The Nuclease A Inhibitor Represents a New Variationof the Rare PR-1 Fold

Thomas W. Kirby1, Geoffrey A. Mueller1, Eugene F. DeRose1

Mark S. Lebetkin1, Gregor Meiss2, Alfred Pingoud2 andRobert E. London1*

1National Institute ofEnvironmental Health SciencesP.O. Box 12233, MD MR-01Research Triangle Park, NC27709, USA

2Institut fur Biochemie, FB08Justus-Liebig-UniversitatHeinrich-Buff-Ring 58D-35392 Giessen, Germany

Nuclease A (NucA) from Anabaena sp. is a non-specific endonuclease ableto degrade single and double-stranded DNA and RNA. The endonucleo-lytic activity is inhibited by the nuclease A inhibitor (NuiA), which bindsto NucA with 1:1 stoichiometry and picomolar affinity. In order to betterunderstand the mechanism of inhibition, the solution structure of NuiAwas determined by NMR methods. The fold of NuiA is an a–b–a sand-wich but standard database searches by DALI and TOP revealed no struc-tural homologies. A visual inspection of a–b–a folds in the CATHdatabase revealed similarities to the PR-1-like fold (SCOP nomenclature).The similarities include the ordering of secondary structural elements, asingle helix on one face of the a–b–a sandwich, and three helices on theother face. However, a major difference is in the IV helix, which in thePR-1 fold is short and perpendicular to the I and III helices, but in NuiAis long and parallel to the I and III helices. Additionally, a strand insertionin the b-sheet makes the NuiA b-sheet completely antiparallel in organiz-ation. The fast time-scale motions of NuiA, characterized by enhancedflexibility of the extended loop between helices III and IV, also show simi-larities to P14a, which is a PR-1 fold. We propose that the purpose of thePR-1 fold is to form a stable scaffold to present this extended structurefor biological interactions with other proteins. This hypothesis is sup-ported by data that show that when NuiA is bound to NucA significantchanges in chemical shift occur in the extended loop between helices IIIand IV.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: nuclease A inhibitor; NuiA; nuclease A; NucA; Anabaena*Corresponding author

Introduction

Structural studies of nuclease inhibitors and thecorresponding inhibitor–nuclease complexes canprovide fundamental insight into the requirementsfor strong protein–protein binding interactions, aswell as practical clues for the development ofinhibitors of potential chemotherapeutic signifi-cance. In comparison with the literature related toproteinase inhibition, there is considerably lessstructural information available for nuclease

inhibitors. Significant exceptions include the Bacil-lus amyloliquefaciens RNase inhibitor barstar, theRNaseA inhibitor (RI), and the colicin endonu-clease E9 inhibitor Im9.1 – 3 NuiA, a protein with noknown homologs, is a potent inhibitor of nucleaseA (NucA) derived from Anabaena sp.4 The targetnuclease, NucA, is a member of a recently discov-ered class of endonucleases with many analogsand a broad range of biological functions.5,6 Prokar-yotic endonucleases are typically secreted to serveeither as part of the nucleotide and phosphate sal-vage pathway or as bacteriocides similar to theEscherichia coli colicins.7 – 9 The related DNA-entrynuclease EndA was shown to be important for effi-cient transformation of Streptococcus pneumoniae.10

The eukaryotic nucleases serve still other func-tions. Nuc1 constitutes the main mitochondrialnuclease activity of Saccharomyces cerevisiae and isinvolved in DNA repair and recombination,11,12

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

These authors contributed equally to this paper.

E-mail address of the corresponding author:london@niehs.nih.gov

Abbreviations used: NuiA, nuclease A inhibitor;NucA, nuclease A; NOE, nuclear Overhauser effect;NOESY, NOE spectroscopy; TOCSY, total correlatedspectroscopy; CSI, chemical shift index; HSQC,heteronuclear single quantum coherence.

doi:10.1061/S0022-2836(02)00460-6 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 320, 771–782

while the related bovine endo G generates primersfor mtDNA replication, and is involved in apopto-tic DNA degradation.6,13,14 Endo G also possessesRNase H activity, rendering it a potential modelsystem for this type of nuclease as well.13

Unregulated non-specific DNA and RNA endo-nucleases are generally toxic to cells, so theiractivity must be carefully controlled. The non-specific DNA/RNA endonuclease from Anabaenasp., NucA, was shown to be inhibited by a proteinnamed NuiA that is encoded immediately adjacentto the NucA gene on the opposite DNA strand.4,15

The close proximity of the genes for the nucleaseand its inhibitor is reminiscent of that observedfor the genes of the E. coli immunity proteins,which are located in a polycistronic operontogether with the colicin DNases that they inhibit.8

Similarly, barnase from Bacillis sp. is expressedtogether with its inhibitor, barstar, in order to clo-sely regulate the nuclease activity.16 However,despite the similarity in function there are nosequence homologies to NuiA currently in thedatabases. Functionally, NuiA is probably similarto the low molecular mass immunity proteins thatprotect E. coli from the DNase activity of the coli-cins, a group of stress-induced proteins withnucleolytic activity which target sensitive cells inthe environment. Furthermore, the colicin E9DNase and NucA share a similar active site archi-tecture and are grouped together in the superfam-ily of His-Me finger endonucleases (SCOPclassification).17,18 However, as shown here, thestructure of NuiA is unrelated to the small E. coliimmunity proteins which lack b-sheet structureand are predominantly helical.19,20 There are nogood sequence homologs of NuiA, and our exam-ination of available databases suggests there arevery few structural homologs.

Here we present the solution structure of NuiAdetermined using NMR methodology. The unique-ness of the nuiA gene is matched by the uniquenessof the protein fold, which is shown to be similar,but significantly different from the rare PR-1 fold.Furthermore, chemical-shift mapping experimentsof the complex of NuiA and NucA allow us tounderstand better the interactions of the two pro-teins and suggest a molecular basis for the inhibition.

Results

The structure determination of NuiA by solutionNMR methods was straightforward. The proteinexhibits good chemical shift dispersion in the 15NHSQC spectra and all the assignment and NOEspectra were collected on a single 3.3 mM 1H, 13C,and 15N-labeled sample at 30 8C; signal-to-noisewas not limiting. Note that the amino acid number-ing in this paper refers to the protein with an N-terminal extension of (MetHis6GlySer) making theconstruct 143 amino acid residues long. It was pre-viously shown that this construct has the sameinhibitory activity as the wild-type.21

Nearly complete resonance assignments weremade beginning at Gly8 after the N-terminal 6-Histag. Missing from the assignments were His51 andthe backbone amide for Gln52, likely due toexchange broadening. Of note in the assignmentswas an extremely high upfield shift of a Gln82 Hb(20.47 ppm) and the Gln82 HN (5.2 ppm). Theseatoms are in close proximity to the aromatic ringof Trp84 as evidenced by NOEs to the indole H11

(data not shown). These NOE assignments areunambiguous because the lowest downfieldshifted amide in the protein is the H11 of Trp84(10.3 ppm), so these unique chemical shifts elimin-ate ambiguity.

After sequential assignments, the chemical shiftindex predictions (CSI) showed clear, continuousregions of secondary structure that included five

Table 1. Statistics for the structure ensembles

Initial structuresNOEs total 877

Ensemble RMSD (A)a

Backbone (residues 9–143) 3.76 ^ 1.19Heavy atoms (residues 9–143) 4.57 ^ 1.16

Final structuresb

NOEsH–CH 3576H–NH 1694HC–NH 1014Unambiguous 3320Ambiguous 1436Totalc 4756

Ensemble RMSD (A)Secondary structure (backbone)b 0.43 ^ 0.19Secondary structure (heavy)b 0.85 ^ 0.36Backbone (residues 9–143)a 1.20 ^ 0.27Heavy atoms (residues 9–143)a 1.81 ^ 0.27

Average violations per structureb

NOEs and/or H-bonds 10.7 ^ 2.2Dihedrals 8.6 ^ 2.7

RMSD (experimental restraints)b

NOEs (A) 0.0893 ^ 0.0097H-bonds (A) 0.1602 ^ 0.0478Dihedral angles (deg.) 2.14 ^ 0.38

RMSD (covalent geometry)b

Bonds (A) 0.0053 ^ 0.0004Angles (deg.) 0.7267 ^ 0.0436Impropers 0.6410 ^ 0.0297

Ramachandran space (%)d

Most favored region 66.4Additionally allowed 22.4Generously allowed 8.0Disallowed 3.2

a Calculated with MOLMOL.45

b Output by ARIA,26 calculated by CNS25 using the ensembleof the seven lowest energy structures.

c Total refers to the sum of the unambiguous and ambiguousrestraints, which is not the same as the total NOEs assignedfrom the individual experiments. Ambiguous restraints canhave multiple assignments, and redundant assignments fromthe individual experiments are filtered in this reported total.

d Calculated with PROCHECK.61 Note that this percentage isover all seven lowest energy structures, not individualstructures.

772 NMR Structure of NuiA

b-strands and four a-helices.22,23 Analysis of main-chain NOEs quickly revealed the connectivity ofthe five predicted b-strands that formed a comple-tely antiparallel b-sheet. The program TALOS,24

which matches chemical shift and primarysequence with a database to predict f and cangles, confirmed a similar secondary structureconsistent with the strands and helices of the CSI.

Initial structures were calculated using the pro-gram CNS25 with manually assigned NOEs asdescribed in Materials and Methods. The inputdata included 877 NOEs, 98 hydrogen bonds, and212 dihedral restraints. The ten best structuresfrom this set showed a 3.5 A backbone RMSD forresidues 9–143. These ten structures were pro-vided as the initial structures for subsequent anal-ysis by ARIA.26 The final analysis by ARIA wasable to assign 4756 NOEs as detailed in Table 1.Examples of ARIA NOE assignments are shownin Figure 1. Figure 1 illustrates the high quality of

the spectra and the completeness of the assignmentanalysis by ARIA. As given in Table 1, the elementsof secondary structure show a backbone pairwiseRMSD value of 0.43(^0.19) A and all backboneatoms excluding the 6-His tag show an average of1.2(^0.16) A RMSD. The ensemble of the sevenlowest energy structures from ARIA are shownaligned for residues 9–143 in Figure 2. The lowRMSD value of the bond, angle, and improper geo-metry (Table 1) is indicative of the high quality ofthe structures.

To test if the input structures biased the ARIAcalculation, new structures were calculated withthe same input spectra but with no hydrogenbond or initial structures. These new structures,calculated starting from an extended chain, have ahigh RMSD value (5.8 A). It is not uncommon forARIA to have difficulties starting from an extendedchain structure. Nevertheless, these structures havethe same pattern of interaction among the strands

Figure 1. NOESY strip plots. Strip plots from the H–CH cube of the CN NOESY-HSQC are shown for (a) Leu27-HD1, (b) Val112-HG1 and (c) Ile136-HD1. ARIA assignments of the cross-peaks are indicated. The labels for the diag-onal peak are bold, and long-range peaks that connect elements of secondary structure are underlined. The peakslabeled with an asterisk ( p ) in (c) were assigned by ARIA to greater than four possibilities. Most are short-range inter-actions to degenerate b-protons.

NMR Structure of NuiA 773

of the b-sheet, but variable positioning of thehelices lead to the high RMSD value (data notshown). Therefore, the initial structures did biasthe ARIA calculation. However, it should be notedthat the ten initial structures show an average5.2 A backbone RMSD to the final minimized aver-age structure from ARIA, indicating that the calcu-lation parameters were not so restrictive as to relycompletely on the initial structure for assignments.

To confirm that the helices are positioned cor-rectly, we analyzed the ARIA-assigned NOEs fromthe NuiA calculations with initial structures. Thenomenclature of the secondary-structure elementsis illustrated in Figure 3(b). Figure 1(a) shows thatLeu27, which is adjacent to helix I, interacts withPhe96 of helix IV and Phe37 of strand A. TheNOEs in Figure 1(b) show that Leu112 of strand Cinteracts with residues from the adjacent strand Dsuch as Val122 and, importantly, there are also

interactions with helix III such as those to Phe74.NOEs from Ile136 on strand E to Phe75 on helixIII (Figure 1(c)) indicate the proximity of thesestructural elements. Similarly, NOEs from Ile136 toresidues 20, 21, and 24 of helix I also position thishelix near strand E. The NOEs illustrated in Figure1 were instrumental in the positioning of thehelices with respect to the b-sheet.

Fold classification

The average minimized structure of NuiA wassubmitted to the fold recognition programs DALIand TOP.27,28 DALI attempts to match a-carbontraces with the family of structurally similar pro-teins (FSSP) which contains representative folds.29

Only one protein, 1e42.pdb, matched with a Z-score of 2.7, which is above the published criticalvalue of 2.0. For comparison, the protein matches

Figure 3. Topology diagrams of (a) P14a and (b) NuiA. The ordering and interactions of the secondary structureelements are displayed in a manner similar to that previously described for P14a.32 Strands are labeled with lettersand helices with roman numerals. Highlighted in red is helix IV. The residues at the beginning and end of secondarystructure elements are indicated for NuiA.

Figure 2. Convergence ofNuiA structures. The back-bone traces of the seven low-est energy structures ofNuiA calculated by ARIA aredisplayed as a wall-eyedstereo diagram aligned overresidues 9–143 by MOLMOL.Helices are colored red andstrands are colored blue.Residues 9–143 aredisplayed.

774 NMR Structure of NuiA

to itself with a Z-score of 23.0. The match to1e42.pdb was only over 69 residues and afterinspection it was determined that the topologieswere dissimilar. A second protein matched byDALI had a Z-score of 1.9 over 65 residues that,upon inspection, was also of a different topology.The results of the TOP search of the SCOPdatabase30 returned a number of proteins with avery high “structural diversity” score. In this com-parison, lower scores correspond to greater simi-larity. The best scores were 27.9 and 31.0 and allthe rest were above 32.4. For reference, proteinswithin the same class (SCOP nomenclature) havean average structural diversity of 32.4 and withinthe same fold 14.0. The ten best hits from TOPwere inspected for similarities but none matchedwell. These ten best structures correspond tomany major protein classes, i.e. all a, all b, andab. This indicates that neither TOP nor DALIwere able to classify this fold with their scoringalgorithms.

A visual inspection of the CATH database pro-vided the first clue as to the fold classification ofthe NuiA.31 A comparison with the 20 representa-tive a–b–a sandwich proteins showed similaritiesto the protein P14a or 3.40.33 (CATH numbering),which is a PR-1-like fold (SCOP nomenclature).The topology diagram of Figure 3(a) is adaptedfrom Figure 5(a) of Fernandez et al.32 The diagramwas drawn in this way to illustrate how, in threedimensions, helices I, III, and IV interact with one“face” of the b-sheet and helix II interacts with theopposite face. The topology diagram also high-lights how helix IV (shown in red) is perpendicularto helices I and III in space. This is better illustratedby the three-dimensional rendering of the ribbon

diagrams in Figure 4. In Figure 3(b) the topologydiagram of NuiA is drawn in a similar way to thatof P14a, in order to highlight both the similaritiesand distinct differences. We note the insertion ofan additional strand in Figure 3(b) makes thesheet completely antiparallel in character,although the order of all other secondary-structuralelements is similar, indicating that the folds arerelated.

Dynamics

In order to characterize the dynamic behavior ofNuiA, a series of T1, T2, and NOE measurementswas performed. The results of the heteronuclearNOE experiments are displayed as ratios of theintensities determined with and without pre-satur-ation of the protons (Figure 5). A low ratio isindicative of fast internal motion.33 For residues10, 34, 84, 85, 87, and 143 the NOE ratio is belowone standard deviation from the mean. Consistentwith these results, the ratio for the indole of Trp84is also more than one standard deviation belowthe mean. Low ratios are typical for residues atthe termini or in flexible regions of proteins. Thiswould account for the low ratio of both the C-term-inal residue 143, and residue 10, which is adjacentto the unassigned, and presumably flexible, N-terminal His-tag. The amide of residue 34 is sol-vent-exposed in the loop preceding strandA. Residues 84, 85, and 87 are all in the loopbetween helices III and IV. What is notable is thatthe corresponding loop region of P14a, exhibitedsome of the lowest NOE ratios as well. However,there is no strong correlation between the T1 andT2 values of the corresponding portions of NuiA

Figure 4. Ribbon models of P14a and NuiA. Ribbon diagrams of (a) P14a and (b) NuiA were rendered withMOLMOL.45 The strands that are similar between the proteins are colored cyan and similar helices are colored redand yellow. The additional gray ribbons are loops that are in close association that were classified as strands byMOLMOL but are only two residues in length. Roman numerals label the helices. The proteins were aligned visuallyto best illustrate the similarities of the fold. Arrows indicate the location of important residues for P14a. The arrowsin (a) indicate the residues proposed to be involved in metal ion binding.36

NMR Structure of NuiA 775

and P14a. The T1 and T2 measurements were usedprimarily to estimate the rotational correlationtime (9.5 ns) for calibrating the NOE distances inARIA. The dynamic behavior of the loop connect-ing helices III and IV is interesting. As discussedbelow, chemical shift mapping studies suggestthat this loop is involved in the interaction withthe target NucA.

Interaction with NucA

In order to assess which residues of NuiA inter-act with NucA, we compared the 1H–15N HSQCspectra of free NuiA with NuiA bound tounlabeled NucA, as shown in Figure 6(a). SinceNucA does not have the robust stability of NuiA,the complex was studied at the lower concen-tration of 180 mM NuiA and NucA. Given the pico-molar affinity, the NuiA should be completelycomplexed at this concentration.15 Consistent withthis expectation, there was no evidence for mul-tiple resonances arising from a mixture of NucA-complexed and uncomplexed NuiA. AlthoughNuiA alone gives acceptable spectra at 180 mM,the 1H– 15N HSQC spectrum of the NuiA in com-

plex with NucA exhibited the broader linewidthsand lower signal-to-noise anticipated for a complexwith molecular mass greater than 40 kDa. The1H– 15N HSQC spectrum was acquired with 40hours of signal averaging. For a slowly exchangingsystem, the only approach to connect the reson-ances of the complex with those of free NuiA is toreassign the resonances in the complex. However,it is possible to compare the shift positions forsome of the more strongly shifted peaks located inrelatively sparse regions of the HSQC spectrum.Of these, the resonances corresponding to residues28, 30, 76, 82, 84, 115–117, 123, and 141–142 appearto be shifted significantly in the complex; the peakscorresponding to these amides are circled in Figure6(a). In the more congested region between 8 to9 ppm (1H) and 119 to 122 ppm (15N), there arealso some significant shifts, although it is difficultto diagnose which residues are involved. Possibly,one should include residues 83 and 96 as shifting.This does not purport to be a complete analysis ofthe shifts. Figure 6(b) shows where, on the struc-ture of NuiA, the residues with significant shiftsare located. Interestingly, most of the affected resi-dues are roughly clustered at the top of the mol-ecule in this orientation.

Figure 5. Heteronuclear NOE data. The results of the heteronuclear NOE experiments are shown as the cross-peakintensity ratio with and without pre-saturation. The x-axis indicates backbone amide residue number; at the far rightthe results for the indole of Trp42, and Trp84 are shown. A value of zero indicates no data for that residue. A blackline is drawn at the mean and the dotted lines are drawn plus and minus one standard deviation from the mean. Thesecondary structure is drawn above the diagram as assigned by PROCHECK.61 Rectangles indicate a-helices andarrows indicate b-strands.

776 NMR Structure of NuiA

Figure 6. Chemical shift changes in NuiA due to complex formation with NucA. (a) The HSQC spectra of NuiAbound to NucA (red) is overlayed with free NuiA (black). Residues for which the shift change was significant are high-lighted with blue circles and labeled. The most likely new chemical shift is indicated with an arrow for a few cases. Thebroken lines from the 83, 96, and 117 labels to the free NuiA peaks do not indicate a possible new chemical shift forthose residues. (b) The circled residues from (a) are colored red on the backbone trace of NuiA and labeled. The orien-tation is rotated slightly from that in Figures 2 and 4(b) to illustrate better which residues experienced a chemical shiftchange.

NMR Structure of NuiA 777

Discussion

Relationship to other structures

The relationship of the NuiA protein structure tothe PR-1-like fold was an unanticipated discovery.There are only two experimentally determinedstructures with this fold, P14a (1CFE) and Ves v 5(1QNX) from tomato and wasp, respectively.32,34

These two proteins are highly homologous insequence and structure34 so for simplicity we willfocus our analysis on P14a. A comparison of theribbon diagrams in Figure 4 illustrates that themajor difference between the NuiA and PR-1 foldsis the positioning of helix IV. Helix IV is the longesthelix in NuiA (residues 89–101) and is positionedbetween helices I and III in a roughly parallelarrangement. In contrast, helix IV in P14a isshort and perpendicular to the correspondinghelices. In fact, in NuiA helices I and III do notcontact each other due to the presence of helix IVpositioned in between, while in P14a helices Iand III are intimately associated. However, inboth structures there is an extended loop of non-regular secondary structure between helices IIIand IV that is restrained by the positions of thehelices.

In the analysis of the structure of P14a, Fer-nandez et al.32 proposed four structural motifsthat would be responsible for the similar foldsfor members of the PR-1 family. We have evalu-ated the structure of NuiA in light of these fourproposed motifs. (1) There is a strict conservationof six cysteine residues in the PR-1 family andpresumably the disulfide bonds that were foundin P14a. However, there are no cysteine residuesin NuiA. (2) There is a conservation of corehydrophobic residues. However, there is nosequence identity between NuiA and PR-1 familymembers35 and also very little sequence hom-ology to anything currently in the databases. (3)Proline-125 of P14a adopts a cis-peptide bondconformation, which is critical for the positioningof the C-terminal b-strand. Given the highdegree of conservation of this proline in the PR-1 family, the authors suggested this was alsocritical to the fold. However, there are no prolineresidues in the loop preceding the C-terminal b-strand in NuiA. Interestingly, the NuiA structuredoes have a cis-proline imide bond for thePro47-Pro48 sequence, which is located betweenstrand A and helix II. (4) There is a high degreeof conservation of local structural motifs such asthe short helix IV among members of the PR-1family, and these motifs were proposed to bethe folding nuclei of P14a. However, helix IV ofNuiA is mostly solvent exposed, while in P14ait is completely buried. This comparison is notintended to deconstruct the analysis of Fernan-dez et al.32 which was based on sequential com-parisons among the PR-1 family proteins; rather,it has been made to provide further insight intothe fold classification of NuiA. The analysis sup-

ports the conclusion that NuiA represents a con-siderably different fold.

We note also that the PR-1 proteins identified bySzyperski et al.36 were suggested to be character-ized by a common set of four residues. Two gluta-mate, E53 and E74, and two histidine residues,H48 and H93 (using the numbering for P14a),were proposed to constitute an active site for thesesystems. It was suggested that these might func-tion as metal ion ligands. No similar metal ionbinding motif is present in NuiA. The two histidineresidues of NuiA, H51 and H60, are both locatedon the surface of the protein in a region whichdoes not appear to be significantly perturbed bythe addition of NucA. The two Nd1 nitrogen atomson these histidine residues are 10.7 A apart in theaverage minimized structure, and the Ne2 atomsare even further apart. Hence, there is no supportfor assignment of NuiA to the PR-1 fold group onthe basis of a conserved active site with metal ionbinding characteristics.

Szyperski et al.36 suggested that the PR-1 foldmay have general significance in the pathogenesisof plants and animals. P14a is upregulated intomato plants in response to fungal and viralinfection, while GliPR, a protein with highsequence homology to P14a and with a similarfold, is highly expressed in the tumor glioblastomamultiform.37 – 39 It was suggested that this rep-resented a link between the human and plantdefense systems.36 Whether the NuiA structureconnects these proteins to the bacterial defense sys-tem is open for debate. The folds of GliPR and P14aare similar, yet distinctly different from that ofNuiA as outlined above, and there is no sequencehomology. The bacterial toxin colicins have anassociated “immunity” protein that protects thebacteria producing the toxin against the effects ofthe DNase. These immunity proteins would seemmore closely related to the function of NuiA. How-ever, the structure of the immunity proteins, forexample Im9, are all helical and not similar toNuiA.40 We are unable to make a direct biologicalconnection between the PR-1 fold and NuiA.Given the differences in the structure andsequence, and the very few structures with PR-1folds, this is perhaps not surprising.

So far we have focused on the question of whatinsights the PR-1 proteins can provide aboutNuiA. It should be mentioned that the moleculartargets for PR-1 proteins are not known. Therefore,NuiA may be able to inform us about the functionof the PR-1 proteins. It is conceivable that some ofthe targets are nucleases (which are produced andsecreted by many fungi) and that some PR-1 pro-teins act as inhibitors of these nucleases. Similarly,it could be speculated that the PR-1-like proteinGliPR, which is produced in high amounts in cer-tain glioblastomas, is directed against endonu-clease G, which is a nuclease related to NucA.One function of endonuclease G is that of an apop-totic nuclease and inhibition of apoptosis is a hall-mark of the malignant phenotype. While the role

778 NMR Structure of NuiA

of the PR-1 proteins as nuclease inhibitors is highlyspeculative, it is a testable hypothesis that weintend to pursue.

NuiA inhibition of NucA

NuiA is known to be a potent inhibitor of NucA.Our examination of the spectra of labeled NuiAbound to unlabeled NucA provides informationon which residues in NuiA are involved in theinteraction. We initially suspected that the regionbetween helices III and IV was a good candidatefor the interaction with NucA because in NuiAand P14a this region has the lowest heteronuclearNOE ratios.32 Possibly, the similar dynamic beha-vior is correlated with a similar functional role forthe corresponding regions of the two proteins.Potentially, this loop in NuiA could interact withthe target protein NucA.

Figure 6(b) shows the location of the NuiA resi-dues for which the amide resonances are signifi-cantly shifted upon complexation with the targetNucA. As expected, there are significant shifts forresidues on the loop between helices III and IV.Those residues are 82, 84 (both main-chain andindole), and possibly 83. There are also shifts inthe C terminus (residues 141 and 142) and theloop between strands C and D (residues 115–117).In general, the chemical shift pattern suggests thatthe parts of the NuiA molecule located at the topin the orientation shown in Figure 6(b) representthe main interaction surface between the inhibitorand the nuclease. Residues 28 and 30 are relativelyclose to Trp84 and their chemical shifts could alsobe perturbed by changes in the tryptophan ringcurrents resulting from movement of this residuein the complex. Phe96 is also an exposed aromaticresidue, and is a good candidate for interactionwith the NucA target, and Ser76 is in the same gen-eral area of NuiA. Hence, although the data arevery incomplete, the observed shift perturbationstend to be clustered in specific regions of the pro-tein and involve residues such as the exposedTrp84 and Phe96, which are good candidates forinvolvement in protein–protein interactions.

In Figure 6(a) we suggest that the chemical shiftof the indole nitrogen atom of Trp84 changes byalmost 8 ppm. Such a large shift is most likely dueto a hydrogen bonding interaction and perhapsring current shifts produced by aromatic residuesin NucA. The active site of NucA contains a trypto-phan and a histidine residue41 that may interactwith Trp84. Although the lack of a continuoustitration curve makes these assignments tentative,He1 of Trp84 is the most strongly downfield-shiftedproton in the free protein and that was our basisfor the new assignment. Further, Trp84 is highlysolvent exposed and that carries an energeticpenalty. Since aromatic residues are common atprotein–protein interfaces, this solvent-exposedtryptophan is a likely candidate for interactionwith NucA. The evidence that Trp84 shows highmobility in the free protein and the change in

chemical shift when complexed with NucAsuggests that this is an important residue forNucA inhibition.

In conclusion, the NuiA protein fold is related tothe PR-1 fold. However, there are differences in theb-sheet structure, as well as in the positioning ofhelix IV, which is the hydrophobic core in P14abut solvent-exposed in NuiA. The structural basisof NuiA inhibition of NucA has been localized toone end of the NuiA molecule. Of special interestis the Trp84 that shows dynamic characteristics inthe free protein, and probably a substantial chemi-cal shift change in the complex. Clearly, the com-plete structure of the complex would be of greatvalue in understanding the basis for the inhibition.Further investigations of the NucA–NuiA complexare currently in progress.

Materials and Methods

Purification

NuiA protein was overexpressed in E. coli and puri-fied by a modification of the previous method.21 PlasmidpHisnuiA21 was propagated in strain BL21 containing thecompatible plasmid pRK248cIts, which expresses a tem-perature-sensitive cI repressor,42 by selecting for bothtetracycline resistance and ampicillin resistance. Cellswere grown to mid-log phase ðA600 ¼, 0:6Þ at 30 8C inM9 medium containing 15N-labeled ammonium chloride,50 mg/ml carbennicillin, 20 mg/ml tetracycline, andeither 13C-labeled glucose or unlabeled glucose. NuiAprotein expression was induced by shifting the tempera-ture to 42 8C for 2.5 hours. Cells were harvested by cen-trifugation (at 7000 g ), resuspended in 10 mM Tris–HCl(pH 8.2), and lyzed by sonication in a Branson Sonifier200 using a microtip probe at output level of 6 for10 £ 30 seconds with 30 seconds cooling. The lysate wascentrifuged at 30,000 g for 40 minutes then the super-natant was applied to a 25 ml IMAC Ni-column of Ni-NTA (Qiagen) and eluted with 10 mM Tris–HCl (pH8.2). The Ni-NTA resin retarded NuiA protein but noimidazole was needed to elute it. The fraction containingNuiA protein was brought to 90% saturation withammonium sulfate to precipitate the protein, and then itwas centrifuged for 15 minutes at 30,000 g. The proteinpellet was redissolved in 100 mM Tris–HCl (pH 7),applied to a 2.6 £ 65 cm column of Sephacryl S-100 andeluted with 100 mM Tris–HCl (pH 7). The major peakof absorbance at 280 nm was found to be pure NuiA pro-tein as judged by SDS/polyacrylamide gel electrophor-esis. The peak fractions were concentrated by use of aMillipore Centriprep YM-3 concentrator and the bufferwas exchanged to 90 mM deuterated Tris–HCl (pH 7),10% 2H2O. Samples for NMR had 2–3 mM protein andremained stable at 30 8C for months. The His-tag waspreviously shown not to interfere with the inhibitoryactivity of NuiA.15

Spectroscopy

NMR spectra were recorded on Varian INOVA 500and 600 MHz instruments. The spectra were processedwith NMRPIPE43 and analyzed with NMRVIEW44 soft-ware on LINUX workstations running Red Hat Linux v.7.1. Note that Fortran and C programs were compiled

NMR Structure of NuiA 779

with the Portland Group compilers† to avoid problemswith gcc/g77 version 2.96, which is distributed withRed Hat 7.1. Molecules were visualized and alignedwith MOLMOL.45

Sequential assignments were made primarily from theHNCACB and CBCACONH experiments46 and in a fewcases of weak intensity peaks analysis of the CNNOESY-HSQC was able to confirm the assignments.47

Proton-nitrogen and proton-carbon correlation spectrawere acquired with the Varian’s gNhsqc and gChsqc (inconstant time mode) experiments, respectively. Side-chains were assigned primarily from the H(CCO)NHand (H)C(CO)NH TOCSY experiments,48,49 again withoccasional assistance from analysis of the NOESY spec-tra. Aromatic side-chains were assigned using the(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE50 and thearomatic region HSQC. NOE experiments that were ana-lyzed include the simultaneously acquired CN NOESY-HSQC47 and the 4D HC–NH NOESY.51,52 The 1H, 13C,and 15N resonances were almost completely assignedafter the 6-His tag beginning at Gly8. The assignmentshave been submitted to the BMRB, entry 5261.

Data for structure calculations were determined fromNOE experiments and analysis of the assignments.Results of the CSI predictions and TALOS were analyzedfor secondary structure information.22 –24 Note that carbo-nyl chemical shift assignments were obtained from theHNCO experiment.46,53 TALOS prediction of backbone fand c angles were used as restraints for structure calcu-lations when they met the confidence criteria establishedby the authors. Angles were restrained to at least ^208 ortwice the standard deviation of the prediction by TALOS.NOE, dihedral angle, and hydrogen bond restraints(described in Results) comprised the data used in thestructure calculation. Dynamics experiments to measureT1, T2, and NOE values were performed according to Far-row et al.54 at 600 and 500 MHz.

Structure calculations

Structure determination proceeded as follows. Conser-vative manual assignment of NOEs from backbone,methyl and aromatic atoms where there were clear sym-metry related cross-peaks in the spectra produced 850NOE assignments. Structures were calculated with theseNOEs, hydrogen bond restraints based on the CSI pre-dictions and identified b-sheets, and dihedral angle fand c restraints based on TALOS predictions. The CNSprotocols used simulated annealing with torsion angleand Cartesian space dynamics using the defaultparameters.55,56 A few mis-assignments were removedbased on frequent violations in the first set of calculatedstructures. After removing the mis-assigned NOEs,structures were calculated that frequently contained noviolations. These structures were used as the startingpoint for subsequent calculations and analysis byARIA.26,57 – 59

The program ARIA allows ambiguous restraints thatare assigned by iterative calculations of structures usingCNS.25,26,57 – 59 All NOE cross-peaks from the CN NOESY-HSQC (divided into 3D H–CH and 3D H–NH datasets) and 4D HC–NH NOESY spectra were input toARIA as unassigned and uncalibrated with respect todistance. No assigned NOEs were provided as inputalthough the initial structures were calculated with themanually assigned NOEs. The only manual intervention

in the spectral data provided to ARIA was to removeartifacts, primarily in the H–CH part of the CN NOESY-HSQC, due to water and apodization of the signal froma few very intense methyl groups. The other data inputto ARIA included the previous hydrogen bondrestraints, and the dihedral restraints from TALOS.There were only a few modifications to the default par-ameters provided by ARIA. Due to the wealth of NOEinteractions in such a concentrated sample the numberof relaxation matrix doublings had to be increased to 50for consistent stable diagonalization of the matrix. Theviolation tolerance was initially set slightly higher thandefault to potentially allow ARIA to correct any manualmis-assignments. The variable for violation tolerancewas arrayed starting with the first iteration: 5.0, 2.5, 1.0,0.25, 0.25, 0.25, 0.25, and 0.25. The value for rotationalcorrelation time was calculated from the trimmed-meanof R1/R2 and was 9.5 ns.60 As a verification of the fold ofthe protein, the ARIA calculations were repeated withoutthe initial structures, and hydrogen bond restraints asdescribed in Results. The structures calculated usingARIA with the initial structure determined by the manu-ally assigned NOEs were submitted to the RSCB, PDB IDcode 1KTU.

The final list of ARIA-assigned NOEs can be categor-ized as follows: 29% are intra-residue, 23% involvesequential residues, and 48% involve residues separatedby at least two in the primary sequence. Among theintra-residue NOEs, 5% involve geminal protons, 39%involve vicinal protons, and 56% involve protons separ-ated by more than two bonds. We included NOE assign-ments between the methyl protons of valine and leucinein the geminal category because the methyl carbonatoms are geminal and their assignment should beregarded as equally trivial. In total, the geminal and vic-inal NOE assignments were 44% of the intra-residueand 13% of the total NOEs. An examination of the outputfrom ARIA revealed that 78% of the manually deter-mined NOEs were confirmed, while another 22% (192)were not assigned. In spot-checking the latter, we foundthat ARIA had problems when there was a significantdegree of overlap. In general, we found that ARIA hadmade reasonable decisions in either not assigning thepeak or assigning the cross-peak to an equally probable,alternative set of residues. In some cases, we were ableto make more complete assignments than ARIA byusing additional information, particularly: (1) use ofsymmetry-related cross-peaks (symmetry effects areignored by ARIA); and (2) use of other NOE assignmentsfor the same residue that were consistent with a particu-lar assignment. Despite these limitations, ARIA was ableto assign a very large number of cross-peaks (4756) andto obtain excellent convergence even without reassigningall of the initial NOEs. Most importantly, after correctingthe manually assigned NOE data for errors and possiblealternate assignments, we found no examples of manu-ally assigned NOE peaks that were not consistent withthe final structure.

Acknowledgments

The authors thank Robert Bass and Mark Rafferty fortheir help setting up and maintaining the computer sys-tems. We also acknowledge the helpful discussions ofDr Lars Pedersen and Dr Traci Hall, NIEHS and Dr RonVenters, Duke University.† www.pgroup.com

780 NMR Structure of NuiA

References

1. Lubienski, M. J., Bycroft, M., Freund, S. M. & Fersht,A. R. (1994). Three-dimensional solution structureand 13C assignments of barstar using nuclear mag-netic resonance spectroscopy. Biochemistry, 33,8866–8877.

2. Kobe, B. & Deisenhofer, J. (1993). Crystal structure ofporcine ribonuclease inhibitor, a protein with leu-cine-rich repeats. Nature, 366, 751–756.

3. Osborne, M. J., Breeze, A. L., Lian, L. Y., Reilly, A.,James, R., Kleanthous, C. & Moore, G. R. (1996).Three-dimensional solution structure and 13C nuclearmagnetic resonance assignments of the colicin E9immunity protein Im9. Biochemistry, 35, 9505–9512.

4. Muro-Pastor, A. M., Herrero, A. & Flores, E. (1997).The nuiA gene from Anabaena sp. encoding an inhibi-tor of the NucA sugar-non-specific nuclease. J. Mol.Biol. 268, 589–598.

5. Fraser, M. J. & Low, R. L. (1993). Fungal and mito-chondrial nucleases. In Nucleases (Linn, S. M.,Lloyd, R. S. & Roberts, R. J., eds), 2nd edit.,pp. 171–208, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY.

6. Fraser, M. (1994). Endo-exonucleases: enzymesinvolved in DNA repair and cell death? BioEssays,16, 761–766.

7. Benedik, M. J. & Strych, U. (1998). Serratia marces-cens and its extracellular nuclease. FEMS Microbiol.Letters, 165, 1–13.

8. Bisno, A. L. (1995). Molecular aspects of bacterialcolonization. Infect. Control Hospital Epidemiol. 16,648–657.

9. James, R., Kleanthous, C. & Moore, G. R. (1996). Thebiology of E colicins: paradigms and paradoxes.Microbiology, 142, 1569–1580.

10. Puyet, A., Greenberg, B. & Lacks, S. A. (1990). Gen-etic and structural characterization of endA. A mem-brane-bound nuclease required for transformation ofStreptococcus pneumoniae. J. Mol. Biol. 213, 727–738.

11. Zassenhaus, H. P., Hofmann, T. J., Uthayashanker, R.,Vincent, R. D. & Zona, M. (1988). Construction of ayeast mutant lacking the mitochondrial nuclease.Nucl. Acids Res. 16, 3283–3296.

12. Dake, E., Hofmann, T. J., McIntire, S., Hudson, A. &Zassenhaus, H. P. (1988). Purification and propertiesof the major nuclease from mitochondria of Saccharo-myces cerevisiae. J. Biol. Chem. 263, 7691–7702.

13. Cote, J. & Ruiz-Carrillo, A. (1993). Primers for mito-chondrial DNA replication generated by endonu-clease G. Science, 261, 765–769.

14. Li, L. Y., Luo, X. & Wang, X. (2001). Endonuclease Gis an apoptotic DNase when released from mitochon-dria. Nature, 412, 95–99.

15. Meiss, G., Franke, I., Gimadutdinow, O., Urbanke, C.& Pingoud, A. (1998). Biochemical characterizationof Anabaena sp. strain PCC 7120 non-specific nucle-ase NucA and its inhibitor NuiA. Eur. J. Biochem.251, 924–934.

16. Hartley, R. W. (1988). Barnase and barstar.Expression of its cloned inhibitor permits expressionof a cloned ribonuclease. J. Mol. Biol. 202, 913–915.

17. Kuhlmann, U. C., Moore, G. R., James, R.,Kleanthous, C. & Hemmings, A. M. (1999). Structuralparsimony in endonuclease active sites: should thenumber of homing endonuclease families be rede-fined? FEBS Letters, 463, 1–2.

18. Pommer, A. J., Cal, S., Keeble, A. H., Walker, D.,Evans, S. J., Kuhlmann, U. C. et al. (2001). Mechanism

and cleavage specificity of the h–N–h endonucleasecolicin e9. J. Mol. Biol. 314, 735–749.

19. Kleanthous, C., James, R., Hemmings, A. M. &Moore, G. R. (1999). Protein antibiotics and theirinhibitors. Biochem. Soc. Trans. 27, 63–67.

20. Kleanthous, C., Kuhlmann, U. C., Pommer, A. J., Fer-guson, N., Radford, S. E., Moore, G. R. et al. (1999).Structural and mechanistic basis of immunity towardendonuclease colicins. Nature Struct. Biol. 6, 243–252.

21. Meiss, G., Bottcher, U., Korn, C., Gimadutdinow, O.& Pingoud, A. (2000). Vectors for dual expression oftarget genes in bacterial and mammalian cells. Bio-techniques, 29, 476–478.

22. Wishart, D. S., Sykes, B. D. & Richards, F. M. (1992).The chemical shift index: a fast and simple methodfor the assignment of protein secondary structurethrough NMR spectroscopy. Biochemistry, 31,1647–1651.

23. Wishart, D. S. & Sykes, B. D. (1994). The 13C chemi-cal-shift index: a simple method for the identificationof protein secondary structure using 13C chemical-shift data. J. Biomol. NMR, 4, 171–180.

24. Cornilescu, G., Delaglio, F. & Bax, A. (1999). Proteinbackbone angle restraints from searching a databasefor chemical shift and sequence homology. J. Biomol.NMR, 13, 289–302.

25. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano,W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998).Crystallography and NMR system: a new softwaresuite for macromolecular structure determination.Acta Crystallog. sect. D, 54, 905–921.

26. Nilges, M. (1995). Calculation of protein structureswith ambiguous distance restraints. Automatedassignment of ambiguous NOE cross-peaks and dis-ulphide connectivities. J. Mol. Biol. 245, 645–660.

27. Lu, G. (2000). TOP: a new method for protein struc-ture comparisons and similarity searches. J. Appl.Crystallog. 33, 176–183.

28. Holm, L. & Sander, C. (1993). Protein structure com-parison by alignment of distance matrices. J. Mol.Biol. 233, 123–138.

29. Holm, L. & Sander, C. (1996). The FSSP database:fold classification based on structure–structurealignment of proteins. Nucl. Acids Res. 24, 206–209.

30. Murzin, A. G., Brenner, S. E., Hubbard, T. & Chothia,C. (1995). SCOP: a structural classification of proteinsdatabase for the investigation of sequences andstructures. J. Mol. Biol. 247, 536–540.

31. Orengo, C. A., Michie, A. D., Jones, S., Jones, D. T.,Swindells, M. B. & Thornton, J. M. (1997). CATH—ahierarchic classification of protein domain structures.Structure, 5, 1093–1108.

32. Fernandez, C., Szyperski, T., Bruyere, T., Ramage, P.,Mosinger, E. & Wuthrich, K. (1997). NMR solutionstructure of the pathogenesis-related protein P14a.J. Mol. Biol. 266, 576–593.

33. Kay, L. E., Torchia, D. A. & Bax, A. (1989). Backbonedynamics of proteins as studied by 15N inversedetected heteronuclear NMR spectroscopy: appli-cation to staphylococcal nuclease. Biochemistry, 28,8972–8979.

34. Henriksen, A., King, T. P., Mirza, O., Monsalve, R. I.,Meno, K., Ipsen, H. et al. (2001). Major venom aller-gen of yellow jackets, Ves v 5: structural characteriz-ation of a pathogenesis-related protein superfamily.Proteins: Struct. Funct. Genet. 45, 438–448.

35. Tatusova, T. A. & Madden, T. L. (1999). BLAST 2sequences, a new tool for comparing protein andnucleotide sequences. FEMS Microbiol. Letters, 174,

NMR Structure of NuiA 781

247–250. Erratum appears in FEMS Microbiol. Letters(1999), 177, 187–188.

36. Szyperski, T., Fernandez, C., Mumenthaler, C. &Wuthrich, K. (1998). Structure comparison of humanglioma pathogenesis-related protein GliPR and theplant pathogenesis-related protein P14a indicates afunctional link between the human immune systemand a plant defense system. Proc. Natl Acad. Sci.USA, 95, 2262–2266.

37. Murphy, E. V., Zhang, Y., Zhu, W. & Biggs, J. (1995).The human glioma pathogenesis-related protein isstructurally related to plant pathogenesis-relatedproteins and its gene is expressed specifically inbrain tumors. Gene, 159, 131–135.

38. Van Loon, L. C. (1985). Pathogenesis-related proteins.Plant Mol. Biol. 4, 111–116.

39. Bol, J. F., Linthorst, H. J. M., & Cornelissen, B. J. C.(1990). Plant pathogenesis-related proteins inducedby virus infection. Annu. Rev. Phytopathol. 28,113–138.

40. Boetzel, R., Czisch, M., Kaptein, R., Hemmings,A. M., James, R., Kleanthous, C. & Moore, G. R.(2000). NMR investigation of the interaction of theinhibitor protein Im9 with its partner DNase. ProteinSci. 9, 1709–1718.

41. Meiss, G., Gimadutdinow, O., Haberland, B. & Pin-goud, A. (2000). Mechanism of DNA cleavage bythe DNA/RNA-non-specific Anabaena sp. PCC 7120endonuclease NucA and its inhibition by NuiA.J. Mol. Biol. 297, 521–534.

42. Bernard, H. U. & Helinski, D. R. (1979). Use of thelambda phage promoter PL to promote geneexpression in hybrid plasmid cloning vehicles.Methods Enzymol. 68, 482–492.

43. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfei-fer, J. & Bax, A. (1995). NMRPipe: a multidimen-sional spectral processing system based on UNIXpipes. J. Biomol. NMR, 6, 277–293.

44. Johnson, B. A. & Blevins, R. A. (1994). NMRVIEW: acomputer program for the visualization and analysisof NMR data. J. Biomol. NMR, 4, 603–614.

45. Koradi, R., Billeter, M. & Wuthrich, K. (1996). MOL-MOL: a program for display and analysis of macro-molecular structures. J. Mol. Graph. 14, 51–55.

46. Muhandiram, D. R. & Kay, L. E. (1994). Gradient-enhanced triple-resonance three-dimensional NMRexperiments with improved sensitivity. J. Magn.Reson. ser. B, 103, 203–216.

47. Pascal, S. M., Muhandiram, R. D., Yamazaki, T., For-man-Kay, J. D. & Kay, L. E. (1994). Simultaneousacquisition of 15N- and 13C-edited NOE spectra ofproteins dissolved in H2O. J. Magn. Reson. ser. B, 103,197–201.

48. Grzesiek, S., Anglister, J. & Bax, A. (1993). Corre-lation of backbone amide and aliphatic side-chainresonances in 13C/15N-enriched proteins by isotropic

mixing of 13C magnetization. J. Magn. Reson. ser. B,101, 114–119.

49. Logan, T. M., Olejniczak, E. T., Xu, R. X. & Fesik, S. W.(1993). A general method for assigning NMR spectraof denatured proteins using 3D HC(CO)NH-TOCSYtriple resonance experiments. J. Biomol. NMR, 3,225–231.

50. Yamazaki, T., Forman-Kay, J. D. & Kay, L. E. (1993).Two-dimensional NMR experiments for correlatingC-13-beta and H-1-delta/epsilon chemical shifts ofaromatic residues in C-13-labeled proteins via scalarcouplings. J. Am. Chem. Soc. 115, 11054–11055.

51. Grzesiek, S., Wingfield, P., Stahl, S., Kaufman, J. D. &Bax, A. (1995). Four-dimensional N-15-separatedNoesy of slowly tumbling perdeuterated N-15-enriched proteins—application to Hiv-1 Nef. J. Am.Chem. Soc. 117, 9594–9595.

52. Venters, R. A., Metzler, W. J., Spicer, L. D., Mueller, L.& Farmer, B. T. (1995). Use of H-1(N)-H-1(N) Noes todetermine protein global folds in perdeuterated pro-teins. J. Am. Chem. Soc. 117, 9592–9593.

53. Kay, L. E., Xu, G. Y. & Yamazaki, T. (1994). Enhanced-sensitivity triple-resonance spectroscopy with mini-mal H2O saturation. J. Magn. Reson. ser. A, 109,129–133.

54. Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal,S. M., Kay, C. M., Gish, G. et al. (1994). Backbonedynamics of a free and phosphopeptide-complexedSrc homology 2 domain studied by 15N NMR relax-ation. Biochemistry, 33, 5984–6003.

55. Nilges, M., Gronenborn, A. M., Brunger, A. T. &Clore, G. M. (1988). Determination of three-dimen-sional structures of proteins by simulated annealingwith interproton distance restraints: application tocrambin, potato carboxypeptidase inhibitor and bar-ley serine proteinase inhibitor 2. Protein Eng. 2,27–38.

56. Stein, E. G., Rice, L. M. & Brunger, A. T. (1997). Tor-sion-angle molecular dynamics as a new efficienttool for NMR structure calculation. J. Magn. Reson.124, 154–164.

57. Nilges, M. & O’Donoghue, S. (1998). AmbiguousNOEs and automated NOE assignment. Prog. NMRSpectrosc. 32, 107–139.

58. Nilges, M. (1997). Ambiguous distance data in thecalculation of NMR structures. Fold. Des. 2, S53–S57.

59. Linge, J. P. & Nilges, M. (1999). Influence of non-bonded parameters on the quality of NMR struc-tures: a new force field for NMR structure calcu-lation. BMJ, 13, 51–59.

60. Tjandra, N., Feller, S. E., Pastor, R. W. & Bax, A.(1995). Rotational diffusion anisotropy of human ubi-quitin from N-15 NMR relaxation. J. Am. Chem. Soc.117, 12562–12566.

61. Laskowski, R. A., MacArthur, M. W. & Thornton,J. M. (1998). Validation of protein models derivedfrom experiment. Curr. Opin. Struct. Biol. 8, 631–639.

Edited by M. F. Summers

(Received 21 January 2002; received in revised form 29 April 2002; accepted 6 May 2002)

782 NMR Structure of NuiA