A novel method to identify nucleic acid binding sites in proteins by

7995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 14 2579-2583

A novel method to identify nucleic acid binding sitesin proteins by scanning mutagenesis: application toiron regulatory proteinBarbara Neupert, Eric Menotti and Lukas C. Kiihn*

Swiss Institute for Experimental Cancer Research, Genetics Unit, Chemin des Boveresses, CH-1066 Epalinges,Switzerland

Received May 15, 1995; Accepted May 31, 1995

ABSTRACT

We describe a new procedure to Identify RNA or DNAbinding sites in proteins, based on a combination ofUV cross-linking and single-hit chemical peptldecleavage. Site-directed mutagenesis is used to createa series of mutants with single Asn-Gly sequences inthe protein to be analysed. Recombinant mutantproteins are incubated with their radiolabelled targetsequence and UV Irradiated. Covalently linked RNA- orDNA-proteln complexes are digested with hydroxyl-amine and labelled peptides identified by SDS-PAGEand autoradiography. The analysis requires only smallamounts of protein and is achieved within a relativelyshort time. Using this method we mapped the site atwhich human iron regulatory protein (IRP) is UVcross-linked to iron responsive element RNA to aminoacid residues 116-151.

INTRODUCTION

The iron regulatory protein (IRP), previously called iron regula-tory factor (IRP) (1) or iron-responsive element binding protein(IRE-BP) (2), is a 98 kDa cytoplasmic RNA binding proteinwhich coordinately regulates cellular iron uptake, storage andutilization. IRP binds with high affinity to RNA hairpinstructures, termed iron-responsive elements (IREs) (1 —4). presentin at least five different mRNAs: single copies exist in the5'-untranslated region (UTR) of ferritin L- and H-chain mRNA(5,6), the erythroid 5-aminolevulinic acid synthase (eALAS)mRNA (7,8) and mitochondrial aconitase mRNA (8), whereasfive copies are present in the 3'-UTR of transferrin receptor (TfR)mRNA (9). The RNA binding activity of IRP is induced by irondeprivation and inactivated by high cellular iron levels. Bindingof IRP prevents ferritin and eALAS mRNA translation (10-13)and protects TfR mRNA from degradation, thereby enhancingreceptor synthesis (1,14,15). This control of cellular ironhomeostasis by IRP has provided a model system for the study ofRNA-protein interactions and post-transcriptional regulatorymechanisms of gene expression in eukaryotic cells.

IRP reveals striking sequence homology with mitochondrialand bacterial aconitases (16—18) and is identical in primary aminoacid sequence to cytoplasmic aconitase (19), a Fe-S proteinwhich catalyzes conversion of citrate to isocitrate. It has beendemonstrated that IRP can function as an enzyme at the expenseof its RNA binding properties (20). The switch from die RNAbinding apo-form to cytoplasmic aconitase has been shown toresult from insertion of an iron-sulfur cluster (21-25).

By using partial V8 protease digestion we have previouslyshown that an N-terminal 67 kDa fragment can covalentlycross-link to 32P-labelled IRE RNA (26), whereas deletionstudies have indicated that the C-terminal domain is alsonecessary for IRE binding (26), suggesting that both the N- andC-terminal regions of IRP participate in the RNA-protein bindingreaction. Thus, unlike hnRNA binding proteins (27), IRP does notseem to contain a distinct RNA binding domain.

In the present study we investigated which regions of IRP areinvolved in RNA binding, using a novel approach based on UVcross-linking of RNA-protein complexes and chemical cleavageat defined positions of IRP. To this end we have introduced singlehydroxylamine cleavage sites throughout the open reading frameof recombinant IRP, such that digestion of the protein with thechemical reagent yields two peptides which can be identified bySDS-PAGE.

MATERIALS AND METHODS

Site-directed mutagenesis

In order to create hydroxylamine cleavage sites, point mutationscreating Asn-Gly sequences were introduced into human IRPcDNA by site-directed mutagenesis of plasmid pGEM-hlRF (26)deleted of its 3'-terminal Kpn\ fragment. The plasmid wastransformed into Escherichia coli strain CJ236, according to themethod of Kunkel et al. (28), and single-stranded DNA preparedusing the helper phage VCS13 (Stratagene, La Jolla, CA).Oligonucleotides (19- or 20mers) carrying specific mismatcheswere hybridized to the single-stranded DNA in order to introducethe following amino acid changes: Thr89—»Asn89(ACG->AAQ, Proll6->Glyll6 (CCT->GGT), Argl51-»Glyl51 (AGA->GGA), Asp212->Asn212 (GAT->AAT),

* To whom correspondence should be addressed

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2580 Nucleic Acids Research, 1995, Vol. 23, No. 14

Ile243-»Asn243 (ATT->AAT), Phe281 ->Asn281 (TTC->AAC),Thr323->Asn323 (ACA->AAC), Asp347^Gly347 (GAC^GGC), Ser372->Asn372 (AGT-+AAT) and Ser510->Asn510(AGT—»AAT). The second strand was synthesized using 1 U T4DNA polymerase (Boehringer, Mannheim, Germany) in thepresence of 0.5 mM dATP, dCTP, dGTP and dTTP, 3 mM MgCl2,2 mM dithiothreitol, 1 mM ATP, 3 U T4 DNA ligase (Boehringer),50 mM NaCl and 20 mM Tris-HCl, pH 7.6. This double-strandedDNA was re-transformed into E.coli strain BZ234. Single-stranded DNA was then prepared from several independentcolonies and the presence of desired mutations confirmed bysequencing (29). The 3'-terminal Kpn\ fragment was subsequentlyre-inserted into the cDNA vector.

Expression and purification of recombinant IRP

In order to express human IRP (26) in E.coli, the entire openreading frame was cloned into the bacterial expression vectorpT7-His to generate IRP with an N-terminal (His)6 tag (30). Thisplasmid was transformed into E.coli strain BL21 (DE3) andamplified overnight at 25°C in the absence of IPTG as described(30). Cells were harvested and lysed by freeze/thawing andsonication in 250 mM NaCl, 0.5% NP-40, 20 mM Tris-HCl, pH8.0. After removal of cellular debris by centrifugation, thesupernatant was applied to a Ni2+-NTA-agarose column(Qiagen, Hilden, Germany) and recombinant IRP was eluted with50 mM imidazole (Sigma, St Louis, MO) as described (30). Therecovered IRP was -50% pure as judged by SDS-PAGE. IRP wasfurther purified on a Mono Q HR column (Pharmacia, Uppsala,Sweden) equilibrated in 20 mM Tris-HCl, pH 8.0, 5% glyceroland eluted with a linear salt gradient (0-1000 mM KC1). IRPelutes at 87 mM KC1 as a pure protein fraction as judged bySDS-PAGE (data not shown).

Preparation of in vitro transcripts and RNA-proteinband shift assays

Radiolabelled ferritin IRE transcript from plasmid pSPT-fer (1)was synthesized in vitro using T7 RNA polymerase (Promega,Madison, WI) from linearized plasmid DNA (1 u.g) in thepresence of 100 ^Ci [a-32P]CTP (800 Ci/mmol) (Amersham,Little Chalfont, UK), 2.5 mM ATP, GTP and UTP (Pharmacia)and 20 U T7 RNA polymerase (Boehringer) in a 20 \i\ reactionvolume. Samples were incubated for 1 h at 37°C. Unlabelledtranscripts of the wild-type ferritin IRE (clone 42) (31) or the HI VTAR stem-loop from plasmid pBTO (32) were synthesized andpurified on a 3% NuSieve agarose gel as described (33).RNA-protein complexes were analysed as described previously(1,3). For competition assays similar amounts of purified IRPs(0.5 ng) were incubated with equimolar amounts of 32P-labelIedferritin IRE (0.1 ng, 5 x 104 c.p.m.) and varying molar excessesof unlabelled RNAs. RNA-protein complexes were treated with5 mg/ml heparin and resolved on a 6% non-denaturing polyacryl-amide gel.

UV cross-linking of the RNA-protein complex

Purified IRPs (~5 fig wild-type and NG mutants, but 1 u:g mutantNG116) were incubated with 2 x 106 c.p.m. [32P]CTP-labelledIRE probe in 100 uJ 20 mM Tris-HCl, pH 8.0,40 mM KC1, 5%glycerol. The binding reaction was carried out for 20 min at roomtemperature as described previously (3). Subsequently, unpro-

tected RNA was digested for another 10 min with 10 U RNase T1.The reaction was then UV irradiated on ice for 45 min in a UVStratalinker 2400 (Stratagene) at a 12 cm distance from theirradiating source (254 nm wavelength), after which RNase A (1ng/u.1; Boehringer) was added for 30 min at 37°C.

Hydroxylamine digestion of the radiolabelled complex

The nucleophilic compound hydroxylamine cleaves the cyclicimide derivative of asparaginyl-glycyl peptide bonds (34).Covalently linked RNA-protein complexes (or recombinantprotein without RNA) were precipitated overnight at -20°C with6 vol. acetone (if necessary, 0.2 (ig/(il BSA can be added as acarrier for precipitation) and then resuspended in 20 (il 1 % SDSat 45°C for 30 min in order to denature the protein prior tochemical cleavage (35). Digestion was carried out in the presenceof 1 M hydroxylamine in 0.1 M K2CO3 (pH 10) for 3 h at 45 °Cin a 40 jxl reaction volume, as described (35). The peptides wereagain acetone precipitated and analysed by SDS-PAGE.

RESULTS AND DISCUSSION

In order to localize the regions of IRP which contact RNA, wemade site-directed mutants which can be cleaved at Asn-Glyresidues by hydroxylamine. Human IRP has one hydroxylaminecleavage site at its very C-terminus and we have introduced asecond site at various positions in the protein, such that in eachcase chemical cleavage generates two fragments of distinct sizes(Fig. 1). Wild-type and mutant IRPs were expressed with a (His)6tag in E.coli and affinity-purified as described in Materials andMethods. IRP fractions were then incubated with radiolabelledIRE transcript and UV irradiated. Covalently linked RNA-protein complexes were digested with hydroxylamine and theradiolabelled digestion products analysed by SDS—PAGE (Fig.2). The contribution of the protected RNA to the apparentmolecular weight of the complexes is negligible, due to RNase Adigestion of the complex after UV cross-linking. The size of thelabelled peptides indicated a cross-link of the IRE probe to theN-terminal fragment of mutants NG151 (19 kDa), NG212(26 kDa), NG243 (29 kDa) NG281 (33 kDa), NG323 (38 kDa),NG347 (41 kDa), NG372 (43 kDa) and NG510 (58 kDa) (Fig.2 A) and the C-terminal fragment of mutants NG89 (87 kDa) andNG116 (84 kDa) (Fig. 2B). In agreement, no labelled N-terminalfragments of -12 or 15 kDa, respectively, were formed bydigestion of mutants NG89 or NG 116. Similarly, the C-terminalfragments of mutants NG151-NG51O did not cross-link to theRNA (Fig. 2A). In order to verify that the binding affinity andspecificity for the IRE was not affected by the point mutationsgenerating new cleavage sites, we carried out competition studies.Wild-type or mutant IRPs (NG89, NG116 and NG151) wereincubated with radiolabelled ferritin IRE and various concentra-tions of unlabelled ferritin IRE, HIV TAR stem-loop RNA ortRNA (Fig. 3). Whereas binding to each of the IRPs wassignificantly competed by a 10-fold excess of IRE, no competi-tion was observed with 500-fold excesses of the other RNAs. Thisis in agreement with previous studies on human placental IRP thatindicated a high specificity of IRE-IRP interaction and cross-linking (1).

Together our results define the UV cross-link site betweenamino acids 116 and 151, in agreement with our previous resultof a UV cross-link to the N-terminal region (26). The same RNA


Nucleic Acids Research, 1995, Vol. 23, No. 14 2581

Predicted sizeof fragments

(kDa)

97.1 +1.5

12.0 + 86.6 + 1.5

14.8 + 83.8 + 1.5

19.0 + 79.8 + 1.5

26.0 + 72.6 + 1.5

29.0 + 69.6 + 1.5

33.0 + 85.6 + 1 .5

37.7 + 60.9 + 1.5

40.6 + 58.0 + 1.5

43.3 + 55.3 + 1.5

58.0 + 40.6 + 1.5

N H 2

N H 2

N H 2

N H 2

N H 2

N H 2

N H 2

N H 2

N H 2

N H 2

N H 2

NGaMiiiiiimiuiminiiiiiiiiiiiiiiiiiimiimiiiiiimiiiiiiim iniuiiiiiiiiiiiiii mini iiiiiiniiiii iiiitiiiimiiij inmit- COOH HIS"hIRP

1 NQ89 889HZiiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiiiiiiiiiiiiriiiiiiiiniiiiiiiiiiiiiiiiiiiiiiiiiiiiiiriiiiiiiiiiiiiuiiHiiiiiiiiHHiiiiiiiuiiiiiiiiiiiiiiiiiiiiiiiiiiiniiiii- COOH

NG116H^iiiiiiiiiiiiiiiiiiiniiiiiiiiiiuiiiiiiiiiiiiiiiiiiiiuiiuiiiiiiiiiiiiiiiiiiiiiiiiiiiuiiiiuiiiiiiniiiiiiiiiiiiiiiiiiiiMiiiiiiiiiMiiiiiiiiiiiiiiiiiiniiiii- COOH

NG151a MI mnuimiiiii 1111111.111..1 i in. miniv'i- COOH

NG212'CSiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiiiiiuiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiniiiit- COOH

NG243•ciiiiiiiiiiiiiiiiiiiiniiiniiiiiiiiiiiiiiiifiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii- COOH

NG281•OIIIIIIIIIIIIIIIIIIIIMIIIIIIIIIIIIIIIIHIIIIIIIIIllllllHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIUIIIIIIIIIIIIIIIIIIIIIIIIllllllllllllllllllllllinillllllllilllll- COOH

NQ323oiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiuiiiiuiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiMiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiiiiiiniuii- COOH

NG347<=• iiiiiii iiiiiiiiiniiiiiniiiiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiiiiiiiiiiiiitiiiiiii iiuiiiiiiiiiiiimiiiiiiiimiiiiiiiimiiiimiiiiiii- COOH

NQ372KZllllllllllllllllllllllllllllllllll IIIIIIIIIHHIIIIIinillllllllllllllllllllllltlllllllllllllllllllllllllllllllllllllllllllllllllllOlllllllllilllll- C O O H

NQ510KZIIIIIIIIIIIIIIIIIIIII IIIIIII IIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIMIIIIIIIIIIIIMIIIIIIIIIIIIIIIIIIIIIIIIIIII 111 Illlllll Illllli Illll- COOH

Figure 1. Schematic representation of His-hIRP and mutants with hydroxylamine cleavage sites. Human IRP has 889 amino acids (broken line) (26) and a naturalhydroxylamine cleavage site at position 876. The (His)ft-tagged fusion protein (His-hIRP) has 14 additional amino acids at its C-terrrunus (open box) and a predictedmolecular weight of 98.6 kDa. Hydroxylamine cleavage sites with the sequence Asn-Gly (NG) were introduced by site-directed mutagenesis and are located at aminoacid positions 89, 116, 151, 212, 243, 281, 323, 347, 372 or 510 of human IRP. The predicted sizes of the N-terminal and C-terminal fragments after hydroxylaminedigestion are indicated on the left.

NGB

NG

WT 89 116 151 212 243 281 323 347 372 510 k n a 89 116 151 WT kDa- 200 . 200

-46

-30

Figure 2. Identification of hydroxylamine-cleaved fragments cross-linked toRNA. Hydroxylamine mutants were UV cross-linked to a radiolabelledIRE-containing RNA and subjected to hydroxylamine digestion as described inMaterials and Methods. Labelled peptides were identified by autoradiographyafter separation by electrophoresis in a 17.5% SDS-polyacrylamide gel (A).Cleavage products of wild-type IRP (WT) and of Asn-<ily mutants at positions89, 116, 151, 212, 243, 281, 323, 347, 372 and 510 are shown. The arrowindicates undigested RNA-protein complexes and closed circles indicatedegradation products present in both wild-type and mutant lanes. Each of themutants 151-510 showed one additional peptide derived from the covalentRNA-protein complex and corresponding to the N-terminal hydroxylaminedigestion fragment (see Fig. 1). However, the corresponding 12 and 14.8 kDaN-termina) fragments of mutants 89 and 116 respectively were not cross-linked.When the same RNA-protein complexes of mutants 89, 116 and 151, alongwith wild-type IRP, were analysed in an 8% SDS-polyacrylamide gel (B),unique labelled RNA-protein complexes corresponding to the C-terminalhydroxylamine fragments of mutants 89 and 116 were visible (marked by anarrow).

binding region has recently been identified by Basilion et al. (36)by a different approach. The authors localized the UV cross-linksite to amino acids 121-130, a region predicted to be within theactive site cleft. However, a different RNA binding element hasbeen located within the C-terminal part of rabbit IRP, betweenresidues 480 and 623, by partial chymotrypsin digestion of UVirradiated RNA-protein complexes (37). This discrepancy maybe due to minor structural differences between human and rabbitIRPs.

Although deletion studies from the C-terminus of IRP havesuggested that RNA binding is not restricted to a single domain(26), we are able to identify only one region of IRP thatcross-links to the IRE probe. It is generally accepted that all aminoacids are potential candidates for covalent linkage with nucleicacids and pyrimidines are thought to cross-link more efficientlythan purines (38). Therefore, the binding site identified by UVcross-linking may reflect the region of IRP which most closelycontacts the RNA, while other parts of the protein may benecessary to maintain a RNA binding conformation. In addition,steric hindrance may prevent cross-linking of some aminoacid—nucleotide interactions.

The previous approach taken by Basilion et al. (36) was toidentify RNA contact sites by UV cross-linking of recombinantIRP, followed by complete protease digestion of the complex andpeptide sequencing. This strategy is currently used for identifyingRNA or DNA binding sites in proteins which are not structuredin separate domains and lack a distinct nucleic acid binding motif.However, the relatively low cross-linking efficiency (-3%)renders this procedure costly and time consuming, since itrequires milligram amounts of recombinant protein and RNA, aswell as a performant peptide sequencing facility. In addition, thepeptide mixture obtained upon complete protease digestion needsto be separated biochemically prior to amino acid sequencing.


2582 Nucleic Acids Research, 1995, Vol. 23, No. 14

IRE TAR tRNAI I I Io S o o o o o o S x competitor

WT

NG89

NG116

hydroxylamine or cyanogen bromide respectively, are generallyless frequent in proteins and therefore target sequences of choiceto be created or destroyed. Furthermore, hydroxylamine digestionis performed under denaturing conditions, such that all cleavagesites are accessible to the chemical reagent. The small number ofpeptides generated upon protein digestion permits immediateanalytical identification of one or a few affinity-labelled peptideswith a restricted number of mutants. The mapping can then befurther refined down to single amino acid residues. This methodshould prove generally applicable to the analysis of any RNA orDNA binding protein.

ACKNOWLEDGEMENTS

We wish to thank S. Sosolic for technical assistance and B.Henderson and S. Gasser for carefully reading the manuscript.The present work was supported by the Swiss National ScienceFoundation.

NG151

Figure 3. Comparison of relative RNA binding affinities of wild-type andmutant IRPs. Purified protein (0.5 ng) from wild-type IRP and mutants NG89,NG116 and NG151 were incubated with gel-purified 32P-labelled wild-typeferritin IRE (0.1 ng, 5 x I04 c.p.m.) and increasing amounts of unlabelledcompetitor RNA, either the same IRE sequence, the HIV-TAR stem-loop ortRNA. RNA-protein complexes were treated with heparin and analysed in 6%non-denaturing polyacrylamide gels as described in Materials and Methods.

Indeed, the identification of affinity-labelled peptides uponcomplete protein digestion with frequently cutting enzymes, liketrypsin or V8 protease, can be difficult. Alternatively, deletionstudies have been used to localize nucleic acid binding elementsin proteins. However, this strategy is adapted for proteinscontaining a distinct binding domain, whereas any deletion inproteins lacking such domains, like IRP, may alter the overallstructure and thereby indirectly affect the binding properties. Inthe present approach the structural alterations introduced bysingle point mutations had no deleterious effect on the specificityor affinity for the IRE (Fig. 3). The interpretation of resultsobtained seems therefore safe. Of course, even site-directedmutations may hit RNA-contacting residues or amino acidscritical for protein folding and thereby affect the RNA-proteininteraction. However, this can be easily verified by analysingmutants which introduce a new cleavage site nearby.

The present method, we believe, is mainly useful in the initialphase of mapping a new RNA or DNA binding site on a protein.Ultimately, and this is certainly also the case for the IRE-IRPcomplex, determination of the three-dimensional structure bycrystallization should be most informative about the physicalinteraction. However, as there is no generally applicable rationalefor co-crystallization, the method must be adapted for eachparticular case and this may represent a long-term investment.Our present approach requires only small amounts of recombi-nant protein and avoids amino acid sequencing. Its novelty lies inthe creation of protein cleavage sites which are rare or absent inthe protein of interest, by taking advantage of the well-establishedand straightforward method of site-directed mutagenesis (28).Asn-Gly dipeptides or methionines, which are cleaved by

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A novel method to identify nucleic acid binding sites in proteins by