清华大学：《生物核磁共振波谱学》课程教学资源（文献资料，英文版）NMR-Protein-DNA.pdf

NMR studies of protein±DNA interactions N. Jamina,*, F. Tomab a CEA/INSTN, 91191 Gif sur Yvette Cedex, France b DeÂpartement de Biologie, UniversiteÂ d'Evry, bld F. Mitterand, 91025 Evry Cedex, France Received 1 June 2000 Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 2. Overview of techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 2.1. Labeling of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 2.2. Chemical shift changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 2.3. Hydrogen exchange rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 2.4. Isotope editing and isotope ®ltering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 2.5. Deuteration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 2.6. Transverse relaxation-optimized spectroscopy (TROSY) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 2.7. Long-range distance constraints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 2.8. Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 2.9. Hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 3. Selected applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 3.1. The helix-turn-helix motif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 3.1.1. Homeodomain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 3.1.2. Lac repressor headpiece . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3.1.3. Trp repressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 3.1.4. Ets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 3.1.5. Myb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 3.2. Zinc ®ngers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 3.2.1. TFIIIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 3.2.2. ADR1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 3.2.3. GATA-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3.2.4. GAGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3.3. Minor groove-binding architectural proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 3.3.1. SRY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 3.3.2. LEF-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 3.3.3. HMG-I(Y) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 0079-6565/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S0079-6565(00)00024-8 www.elsevier.nl/locate/pnmrs * Corresponding author. Tel.: 133-1-69-08-96-38; fax: 133-1-69-08-57-53. E-mail address: nadege.jamin@cea.fr (N. Jamin)

N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001)83-114 3.4. Recognition using B-sheet 3. 1. Tn916 integrase 3. 4.2. GCC-box binding domain 4. Perspectives 112 References 1. Introduction bound to a 14-mer duplex DNA containing the bs site [1] and the lac repressor headpiece(residues 1 Understanding at a molecular level, the mechan- 56, HP56)complexed with a 11-mer operator [2] isms for the control of genetic information and its This review will describe the use of nmr to obtain replication, packaging and repair necessitates the information on complexes of proteins with their speci elucidation of the detailed interactions between fic DNA targets. Most of the NMR techniques used to proteins and DNA. The last ten years have produced study protein-DNA interactions are also employed a large amount of structural information about for other type of protein complexes. Therefore, for a protein-DNA complexes from both X-ray crystallo- detailed description of the NMr techniques, the graphy and NMR. These data reveal the complexity of reader is referred to recent reviews [3-5]or to specific the DNA recognition process. The absence of a papers referenced in the text recognition code' is particularly evident among the divided in three parts. The first part is hree zinc fingers of the transcription factor TFIIIA as an overview of the NMR techniques commonly used homologue residues in different complexes do not to get information on protein-DNA interactions. It always contact corresponding base pairs. Direct inter- includes a brief description of DNA labeling techni- action between protein side-chains and DNa bases ques, the use of chemical shift or hydrogen exchange not only involve secondary structures like a-helix or changes to find the binding site, the use of hydrogen B-sheet but also flexible loops and arms. Moreover exchange or relaxation data to get dynamics informa residues not involved in specific interactions such as tion on the binding process, the use of the main the linker residues of the three zinc fingers domain of isotope filtering and editing techniques as well as TFIIA can be as important for the protein-DNA transverse relaxation-optimized spectroscopy to interaction as residues making contact with DNA assign the NMR signals, and newly developed tech- bases niques to deal with large complexes or to obtain long NMR makes its unique contribution to the under- range distance restraints. The second part comprises standing of protein-DNA interactions by highlighting applications of these techniques to different protein the dynamic aspects of protein-DNA interactions: DNA complexes. Protein-DNA complexes are clas dynamics of disorder-to-order transitions upon DNa sified according to the protein recognition motif: binding, dynamics at the protein-DNA interface, helix-turn-helix(HT), zinc finger, minor groove dynamics of opening and closing of base-pairs and, binding motif and B-sheet. Finally, the third part measurements of lifetimes of water molecules at th presents the future perspectives that can be inferred protein-DNA interface. from the emerging NMR techniques During the last 10 years, more than 20 structures of specific protein-DNA complexes and numerous data on protein-DNA interactions have been obtained by 2. Overview of techniques NMR thanks to the developments in protein and nucleic acid synthesis, in isotopic labeling techniques Protein-nucleic acids complexes are large entities and in heteronuclear magnetic resonance spectro- and the availability ofC-andN-labeled proteins copy. The first 3D NMR structures of a protein has made the determination of their solution structures DNA complex were obtained in 1993: the Drosophila attainable. Double and triple resonance spectroscopy antennapedia mutant homeodomain(Antp(C39S) facilitates the resonance assignments, the measurement

3.4. Recognition using b-sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.4.1. Tn916 integrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.4.2. GCC-box binding domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 4. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 1. Introduction Understanding at a molecular level, the mechanisms for the control of genetic information and its replication, packaging and repair necessitates the elucidation of the detailed interactions between proteins and DNA. The last ten years have produced a large amount of structural information about protein±DNA complexes from both X-ray crystallography and NMR. These data reveal the complexity of the DNA recognition process. The absence of a `recognition code' is particularly evident among the three zinc ®ngers of the transcription factor TFIIIA as homologue residues in different complexes do not always contact corresponding base pairs. Direct interaction between protein side-chains and DNA bases not only involve secondary structures like a-helix or b-sheet but also ¯exible loops and arms. Moreover residues not involved in speci®c interactions such as the linker residues of the three zinc ®ngers domain of TFIIIA can be as important for the protein±DNA interaction as residues making contact with DNA bases. NMR makes its unique contribution to the understanding of protein±DNA interactions by highlighting the dynamic aspects of protein±DNA interactions: dynamics of disorder-to-order transitions upon DNA binding, dynamics at the protein±DNA interface, dynamics of opening and closing of base-pairs and, measurements of lifetimes of water molecules at the protein±DNA interface. During the last 10 years, more than 20 structures of speci®c protein±DNA complexes and numerous data on protein±DNA interactions have been obtained by NMR thanks to the developments in protein and nucleic acid synthesis, in isotopic labeling techniques and in heteronuclear magnetic resonance spectroscopy. The ®rst 3D NMR structures of a protein± DNA complex were obtained in 1993: the Drosophila antennapedia mutant homeodomain (Antp(C39S)) bound to a 14-mer duplex DNA containing the BS2 site [1] and the lac repressor headpiece (residues 1± 56, HP56) complexed with a 11-mer operator [2]. This review will describe the use of NMR to obtain information on complexes of proteins with their speci- ®c DNA targets. Most of the NMR techniques used to study protein±DNA interactions are also employed for other type of protein complexes. Therefore, for a detailed description of the NMR techniques, the reader is referred to recent reviews [3±5] or to speci®c papers referenced in the text. This review is divided in three parts. The ®rst part is an overview of the NMR techniques commonly used to get information on protein±DNA interactions. It includes a brief description of DNA labeling techniques, the use of chemical shift or hydrogen exchange changes to ®nd the binding site, the use of hydrogen exchange or relaxation data to get dynamics information on the binding process, the use of the main isotope ®ltering and editing techniques as well as transverse relaxation-optimized spectroscopy to assign the NMR signals, and newly developed techniques to deal with large complexes or to obtain longrange distance restraints. The second part comprises applications of these techniques to different protein± DNA complexes. Protein±DNA complexes are classi®ed according to the protein recognition motif: helix-turn-helix (HTH), zinc ®nger, minor groove binding motif and b-sheet. Finally, the third part presents the future perspectives that can be inferred from the emerging NMR techniques. 2. Overview of techniques Protein±nucleic acids complexes are large entities and the availability of 13C- and 15N-labeled proteins has made the determination of their solution structures attainable. Double and triple resonance spectroscopy facilitates the resonance assignments, the measurement 84 N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114

N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38(2001)83-114 Culture of cells withC carbon source [6]. Labeled ribonucleotides are prepared from the and Nitrogen sourc isolation of bacterial rna from in labeled medium, the hydrolysis of RNA and the separation phenolextraction of the ribonucleotides [7]. They are then chemi tized into nucleoside 3-phosphoramidites which DNA and RNA are used for preparing oligonucleotides on a DNA onthe Nucleic acids hydrolysis Using this method, a 14-base pair DNA dupl ully C, n doubly-labeled as well as partially 5 monophosphate nucleotide labeled at those nucleotides that form the prote DNA interface has been prepared to study its inter action with the antennapedia homeodomain [8] The general procedure for the production of uniformly C, N-labeled DNA by enzymatic synth- dNMPs rAMPs esis is described in Fig. 1. Zimmer and Crothers have shown that milligram quantities of material can be synthesized using this procedure [9]. Their method dNTP comprises the production of uniformly,N- labeled deoxynucleotides from enzymatic hydrolysis of the dNA of bacteria grown on 99%CH3OH and >98%NHCI as sole carbon and nitrogen sources DNA oligonucleotid The labeled DNa are then converted enzymatically to the triphosphates and used in a DNA polymerization I General procedure for the enzymatic synthesis reaction that utilizes an oligonucleotide hairpin labeled DNA primer-template containing a ribonucleotide at the 3 terminus. Alkaline hydrolysis of the ribonucleotide of coupling constants and of relaxation parameters not linkage between the labeled DNA and the unlabeled accessible by proton resonance spectroscopy. It is primer-template followed by purification yields the only recently that efficient labeling of DNA [6-9] labeled DNA. More recently variations of this method has been published thus opening applications of have been proposed by two other groups [10,11] heteronuclear spectroscopy to DNA. We will present Masse and coworkers [10] proposed three modifica- briefly the new labeling methods proposed for DNA. tions. First, the mixed dNTPs are separated from one We will also give an overview of the NMr techniques another so that the ratio of the four dNTPs correspond used to extract structural information about protei ing to the sequence of the deoxyoligonucleotide are DNA complexes including chemical shift changes, used in the reaction. Secondly, Taq polymerase is hydrogen exchange rates, isotope editing and filtering used instead of Klenow fragment of DNA polymerase echniques and methods for measuring protein I in the polymerization step. Third, an additional step dynamics to study the changes in protein flexibility is used to remove non-templated addition at the 3 upon binding. end. Louis and coworkers [11] used the same mole cule for the primer and template in the bidirectional 2.1. Labeling of dNA polymerase chain reaction thus obtaining an exponen- tial growth in the length of the double strand that matic methods. The chemical synthesis of DNA oligo- and coworkers [ll]. It comprises the growth of a mers involves the solid-phase phosphoramidite suitable plasmid containing mutiple copies of the

of coupling constants and of relaxation parameters not accessible by proton resonance spectroscopy. It is only recently that ef®cient labeling of DNA [6±9] has been published thus opening applications of heteronuclear spectroscopy to DNA. We will present brie¯y the new labeling methods proposed for DNA. We will also give an overview of the NMR techniques used to extract structural information about protein± DNA complexes including chemical shift changes, hydrogen exchange rates, isotope editing and ®ltering techniques and methods for measuring protein dynamics to study the changes in protein ¯exibility upon binding. 2.1. Labeling of DNA Large quantities of labeled DNA fragments for NMR studies can be synthesized by chemical or enzymatic methods. The chemical synthesis of DNA oligomers involves the solid-phase phosphoramidite method using isotopically labeled monomer units [6]. Labeled ribonucleotides are prepared from the isolation of bacterial RNA from cells grown in labeled medium, the hydrolysis of RNA and the separation of the ribonucleotides [7]. They are then chemically converted to deoxynucleotides and derivatized into nucleoside 30 -phosphoramidites which are used for preparing oligonucleotides on a DNA synthesizer. Using this method, a 14-base pair DNA duplex fully 13C,15N doubly-labeled as well as partially labeled at those nucleotides that form the protein± DNA interface has been prepared to study its interaction with the antennapedia homeodomain [8]. The general procedure for the production of uniformly 13C,15N-labeled DNA by enzymatic synthesis is described in Fig. 1. Zimmer and Crothers have shown that milligram quantities of material can be synthesized using this procedure [9]. Their method comprises the production of uniformly 13C,15Nlabeled deoxynucleotides from enzymatic hydrolysis of the DNA of bacteria grown on 99% 13CH3OH and .98% 15NH4Cl as sole carbon and nitrogen sources. The labeled DNA are then converted enzymatically to the triphosphates and used in a DNA polymerization reaction that utilizes an oligonucleotide hairpin primer-template containing a ribonucleotide at the 30 terminus. Alkaline hydrolysis of the ribonucleotide linkage between the labeled DNA and the unlabeled primer-template followed by puri®cation yields the labeled DNA. More recently variations of this method have been proposed by two other groups [10,11]. Masse and coworkers [10] proposed three modi®cations. First, the mixed dNTPs are separated from one another so that the ratio of the four dNTPs corresponding to the sequence of the deoxyoligonucleotide are used in the reaction. Secondly, Taq polymerase is used instead of Klenow fragment of DNA polymerase I in the polymerization step. Third, an additional step is used to remove non-templated addition at the 30 end. Louis and coworkers [11] used the same molecule for the primer and template in the bidirectional polymerase chain reaction thus obtaining an exponential growth in the length of the double strand that contains two repeats of the desired DNA sequence. An additional method has been presented by Louis and coworkers [11]. It comprises the growth of a suitable plasmid containing mutiple copies of the N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 85 Culture of cells with 13C carbon source and 15N nitrogen source cell lysis phenol extraction DNA and RNA proteins Nucleic acids hydrolysis 5’ monophosphate nucleotide nucleotide separation dNMPs rNMPs dNTPs DNA oligonucleotide Fig. 1. General procedure for the enzymatic synthesis of 13C,15Nlabeled DNA

N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001)83-114 i.e. the dissociation constants Kd are less than 10M GR [R2R3] and detailed information can be obtained on the IMIM,I complexes because of the slow exchange regim between free and bound states (lifetimes greater than I s) at the chemical shift time-scale. The rate of exchange is much less than the difference in the chemical shift between the two states and. at a mole ratio less than the stoichiometric ratio, two sets of resonances are observed corresponding to the free and bound states. Therefore. the resonances of the complex have to be assigned using NMR techniques employed for large molecules and/or edited/filtered Fig. 2 shows the imino region of the H obtained upon addition of different amounts of ESapeolra tion of R2R3 dna binding domain of c-Myb to a solution of mim12 oligonucleotide [12]. On addition of the protein, new resonance lines corresponding to the bound mim12 dodecamer appear. Some of these lines are split into two signals which indicate the enc forms. The lifetimes of these two forms are longer than the inverse of the frequency difference between the free and bound state 14.0 12.0 Chemical shifts are very sensitive probes of the Fig.2. Imino region of the H-NMR 600 MHz spectra obtained local environments of the nucleus but unfortunately upon addition of different amour ding domain of c-Myb to a sol olton of the rzR DNA it is not possible to predict their values from the 20°C. conformation of the complex or conversely to deduce the conformation from their values. Nevertheless they are useful parameters to gain insight into the desired DNA sequence in E. coli with N andc parts of the molecules influenced by the interaction nutrients. These methods have been applied to the Schmiedeskamp and coworkers [13] have shown by synthesis of fully or partiallyC,N orN-labeled analysis of H and Ca chemical shifts that little double strand oligonucleotides of 10-21 base pairs. a change in the structure of the zinc-finger domain 32 base DNA oligonucleotide that folds to form an from the yeast transcription factor ADRI occurs intramolecular quadruplex as well as a 12 base oligo- upon binding to a 14mer DNA containing the UAS nucleotide that dimerizes and folds to form a quadru- half site. A correlation between the protein-DNA plex uniformly C, N-doubly labeled have also been interface mapped by chemical shift changes and that produced for NMR studies. by muta ts was found Both these methods require a high level of exper However, the identification of the dna binding site tise Site specific labeling is more easily attained with using DNA induced chemical shift changes should be the chemical method and is therefore the method of done with care. This approach is not feasible for hoice for the synthesis of site specific labeled DNA. numerous protein-DNA complexes where proteins undergo conformational transitions and dynamics 2. 2. Chemical shift changes changes upon binding that will affect the chemical shifts. This has been recently demonstrated by Foster Interactions of protein with DNA fragment contain- and coworkers [14]. These authors analyzed the corre ing specific binding sites are tight binding interactions lation between the chemical shift changes upon

desired DNA sequence in E. coli with 15N and 13C nutrients. These methods have been applied to the synthesis of fully or partially 13C,15N or 15N-labeled double strand oligonucleotides of 10±21 base pairs. A 32 base DNA oligonucleotide that folds to form an intramolecular quadruplex as well as a 12 base oligonucleotide that dimerizes and folds to form a quadruplex uniformly 13C,15N-doubly labeled have also been produced for NMR studies. Both these methods require a high level of expertise. Site speci®c labeling is more easily attained with the chemical method and is therefore the method of choice for the synthesis of site speci®c labeled DNA. 2.2. Chemical shift changes Interactions of protein with DNA fragment containing speci®c binding sites are tight binding interactions i.e. the dissociation constants Kd are less than 1028 M and detailed information can be obtained on the complexes because of the slow exchange regime between free and bound states (lifetimes greater than 1 s) at the chemical shift time-scale. The rate of exchange is much less than the difference in the chemical shift between the two states and, at a mole ratio less than the stoichiometric ratio, two sets of resonances are observed corresponding to the free and bound states. Therefore, the resonances of the complex have to be assigned using NMR techniques employed for large molecules and/or edited/®ltered techniques. Fig. 2 shows the imino region of the 1 H spectra obtained upon addition of different amounts of a solution of R2R3 DNA binding domain of c-Myb to a solution of mim12 oligonucleotide [12]. On addition of the protein, new resonance lines corresponding to the bound mim12 dodecamer appear. Some of these lines are split into two signals which indicate the simultaneous presence of two forms. The lifetimes of these two forms are longer than the inverse of the frequency difference between the free and bound state resonances. Chemical shifts are very sensitive probes of the local environments of the nucleus but unfortunately it is not possible to predict their values from the conformation of the complex or conversely to deduce the conformation from their values. Nevertheless, they are useful parameters to gain insight into the parts of the molecules in¯uenced by the interaction. Schmiedeskamp and coworkers [13] have shown by analysis of 1 H and 13Ca chemical shifts that little change in the structure of the zinc-®nger domain from the yeast transcription factor ADR1 occurs upon binding to a 14mer DNA containing the UAS half site. A correlation between the protein±DNA interface mapped by chemical shift changes and that mapped by mutagenesis experiments was found. However, the identi®cation of the DNA binding site using DNA induced chemical shift changes should be done with care. This approach is not feasible for numerous protein±DNA complexes where proteins undergo conformational transitions and dynamics changes upon binding that will affect the chemical shifts. This has been recently demonstrated by Foster and coworkers [14]. These authors analyzed the correlation between the chemical shift changes upon 86 N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 Fig. 2. Imino region of the 1 H-NMR 600 MHz spectra obtained upon addition of different amount of a solution of the R2R3 DNA binding domain of c-Myb to a solution of mim12 oligonucleotide at 208C