulting 222 amino

acid protein contained four N-terminal extraneous residues

(GSHM) from the His tag The sequence composition of

purified RICH was confirmed by mass spectrometry

NMR spectroscopy

NMR resonance assignments of the catalytic domain of

RICH were determined previously [23] All NMR

experi-ments were recorded at 307 K NMR samples were 1 mm

protein in 50 mm 4-morpholineethanesulfonic acid buffer,

0.15 m NaCl, 1 mm dithiothreitol at pH 6.0 NMR spectra

were processed with nmrpipe [39] and xwinnmr software

version 3 (Bruker Biospin) and analyzed with xeasy [40]

For titrations, 3¢-AMP and Na2HPO4 were purchased

from Sigma (Saint-Louis, MO, USA) and used without any

additional purification Titrations were monitored by15N-1H

heteronuclear single-quantum correlation spectra following

addition of inhibitors to15N-labeled RICH (172–389) on a

Bruker DRX 600 MHz spectrometer The experiments were

recorded with 128 increments using 4–8 scans and lasted for

10–20 min Chemical shift changes were calculated as

(DHN2+ (0.2*DN)2)1⁄ 2in p.p.m Samples contained 50 mm

4-morpholineethanesulfonic acid, 0.15 m NaCl and 1 mm

di-thiothreitol at pH 6.0 and0.4–0.5 mm RICH at 307 K The

inhibitor concentrations ranged from 0.1 to 60 mm

depend-ing on the affinity and solubility of the inhibitor The

hetero-nuclear single-quantum correlation spectra of complexes

were assigned by monitoring chemical shift changes upon

addition of the substrate, since the binding takes place in the

fast exchange The pH of the NMR samples was monitored

during the titrations and adjusted as needed Chemical shift

changes for individual residues were fit to a one-site binding

equation: d¼ dmaxÆ [L]⁄ (Kd+ [L]) where d is the chemical

shift change, [L] is the total ligand concentration

(uncor-rected for binding to RICH), and Kdis the dissociation con-stant The fitting was carried out using the computer program grafit, version 3.0 (Ericathus Software, Horley, UK) to determine dmaxand the Kdof binding

Structure calculation NOESY constraints for the structure determination were obtained from15N-edited NOESY (mixing time 80 ms) and

13

C-edited NOESY (mixing time 80 ms) 3D experiments acquired on a Varian Unity Inova 800 MHz spectrometer

at the Quebec-Eastern Canada High-Field NMR Facility For the structure determination, a set of ARIA-assigned [41] and manually verified 1388 NOEs were collected from

15

N- and 13C-edited NOESY spectra of RICH (172–389) Three hundred and twenty-two backbone angles resulted from chemical shift index using the TALOS database [42] Hydrogen bonds were predicted from NOE analysis The starting structure was generated with modeller 6v2 [43] using human CNPase crystal structure (PDB code 1WOJ) and was in agreement with manually assigned NOEs One hundred and fifty structures were calculated and refined using standard protocols in cns v.1.1 [44] procheck-nmr [45] was used to check the protein stereochemical geometry The structural statistics for 10 structures are shown in Table 1 The coordinates have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) under PDB accession code 2I3E and the NMR assignments under BMRB accession number 7167

Acknowledgements

We acknowledge Dr M D Uhler (University of Michigan, USA) for a gift of gRICH68 cDNA We thank T Sprules for help in running NMR experi-ments at the Quebec-Eastern Canada High-Field NMR Facility This work was funded by operating grant MOP-43967 to KG and PB from the Canadian Institutes of Health Research

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Supplementary material

The following supplementary material is available online:

Fig S1 Plot of NOE number per residue

Fig S2 Determination of the dissociation constant of 12.0 ± 1.9 mM for orthophosphate binding from the amide chemical shift changes of Gly335

Table S1 Structural statistics for rCNP catalytic domain

This material is available as part of the online article from http://www.blackwell-synergy.com

Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corres-ponding author for the article