The Disordered C-Terminal Domain of Human DNA Glycosylase NEIL1 Contributes to Its Stability via...

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The Disordered C-Terminal Domain of Human DNAGlycosylase NEIL1 Contributes to Its Stability viaIntramolecular Interactions

Muralidhar L. Hegde1,2, Susan E. Tsutakawa3, Pavana M. Hegde1, Luis Marcelo F. Holthauzen1,4, Jing Li5, Numan Oezguen6,Vincent J. Hilser5, John A. Tainer3 and Sankar Mitra1

1 - Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA2 - Department of Neurology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA3 - Department of Life Sciences, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA 94720, USA4 - Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA5 - Departments of Biology and Biophysics, Johns Hopkins University, Baltimore, MD 21218-2685, USA6 - Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA

Correspondence to John A. Tainer and Sankar Mitra: [email protected]; [email protected]://dx.doi.org/10.1016/j.jmb.2013.03.030Edited by R. W. Kriwacki

Abstract

NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that exciseoxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsicallydisordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei.Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficientin vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with otherbase excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in thepurified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residuescauses a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposedenvironment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to thenative enzyme. These observations are consistent with stabilization of the native NEIL1 structure viaintramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355–360) near the C-terminus. Small-angle X-ray scattering (SAXS)analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providingspecificity for NEIL1's multiple, functional interactions.

© 2013 Elsevier Ltd. All rights reserved.

Introduction

Oxidatively damaged bases in the genome,induced both endogenously and by exogenoustoxicants, are repaired by the evolutionarily con-served base excision repair (BER) pathway,1,2

which is initiated by a DNA glycosylase (DG). DGsspecific for oxidized DNA bases in mammalian cellshave dual enzymatic functions: base excisionfollowed by cleavage of the resulting abasic (AP)site via a lyase activity, generating a 1-nt gap at thelesion site. Five mammalian DGs responsible for

repairing oxidized DNA bases belong to two majorfamilies: the Nth and Fpg/Nei, named after theirbacterial prototypes endonuclease III (Nth) andendonuclease VIII (Nei), respectively. NEIL1 [Nei(endonuclease VIII)-like protein 1],3,4 NEIL2,5 andNEIL36 in the Fpg/Nei family are distinct from the Nthfamily members OGG1 and NTH1 with regard toboth structure and AP lyase reaction,7 in spite oftheir overlapping substrate range.1 While NEIL1/2generate 3′ phosphate (P) at the strand break via aβδ-lyase activity, OGG1 and NTH1 generate 3′ α,βunsaturated phosphoaldehyde (dRP) via β-

0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserved. J. Mol. Biol. (2013) 425, 2359–2371


elimination reaction. These 3′ blocking groups areremoved by polynucleotide kinase 3′ phosphataseand AP endonuclease 1, respectively, in the nextrepair step, common with DNA single-strand breakrepair.7 The 1-nt gap at the resulting 3′OH-contain-ing strand break is then filled in with the appropriatenucleotide by a DNA polymerase, followed by nicksealing by a DNA ligase.1,2

NEIL1 is unique among the oxidized base-specificDGs because of its S-phase-specific activation,3

ability to excise substrate bases in single-strandedDNA,8 and functional association with DNA replica-tion proteins.9–12 All of these observations implicateNEIL1 in preferential repair of the replicating mam-malian genome. NEIL1 interacts with downstream,conventional repair proteins, as well as noncanonicalaccessory proteins, invariably via a common inter-action interface (amino acids 312–349) within its C-terminal (~100 residues) domain (CTD).13 How sucha short peptide is involved in more than 20 specificprotein–protein interactions is unclear. The ~78 C-

terminal residues (amino acids 312–389) are dis-pensable for NEIL1's lesion excision and AP lyaseactivities but are required for efficient, overall repairboth in cells and in vitro.13 The disordered structureof the NEIL1's CTD was predicted from PONDRanalysis14 and likely was involved in the failure tocrystallize the full-length protein.15 Notably, thecrystal structure of the NEIL1 polypeptide lacking56 C-terminal residues showed a lack of electrondensity for the 40 C-terminal residues thatremained.15 Thus, no structural information is avail-able for the CTD.In apparent contrast to the data supporting an

intrinsically disordered CTD, we surprisingly founddecreased stability and distinct intrinsic fluorescenceof the CTD-truncated NEIL1 polypeptide comparedto that of the wild-type (WT) protein. As thesefindings are counter to the expectation of a disor-dered extension structurally dissociated from thefolded core domain, we decided to carry out furtherinvestigation of the CTD structure. Based on

Fig. 1. Distinct intrinsic fluorescence emission spectra of WT NEIL1 and its mutants lacking the disordered C-terminus.(a) Coomassie-stained SDS-PAGE of purifiedWT NEIL1 and its deletion or point mutants. (b) The CD spectrum of NEIL1'sC-terminal 78-residue peptide (amino acids 312–389; C78) reveals a prominently disordered structure with random-coilconformation. (c) Locations of four Trp and six Tyr residues within the N-terminal core domain in NEIL1's sequence. (d)Intrinsic fluorescence emission spectra of NEIL1 and its N349 and N311 C-terminal deletion mutants; excitation at 280 nm.

2360 Solution Structure of Human DNA Glycosylase NEIL1


combined biochemical, mutational, and biophysicalstudies, and solution structure analysis, we showhere that the CTD folds back to interact with theglobular domain in native NEIL1 where the dynamicassociation is driven by electrostatic interactions andthat this intramolecular association in native NEIL1 isimportant for maintaining stability and structuralintegrity of the genome.

Results

Distinct fluorescence spectra of NEIL1 and itsC-terminal deletion mutants

A series of studies from our laboratory demon-strated that NEIL1 interacts with downstream repairand other noncanonical accessory proteins via a38-residue common interaction domain in theCTD.9–13,16 We also showed recently that theCTD, dispensable for NEIL1's in vitro DG activity,is required for NEIL1-initiated complete repair andthat deletion of the CTD significantly increases thesensitivity of human cells to oxidative stress.13

However, little information is available about thestructure of this CTD region and its influence on thestructure of NEIL1's core domain. Predominantlyrandom-coil conformation of the CTD (78-residuesegment), initially predicted by PONDR modeling,14

was confirmed by the circular dichroism (CD)spectrum of the purified C78 peptide (Fig. 1b).The purity of WT NEIL1 and its mutant polypeptidesused in this study is shown in Fig. 1a.To gain further insight into the CTD structure in

the context of the native protein, we analyzed theintrinsic fluorescence of the WT NEIL1 and its C-terminally truncated mutants. WT NEIL1 containsfour Trp and six Tyr residues, all of which arelocated in its globular region (amino acids 1–311;Fig. 1c). Excitation at 280 nm (for both Tyr and Trp)showed typical Tyr emission spectrum of WT NEIL1with λmax at 306 nm (Fig. 1d). However, thefluorescence of NEIL1 polypeptides with C-terminaldeletions of 40 aa (N349) or 78 aa (N311) showedmarked red shifts toward the Trp emission spec-trum. The blue shift observed in the fluorescence ofWT NEIL1 compared to its deletion mutantssuggests a more nonpolar environment of theseTrp residues, indicating that they are more shieldedfrom the solvent than in the mutants. The fluores-cence output of the N349 and N311 mutants wasalso about 2-fold higher than that of WT NEIL1.These unexpected observations suggested a pro-found impact of the C-terminal segment on the corestructure, which could be due to intramolecular fold-back of the extended C-terminal tail on the globularcore surface, causing solvent shielding of the Trpresidues.

Propensity of NEIL1's CTD to form an orderedstructure

The specific interactions involving NEIL1's C-terminal region with multiple partner proteins sug-gest that the flexible CTD could lock into distinctconformations with the partners. To test whether thedisordered region alone could form an orderedstructure, we examined the effect of osmolytes

Fig. 2. Folding propensity of NEIL1's disorderedC-terminal segment. CD spectral analysis of the effect ofthe osmolyte TMAO (3 M) on the secondary structure ofWT NEIL1 (a) and its N311 mutant (b) and C78 peptide (c).

2361Solution Structure of Human DNA Glycosylase NEIL1


trimethylamine-N-oxide (TMAO) and sarcosine, well-known protein-folding agents.17–19 While TMAO(3 M) induced folding in WT NEIL1 (Fig. 2a) and itsC78 peptide (Fig. 2c) as monitored by the CDspectra, no change was observed for the N311mutant lacking most of the disordered segment.Although the disordered segment extends to 100 C-terminal residues, the C78 peptide, the product ofendoproteinase Asp-N,12 was studied. The inabilityof TMAO to induce additional folding in N311suggested that the increased secondary structurein WT NEIL1 is attributable to the C-terminalsegment. Sarcosine treatment produced similarresults (data not shown).

The CTD contributes to NEIL1's stability

We reasoned that the NEIL1 CTD contributed tothe structural stability of the WT protein, which wouldaffect NEIL1's urea or heat denaturation. While themidpoint of transition from the native to denaturedstates for WT NEIL1 was observed at 4.3 M urea,50% denaturation of the deletion mutants N349,N311, and N288 occurred at about 2 M urea(Fig. 3a). Furthermore, while the mutants exhibitedan apparently cooperative single transition betweenthe folded and unfolded states,19,20 the transition forthe WT protein appears less cooperative, presum-ably due to the presence of multiple intermediate

Fig. 3. The C-terminal region enhances NEIL1's stability. (a) Denaturation of WT and deletion mutants of NEIL1 byincreasing amounts of urea, measured by CD spectra at 222 nm with a 1-min time scan. The sigmoidal transition curveswere fitted for experimental points of molar ellipticity at 222 nm as a function of urea concentration using ORIGIN software.(b) Activity of WT NEIL1 and N311 mutant with 5′-32P-labeled 5-OHU-containing single-stranded DNA oligo at theindicated temperature. Change in activity as percent product is given as histogram (bottom panel). (c) Kinetics of activity.



states. The Gibbs free energies (ΔG0) of unfoldingwere calculated to be 4.4 kJ/mol for WT NEIL1 and2.9, 2.5, and 1.3 kJ/mol for the N349, N311, andN288 mutants, respectively.To test whether the change in stability affects

enzymatic activity, we compared the base excision/AP lyase activities of WT NEIL1 versus N311 mutantusing a 5-OHU (an oxidative deamination product ofC)-containing single-stranded deoxyoligonucleotidesubstrate as a function of temperature. While theactivity decreased marginally (20%) for WT NEIL1 byraising the temperature from 37°C versus 45°C, theN311 mutant showed ~80% loss of activity (Fig. 3b),strongly suggesting decreased thermal stability of the311 mutant. Taken together, these data demonstratethat the CTD is critical for NEIL1's functional stability.

Intramolecular fold-back involves interactionsbetween NEIL1's C-terminal and core domains

The ~2-fold difference in the stability of WT NEIL1versus its deletion mutants was surprising and iscounter to the common perception of disorderedterminal domains as being structurally independentof the core domain. To gain further structural insightabout this observation and to experimentally test theintramolecular interaction between the C-terminaltail and the globular core surface, we monitored co-elution of purified C-terminal peptides with His-tagged WT NEIL1 and its N311 mutant bound toNi-NTA beads (Fig. 4). The 40-residue C-terminalpeptide (amino acids 350–389) but not an interiorpeptide (amino acids 312–349) stably bound to themutant protein and, to a lesser extent, to the WTprotein. The weak binding of the CTD also to the WTNEIL1 could be due to the dynamic nature ofintramolecular fold-back, which was competitivelydisrupted by the exogenous peptide.

Identification of clustered C-terminal Lysresidues involved in fold-back

The space-filling model of NEIL1's folded domainshowed an acidic residue cluster (indicated in red inFig. 5a), based on its crystal structure.15 Twoclusters of Lys and Arg residues, cluster(i) contain-ing amino acids 295–300 and cluster(ii) containingamino acids 355–360, are present in the C-terminalsegment of NEIL1 (Fig. 5b). We purified two site-specific point mutants of NEIL1 in which all of the Lysresidues in these clusters were substituted with Ala.The fluorescence spectrum of the cluster(i) mutant(K296A/K297A/K298A) was similar to that of WTNEIL1, but the cluster(ii) mutant (K356A/K357A/K360A) exhibited Trp emission spectrum similar tothat of the truncated NEIL1 (Fig. 5c). These resultssuggest that the basic residue cluster(ii) participatesin intramolecular interaction with the acidic cluster inthe core domain. Importantly, Trp280 is surface

exposed in truncated NEIL1 (Fig. 5a), despite theblue-shifted emission spectrum for WT NEIL1. Thissuggests that the emission from Trp280 is likelymasked due to the fold-back in the WT protein.Urea denaturation studies showed a significant

decrease in the stability of the cluster(ii) mutant,compared to the WT NEIL1 (Fig. 5d). Although thetransition midpoint and stability of the cluster(ii) mutantwere comparable to those of the C-terminal deletionmutants, the denaturation pattern and transition char-acteristics were different. While the deletion mutantsshowed an apparent single phase transition (Fig. 3a),the cluster(ii) mutant showed a broad transition likeWTNEIL1. These denaturation data suggest incompletedisruption of the intramolecular interactions in thecluster(ii) mutant. Taken together, these results under-score the contribution of electrostatic interactions instabilizing the fold-back of the CTD.To get further insights into the involvement of

electrostatic forces in the fold-back, we analyzed thesalt dependence of NEIL1's intrinsic fluorescencespectrum, which revealed partial disruption of thefold-back interaction at 800 mM NaCl as indicatedby a shift in fluorescence emission spectra (Fig. 6a).The higher-than-expected NaCl concentration re-quired for disrupting the fold-back suggested that

Fig. 4. Binding of purified C-terminus to peptidestruncated and full-length NEIL1. His-affinity co-elution ofpurified GST-tagged C-terminal peptides (amino acids312–349 and amino acids 350–389) with His-tagged N311and WT NEIL1 bound to N1-NTA beads. A GST-taggedFEN-1 C-terminal peptide9 that binds to NEIL1 via itsC-terminal residues (312–349) was used as a control.



although the initial affinity was provided by electro-static forces, the interaction may be further stabilizedby hydrophobic forces, which would be enhancedwith increasing salt concentration.

DNA binding disrupts fold-back interaction torelease the tail

Fluorescence analysis of NEIL1 in the presence of a50-mer duplex DNA oligo showed partial shift from Tyrto Trp emission spectrum (Fig. 6b), suggesting therelease of NEIL1's C-terminal tail. Interestingly, similarfold-back of the disordered C-terminal segment to thecore and its release after DNA binding was observedfor theDrosophila cryptochromeCRYprotein that was

attributed to DNAmimicry of the flexible tail.21 While asimilar scenario may exist for NEIL1, it is also possiblethat the C-terminal region itself binds independently toDNA, leading to its release from the core. In support ofthe latter, our unpublished observations suggest thatthe C-terminal segment does bind to DNA andfacilitate target DNA search via intra-strand transfer(J. Iwahara et al., unpublished results).

Small-angle X-ray scattering analysis indicatesflexibility of NEIL1's C-terminal region

Small-angle X-ray scattering (SAXS) analysis offull-length NEIL1, combined with its crystal structurerestraints and conformational analyses (Fig. 7),

Fig. 5. Contribution of electrostatic binding in NEIL1's intramolecular interaction involving a basic residue cluster at theC-terminus and a surface-exposed acidic residue cluster in the core domain. (a) Space-filling model of NEIL1's coredomain based on crystal structure coordinates15 displaying a surface-exposed cluster of acidic amino acid residues (inred) within the circle. The Tyr/Trp residues partially or fully surface exposed are indicated. (b) Presence of two basicresidue clusters at NEIL1's C-terminus. (c) Intrinsic fluorescence of cluster(i) and cluster(ii) mutants of NEIL1. (d) Ureadenaturation of WT NEIL1 and cluster(ii) mutant.



provided evidence for flexible conformation andensembles of NEIL1 in solution. SAXS is a suitabletechnique for studying flexible proteins.22–26 Thescattering curve of nativeNEIL1was distinctly differentfrom the crystal structure of the folded domain. Thelinearity of the Guinier plots confirmed lack ofaggregation of NEIL1 samples. The parabolic curveand upward tail in the Kratky analysis, the tail at longerdistances in the electron pair distribution or P(r) plot,and the ab initio shape prediction are all consistentwith a folded domain and an unfolded tail. The radiusof gyration, Rg, independent of sample concentrationto provide an important constraint for possible solutionstructures,22 was 37.8 Å and 40.3 Å from the Guinierand P(r) calculations, respectively (Table 1). Toobjectively evaluate the SAXS results, we used themolecular dynamics CHARMMprogram implementedin the program BilboMD to generate ~5000 atomicmodels where the catalytic NEIL1 core was fixed andthe C-terminal region was allowed to moveflexibly.27,28 CHARMM parameters set up in BilboMDare designed to explore conformational space. Thepredicted scattering curve of each BilboMD modelwas calculated using the program FOXS and com-pared to the experimental scattering curve.29 In aMinimal Ensemble Search (MES),27,30 random com-binations of three predicted scattering curves werethen compared to find the ensemble with the best fit tothe scattering curve, as indicated by the χ2 fit. Additionof the C-terminus improved the χ2 fit from 15 for thecatalytic core to 1.69 for the best single BilboMDmodel. The fit further improved to 1.07 for theensemble. Although the conformations of NEIL1 areobviously not limited to three in solution, the improvedfit of the ensemble to the experimental scattering datasupports our conclusion that any single conformationcannot explain the experimental scattering data andthat multiple conformations exist in solution.

Discussion

Structures of BER proteins have proven particu-larly useful for characterizing structural elements

associated with specific activities31 and generalmechanisms.32 As structure determination is oftenprecluded for proteins with flexible regions, such asNEIL1, SAXS and computational analysis togetherwith other biophysical methods have providedimportant information about structure–function re-lationships including interactions.23,33 Human NEIL1interacts binarily with most downstream repair pro-teins, including Polβ, LigIIIα, and XRCC1, which actin the single nucleotide incorporation (SN)-BERpathway.7,13 NEIL1 also interacts with the replica-tion-associated long-patch (LP)-BER proteins in-cluding flap endonuclease 1 (FEN-1),9 PCNA,10

and RPA.11 Furthermore, NEIL1 stably interacts withseveral noncanonical repair proteins, for example,Werner protein (WRN), a RecQ helicase,12 andhnRNP-U.16 These multiple interactions have beensuggested to play various roles, including stabilizingthe interacting proteins and selecting repair sub-pathways.13 Surprisingly, all of these interactionsinvolve NEIL1's unstructured CTD that includes a38-residue common interaction interface. This CTDis absent in NEIL1's prototype Nei in Escherichia coliand is likely to have been added during mammalianevolution, presumably to cope with higher complex-ity and regulation in mammalian BER relative to E.coli.1,13,14 However, how NEIL1 interacts with atleast 20 partner proteins by using a short commoninteraction segment is not understood. Furthermore,no structural information was available for this C-terminal extension, which needed to be removed forits crystallization.15

The distinct fluorescence spectra of WT NEIL1versus its deletion mutants suggested tight associ-ation between the core domain and CTD and astrong influence of the C-terminal region on NEIL1'sstructure. Based on biophysical, biochemical, andSAXS analysis of native NEIL1 and its mutants, wepropose that NEIL1's C-terminal segment folds backto interact with the core domain in the native proteinin the absence of its partners. The intramolecularfold-back was also supported by our preliminary MDsimulation analysis (N.O., unpublished results). The

Fig. 6. Disruption of NEIL1's in-tramolecular fold-back by salt orDNA binding. Intrinsic fluorescenceemission spectra of WT NEIL1(2 μM) in the presence of NaCl (a)or of equimolar 50-mer duplex DNAoligo (b).



intramolecular interaction between the basic aminoacid cluster near the C-terminus and the acidiccluster in the core domain significantly enhances thestability of free NEIL1. This fold-back was partiallydisrupted in the presence of a DNA oligo as indicatedby the change in its intrinsic fluorescence (Fig. 6b).The intramolecular interaction in native NEIL1 mayregulate its half-life in vivo and prevent the CTD fromnonspecific interactions, which could lead to aggre-gation, and susceptibility to proteolysis. Similar fold-back interaction of the N-terminal region mayregulate interactions of tyrosyl-DNA phosphodies-terase 2.34

The flexibility of the C-terminal segment, sug-gested by SAXS analysis, may also be critical forNEIL1's binding to multiple partners. Our recentstudies have demonstrated the role of NEIL1's C-terminal region in its multiple interactions with otherrepair and noncanonical proteins and its functionalrole in repair.9–14,16 While it is intriguing how a short,~38-residue segment in NEIL1's C-terminus pro-vides binding interface for at least 20 partnerproteins with high affinity and specificity, we predictthat the flexibility of the C-terminus allows specificinduced-fit conformation with each partner and thusfacilitates its multiple interactions. However, further

Fig. 7. SAXS analysis of WT NEIL1 is consistent with the structure of its catalytic core connected to a disorderedC-terminus. (a) The SAXS scattering curve shows that the experimental data of the full-length protein do not match thescattering data calculated from the catalytic core crystal structure butmatch the scattering predicted fromanensemble ofMESmodels of the catalytic core with a disordered C-terminus. The Guinier plot and residuals of the experimental data (below)indicate monodispersity of the sample. (b) The Kratky plot shows a parabolic curve with a rise in the curve at higher q,consistent with the presence of an ordered core and an additional disordered region. (c) The P(r) plot shows a bilobal shapewith an extension, consistent with the crystal structure and an extended tail. The calculated P(r) of the MESmodels is shownfor comparison to the experimental data. (d) The ab initio shape predicted from the experimental data shows not only acompact volume consistent with the crystal structure but also a volumeextending from theC-terminal portion of the protein. (e)BilboMD models and their percentage representation in the population that were selected by the MES fit the experimentalscattering data as an ensemble. The calculated scattering and P(r) from these MES models are shown in (a) and (c).



investigations are needed to experimentally estab-lish this. SAXS has revealed important functionalroles for unstructured regions in other DNA repairproteins, such as NBS1 where the N-terminaldomains are folded but the CTD is unstructured butrequired for binding to its nuclease partner Mre11.35

The ability of the CTD to exist in multiple states withsubtle change in conformation could help lock indistinct conformations specific for each partner.FEN-1 evidently employs analogous local andsubstrate-specific folding of its active-site gatewayand cap regions to aid specific binding of DNAflaps.36 As seen for FEN-1, the initial affinity for aninteracting partner may induce a subtle conforma-tional change in NEIL1 required to stabilize theinteraction. The propensity of the C-terminal seg-ment to fold in the presence of osmolytes supportsthis scenario.

Intrinsic disorder and allosteric coupling

Intrinsically disordered proteins have emerged asan important class of polypeptides37–42 that adoptmultiple structures after interaction with variousligands, suggesting that coupled folding and bindingfacilitate biological functions ranging from catalysis tosignal transduction.43,44 By exploiting region-specificorder-to-disorder transitions, as observed for the CTDof NEIL1, proteins with multiple partners could fine-tune the affinity for the ligand. In the case ofNEIL1, thecoupling between the core and the C-terminal aminoacids [named as the core and the C-terminal region(R)] is schematically illustrated in Fig. 8a. Eachsegment is depicted to have intrinsic stability (ΔGRand ΔGC); the coupling free energy (Δgint) describesthe quantitative influence of the unfolding of onedomain on the stability of the other. In principle43 andin practice, this coupling energy can be positive45 ornegative.46 For NEIL1, the fact that the stability of thefull-length protein is higher than that of the truncatedvariants indicates positive coupling energy. This isfurther evident when comparing the simulated unfold-

ing curve of a two-domain protein (Fig. 8b) with theexperimental data for NEIL1 (Fig. 3a). For allscenarios wherein the R region is disordered(ΔGR b 0) and the core is folded (ΔGC N 0), destabi-lization of the protein through truncation or mutation(Fig. 8b) can only be facilitated with when Δgint N 0.Although it is clear that the intrinsically disordered C-terminal segment stabilizes NEIL1 and protects it fromboth thermal and chemical inactivation, what role thiscoupling plays in controlling NEIL1 function is atpresent unknown. However, the fact that the equilib-rium appears poised at a point where both structuredand unstructured states exist inmeasurable quantitiessuggests that these states are functionally relevant.43

Intrinsically unstructured regions and electrostaticforces are known to be generally important formolecular interactions.40,47 Flexibility appears tofavor sequence-based recognition, consistent withthe NEIL1 CTD, as observed from the recognition ofintact proteins by anti-peptide antibodies.48 Fur-thermore, unstructured regions present in FEN1and DNA ligase are involved in their interaction withthe PCNA partner.49,50 An unstructured motif pro-motes Rad51 assembly into multimers.51 Further-more, electrostatic interactions could providesubstrate guidance,52 orient proteins for functionalinteractions,53 and regulate DNA repair proteins byDNA mimicry.54 However, the role of electrostaticinteraction in promoting stability of an intrinsicallyunstructured region as observed for NEIL1 isunprecedented, to the best of our knowledge.Thus, this study provides significant insight intothe dynamic nature of NEIL1's intrinsically disor-dered CTD, which may be important for functionaland structural roles in multiprotein interactions ingeneral.13,14

Materials and Methods

Expression and purification of NEIL1 and its mutantpolypeptides

Recombinant untagged WT NEIL1 and its truncatedpolypeptides 1–349 and 1–288 were purified to homoge-neity from E. coli BL21 RIPL-enriched cells harboringcorresponding expression plasmids.3,9 The 1–311 mutantclone of NEIL1 was generated by introducing stop codonsafter the 311th amino acid position in a NEIL1 expressionplasmid (pET22b)3 using the QuikChange Site-DirectedMutagenesis Kit (Stratagene) and purified as previouslydescribed.13 The glutathione S-transferase (GST)-fusedNEIL1 CTDs 312–389, 312–349, and 350–389 wereexpressed in E. coli and purified from the cell extracts viaglutathione-agarose affinity chromatography and elutingthe bound proteins with 10 mM reduced glutathione.9,16

The proteins were digested with thrombin to cleave theGST tag, followed by removal of GST by chromatographyon SP-Sepharose. The purity of the protein preparationswas confirmed by SDS-PAGE analysis.

Table 1. SAXS data collection parameters

SAXS data collection parametersBeamline 12.3.1 Advanced Light Source,

Lawrence Berkeley NationalLaboratory

Wavelength (Å) 1.12Q range (Å−1) 0.0154–0.29699Exposure time (s) 7Concentration (mg/ml) 10Temperature (°C) 4

Structural parametersI(0) from P(r) (arbitrary units) 1029Rg (Å) from P(r) 40.3I(0) from Guinier (arbitrary units) 1017Rg (Å) from Guinier 37.8Dmax (Å) 150



The clustered point mutant constructs for NEIL1, K296A/K297A/K298A, and K356A/K357A/K360A were generatedusing the QuikChange Site-Directed Mutagenesis Kit asabove. Mutagenesis reactions were performed using apET16(a) vector harboring NEIL1 cDNA, using appropriateprimers. Mutations were confirmed by direct sequencing.After expression in E. coli transfected with the plasmid, therecombinant untagged proteins were purified according tothe protocol used for WT NEIL1.

Fluorescence spectroscopy

The intrinsic fluorescence of NEIL1 and its mutants wasmeasured in a SPEX Fluoromax spectrofluorimeter (Hor-iba Jobin Yvon). The protein solutions in 10 mM phos-phate-buffered saline (PBS) buffer (pH 7.5) were excitedat 280 nm at 25 °C, and emission was monitored at 300–450 nm. The average spectrum was obtained fromtriplicate measurements.

CD spectroscopy

The CD spectra (195–260 nm) of the proteins (2 μM) in10 mM PBS buffer (pH 7.5) in the absence or presence ofosmolytes TMAO or sarcosine were taken in an AVIV CDspectrometer model 215 using a 1-mm cuvette.55 Four

repetitions of the CD spectra were used for averaging andthen corrected for the contribution from the buffer alone.The osmolytes TMAO and sarcosine were dissolved inpure water at 6 M, treated with granulated activatedcarbon, and filtered with 0.2 μM filter. The final concentra-tion of the osmolyte solutions was calculated from therefractive index.19

Protein denaturation studies

Urea (N99.9% purity, Sigma) was dissolved in 10 mMPBS (pH 7.5) to 8 M, treated with activated charcoal, andthe final concentration of the filtered solution wascalculated from the refractive index.19 To analyze theeffect of urea on NEIL1's folding, we diluted the protein to2 μM into both urea and plain PBS buffer. The twosolutions were then mixed at various proportions to yieldincreasing urea concentrations while maintaining the 2-μMprotein concentration and used for CD measurements at222 nm using time scan (1 min).

DG/AP lyase assay

To produce radiolabeled substrate, we labeled the single-stranded 5-OHU-containing deoxyoligonucleotide se-quence 5′-GCTTAGCTTGGAATCGTATCATGTA(5OHU)

Fig. 8. (a) Schematic represen-tation of the ensemble model ofallosteric regulation in native NEIL1containing the core domain and theregulatory region (R). Each regioncan be in native state or fullyunfolded, resulting in four possiblestates (i.e., N, I1, I2, and U). ΔGi (C)is the free energy of each state inthe presence of urea. ΔGC and ΔGRare the free energies of the fullyunfolded state of the core domainand R region in the absence of urea,with the native state as reference.Δgint is the interaction energy be-tween the coupled core domain andR region in absence of urea. mC,mR, and mint are the slopes of thefree-energy dependence on ureaconcentration for the core domain,R region, and interaction interface,respectively. S.W. is the statisticalweight of each state. Q is thepartition function, which is the sumof the statistical weights of all thestates in the ensemble. Obs is theobservable for each state used inthe simulation. (b) Simulated ureaunfolding curve for NEIL1 WT(black line) composed of the core

and regulatory region, N311 composed of only the core domain, and NEIL1-cluster(ii) mutant composed of the coredomain and mutated regulatory region. The parameters used in generating the simulated curve are as follows: ΔGC =2.5 kcal/mol, ΔGR = 1 kcal/mol, Δgint (WT) = 6 kcal/mol, Δgint [cluster(ii) mutant] = 2 kcal/mol,mC = −1.3 kcal/mol−1 K−1,mR = −0.1 kcal/mol−1 K−1, mint(WT) = −0.5 kcal/mol−1 K−1, mint[cluster(ii) mutant] = −0.1 kcal/mol−1 K−1, Obs1 = 0,Obs2 = 0.8.



ACTCGTGTGCCGTGTAGACCGTGCC-3′ at the 5′-termi-nus with [γ-32P]-ATP using T4-PNK (New England Biolabs).The labeled substrate was then separated from unincorpo-rated radioactivity by chromatography on Sephadex G25.The strand cleavage of substrate DNA by NEIL1 wasanalyzed after incubation of 5′ 32P-5-OHU-containingsubstrate at the indicated temperatures for 5 min in a 10-μlreaction mixture containing 40 mM Hepes–KOH, pH 7.5,50 mMKCl, 1 mMMgCl2, 100 μg/ml bovine serum albumin,and 5% glycerol. The reaction was stopped with theformamide dye mix (80% formamide, 20 mM NaOH,20 mM ethylenediaminetetraacetic acid, 0.05% bromophe-nol blue, and 0.05% xylene cyanol). The products wereseparated by denaturing gel electrophoresis in 20%polyacrylamide containing 8 M urea, in 1× Tris/borate–ethylenediaminetetraacetic acid buffer, pH 8.4.9 The radio-activity was quantitated in a PhosphorImager using ImageQuant software (Amersham Biosciences).

His-affinity co-elution analysis

His-affinity pull-down assays were carried out aspreviously described.9,10 Briefly, NEIL1's C-terminal pep-tides 312–349 and 349–389 (untagged, 20 pmol) weremixed with His-tagged WT NEIL1, or its N-terminalpolypeptide 1–311 (40 pmol), prebound with His-selectmagnetic nickel beads (Sigma) and incubated for 1 h at4 °C with constant rotation in a buffer containing Tris-buffered saline, 5% bovine serum albumin, and 10%glycerol. After washing the beads with Tris-buffered salinecontaining 0.1% Triton X-100 and 400 mM NaCl, thebound proteins were eluted with SDS sample dye andtested for the presence of the C-terminal peptides byimmunoblotting.

SAXS analysis

SAXS data for NEIL1 were collected at the SIBYLS12.3.1 beamline at the Advanced Light Source, LawrenceBerkeley National Laboratory.26,56 Scattering measure-ments were performed on 20 μl of NEIL1 at 10 mg/mldialyzed in PBS buffer and 1 mM DTT at 4 °C, 1.5 m fromthe Mar165 detector. Sequential exposures (7, 7, 35, and7 s) were taken at 11,111 eV. Data from the 10-mg/mlsolution were consistent with those for NEIL1 purified bygel filtration, indicating a lack of aggregation in theconcentrated sample. No radiation-induced aggregationwas observed, and the first and third exposures weremerged. Data were analyzed using the ATSAS programsuite57 and BilboMD.27 BilboMD models were generatedwith Rg between 36 and 44 Å. The envelopes and overlaidpdb were done in Chimera58 and BilboMD models weredone in PyMOL (Shrodinger).

Acknowledgements

The research was supported by U.S. Public HealthService grants R01CA81063, CA158910 (S.M.), andP01 CA92854 (J.A.T. and S.M.); R01 GM046312(J.A.T.) and R01 GM 63747 (V.J.H.); University of

Texas Medical Branch National Institute of Environ-mental Health Sciences Center and pilot grants P30ES006676 (S.M. and M.L.H.); and Alzheimer'sAssociation grant NIRG-12-242135 (M.L.H.). SAXSdata were collected at the SIBYLS beamline 12.3.1(Advanced Light Source, IDAT, Contract DE-AC02-05CH11231). CD and fluorescence experimentswere performed at the biophysical core facility attheUniversity of TexasMedical Branch.We thankDr.David Konkel for carefully editing the manuscript.

Received 15 November 2012;Received in revised form 9 March 2013;

Accepted 13 March 2013Available online 27 March 2013

Keywords:DNA repair;

intrinsically unstructured region;electrostatic interactions;

protein stability;oxidative DNA damage

Abbreviations used:BER, base excision repair; SAXS, small angle X-ray

scattering; NEIL1, Nei (endonuclease VIII)-like protein 1;WT, wild type; TMAO, trimethylamine-N-oxide;

CTD, C-terminal domain; DG, DNA glycosylase;MES, Minimal Ensemble Search; FEN-1,

flap endonuclease 1; GST, glutathione S-transferase;PBS, phosphate-buffered saline.

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