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Mutagenesis Analysis of the Interaction Between the Dorsal Rel Homology Domain and HMG Boxes of DSP1 Protein

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Mutagenesis Analysis of the Interaction Between the Dorsal Rel Homology Domain and HMG Boxes of DSP1 Protein J. Biochem. 134, 583-589 (2003) DOI: 10.1093/jb/mvgl77 Mutagenesis Analysis of the Interaction between the Dorsal Rel Homology Domain and HMG Boxes of DSP1 Protein Davy Martin, Anne Daulny, Martine Decoville and Daniel Locker* Centre de Biophysique Moleculaire, CNRS, conventionne avec l'Universite d'Orleans , rue Charles Sadron, 45071 Orleans cedex 2, France Received June 6, 2003; accepted August 4, 2003 DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various trun cated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region (253KKRK256) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered. Key words: Dorsal, Drosophila, DSP1, HMGB, protein-protein interaction. HMGB1 proteins are an abundant class of mammalian The Drosophila Dorsal switch protein (DSP1) is a chromatin proteins, present in all vertebrate nuclei. member of the HMGB1/2 family (10, 11). Like mamma HMGB1 proteins are characterized by three structural lian HMGB1 proteins, it has two HMG boxes and a C domains: the N-terminal A domain, the central B-domain terminal acidic tail, but also contains an additional N and the terminal C-domain. The two domains A and B terminal glutamine-rich region. The two HMG boxes of comprise 75-80 residues, named box A and box B respec DSP1 are very similar to the two HMG boxes of HMGB1/ tively, with about 30% similarity in amino acid sequence. 2 (12, 13). In addition, the dspl and Hmgbl genes NMR data showed that boxes A and B have a similar fold revealed a highly conserved structure with similar exon with minor differences, in which three alpha helical seg intron boundaries, suggesting that they derive from a ments form an L-shaped structure stabilized by a hydro common ancestor. DSP1 was first isolated as a corepres sor of the Dorsal protein, but it seems to have additional phobic core (1-3). HMGB1 proteins display structure specific rather than sequence specific DNA binding. functions during drosophila development, as absence of DSP1 causes homeotic transformations. Although they bind to double-stranded DNA and facili tate the circularization of DNA restriction fragments, Dorsal belongs to the Rel family of transcription fac they interact with higher affinity with DNA already con tors. This family has been implicated in different physio logical and cellular processes, such as immunity, inflam taining a bend, such as four-way junctions and cisplatin mation, oncogenesis, apoptosis, and embryonic modified DNA (4, 5). development (reviewed in Ref. 14). The members of this They also have the property to distort the DNA struc family are characterized by a conserved 300 amino acid ture quite dramatically by introducing sharp bends upon region, the Rel homology domain (RHD), located at the binding. It is worthy of note that a recurring function pro N-terminus (reviewed in Ref. 15). The RHD domain posed for HMGB1 is the enhancement of the binding of mediates DNA binding, homodimer and heterodimer for various transcription factors like Hox protein (6), p53 (7) mation and contains the nuclear localization signal. Dur or steroid hormone receptors (8). Impaired glucocorticoid ing drosophila embryonic development, Dorsal can act as receptor binding has been provided as an explanation for a transcriptional activator or a repressor depending on the pleiotropic effects on glucose metabolism observed in the promoter content. HMGB1-knock-out mice (9). These observations suggest In a recent study (16), we have shown by affinity chro that HMGB1 could be implicated in the formation of matography and far-western experiments that DSP1 and nucleoprotein complexes that play a role in transcription. Dorsal interact with each other in vitro. Analysis of the Despite numerous studies, it is not clear whether the dif binding of several truncated DSP1 proteins has revealed ferent domains of HMGB1 participate in recognition of two facts: firstly, the HMG boxes of DSP1 are necessary specific proteins. for the interaction; secondly, the basic residues between the two boxes are a prerequisite for the interaction. We have also shown by electrophoretic mobility shift assay that Dorsal binding to DNA is significantly increased by * To whom correspondence should be addressed. Tel: +33-2-38-25-55 82, Fax: +33-2-38-63-15-17, E-mail: [email protected] the presence of DSP1. Vol. 134, No. 4, 2003 583 c 2003 The Japanese Biochemical Society. 584 D. Martin et al. Table 1. Primers" used for construction of DSP1 Dorsal RHD expressing plasmids and DSP1 mutants. *Primers are used on DSP1-162 , F33 or J11 as template except for the quadruple mutant (K253AK254AR255AK256A) where we used the K253A K254A plasmid as template. In order to investigate the mode of interaction between ter refers to the amino acid in the wild-type protein, the Dorsal and DSP1, we undertook a mutational analysis of number refers to the position of this amino acid in the DSP1. Our choice of mutations predated the release of full-length protein DSP1, and the second letter refers to the interaction data between Dorsal and DSP1, notably the amino acid introduced in the mutant (Fig. 1). All con on the fact that HMG boxes of DSP1 are necessary but structions were verified by sequencing. For expression of not sufficient for the interaction. In a mutational analy the proteins, the E. coli strain BL21 (DE3) was trans sis (17), it was reported that mutation of the tryptophan formed by the plasmids. residue in the A domain of HMGB1 affects the protein Purification of Recombinant Proteins-Purification of tertiary structure. We have used this property to obtain the various DSP1 proteins from E. coli crude extract was DSP1 proteins with one or two unfolded domains. In this performed as described elsewhere (18) work, we have compared the interaction between Dorsal Far-Western Experiments-Far-western experiments and various mutated forms of DSP1 (W212 W302 substi were performed as described in (16). Synthesis of radiola tuted by arginine or 253KKRK256substituted by alanine) . belled Dorsal RHD was performed using a pGEM3Zf(+) Our results strongly suggest that mutations altering the clone as template and the TNT coupled reticulocyte folding of the HMG domains altered the protein/protein lysate system (Promega) in the presence of [35S]methio interactions and the stimulation of Dorsal DNA binding . nine. Electrophoretic Mobility Shift Assay (EMSA)-Two MATERIALSAND METHODS 43mer oligonucleotides corresponding to Dorsal strong binding site were purified on a 12% polyacrylamide dena Cloning-The Dorsal RHD coding sequence was ampli turing gel (19:1 acrylamide:bisacrylamide). The corre fied by PCR using the pTrcHisB Dorsal vector (16) as sponding top strands were end-labeled using [ƒÁ-32P]ATP template and the primers described in Table 1. The PCR and T4 kinase and hybridized with their complementary product was inserted into pGEM-3Zf(+) vector using Hin strands. The double-stranded species were purified on a dIII site. The Dorsal RHD coding sequence was excised 6% polyacrylamide gel followed by electroelution. Their using the NdeI and BclI sites and inserted into pET-5a sequences are as follows (the underlined sequences corre vector using NdeI and BamHI sites. For expression , E. spond to the Dorsal binding site): coli BL21 (DE3) strain was transformed by the plasmid . d12 5•Œ-ATCCTCTATAACTGGGAGAAACCCAATCAAT The constructions were verified by sequencing . ATTCGTTCACCC-3 Construction of the plasmids expressing DSP1-162 or 3•Œ-TAGGAGATATTGACCCTCTTTCGGTTAGTTATA DSP1 mutant proteins with different deletions has been AGCAAGTGGG-5•Œ described elsewhere (16) (18). Site-directed mutagenesis DNA (1.3nM) was incubated with increasing quanti was carried out with the corresponding pET 5a DSP1-162 ties of the Dorsal RHD for 15 min on ice in a total reac J11 and F33 plasmids and the appropriate primers tion volume of 10ƒÊl containing 50mM Tris-HCl pH 8, (Table 1), using the QuickChangeTM Site-directed Muta 150mM NaCl, 1mM DTT, 1mM EDTA, 100ƒÊg/ml BSA genesis kit (Stratagene). The name of the mutants was (binding buffer). To this was added 2ƒÊl of 15% Ficoll with chosen according to the following guidelines: the first let- dyes, and the mixture was loaded on a 6% polyacrylamide J. Biochem. Correctly Folded HMG Boxes Are Necessary for Dorsal-DSP1 Interactions 585 Fig. 2. Interaction between Dorsal and mutants DSP1 pro teins monitored by far-western experiment. (A) Coomassie stained SDS-PAGE of the purified proteins. (B) Phosphorimager scan of the membrane after transfer of the proteins and incubation with radiolabelled Dorsal RHD. Lane 1, DSPl-162 W212R W302R ; lane 2, DSPJ-162 W21211; lane 3, DSPJ-162 W302R ; lane 4, DSPJ-162 K253A K254A R255A K256A ; lane 5, DSPJ-162 ; lane 6, molecular weight marker. RESULTS Binding of the Different DSP1 Domains to Dorsal RHD Protein-In a previous work, we have shown that the Dorsal DSP1 interaction was mediated by the HMG boxes of DSP1.
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