! ! CHAPTER 5: RESULTS and DISCUSSION (Biochemical Studies) 5.1 ID2 protein activity To ensure that the purified ID2 protein was functionally active, competitive electromobility shift assays (EMSAs) were performed to look for loss of binding of a Group A bHLH transcription factor to its cy5-labeled DNA probe with an increasing concentration of ID2. E47, a Group A bHLH transcription factor and known binding partner of ID2 was cloned, expressed and purified (Appendix 3) for use in these studies. E47 bound an E-box element found in the DNA sequence of the MCK promoter containing the motif (CANNTG). The sequence GGATCCCCCCAACACCTGCTGCCTGA was ordered as cy5-labeled forward and reverse probes and annealed in a thermal cycler. Negative controls were used to ensure that ID proteins did not bind DNA (Figure 22A) and a mutant e-box element showed that E47 only bound to the wild-type e-box element (Figure 22B). Figure 22: EMSA controls (A) ID proteins did not bind e-box DNA, only E47, a bHLH transcription factor bound to DNA (B) bHLH transcription factors (E47, Mash1, MYOD1) did not bind to mutant e-box DNA. The ID proteins did not bind the mutant e-box probe. ! ! 64! ID2 at different concentrations was incubated with E47 for 10 mins at room temperature in EMSA binding buffer. Cy5-labeled DNA was added and allowed to compete with ID2 for E47 binding for another 15 mins at room temperature to a final reaction volume of 20 µl. The mixtures were loaded on a native gel and scanned for cy5 intensity. Results showed that the ID2 N-HLH82-L protein used in the crystallization experiments was successful in binding E47 and was therefore functionally active (Figure 23). Figure 23: EMSA 6% native gel showing that increasing concentration of ID2 inhibited E47 binding to DNA. Lanes without ID2 (lanes and 2) denoted by “-“. Number of “+” denoted relative concentration of ID2 added. All lanes contained μM E47. This showed that the purified ID2 used for crystallization was active. ! ! ! 65! 5.2 ID heterodimer binding specificity and affinity Although the ID2 and ID3 structures revealed a very similar fold to other classes of bHLH-containing proteins such as E47, MYOD1, NeuroD1 and MAX, previous studies showed that the ID proteins did not bind to any class of bHLH-containing proteins. Instead, they preferentially bound to Group A bHLH-containing proteins. Previous reports showed that ID1 and ID2 bound to E47 and MYOD1 (Group A). Using EMSAs, it was found that ID1, ID2 and ID3 all bound to E47 (Figure 24). ID1 and ID2 but not ID3 bound weakly to MYOD1 (Figure 25). This confirmed the reported observations by Langlands (Langlands, et al., 1997). The E47-MYOD1 heterodimer EMSA experiment (Figure 26) showed the same binding pattern as with E47 and MYOD1 homodimers. This affirmed that IDs preferentially bound E47 and likely inhibited MYOD1 indirectly by binding to MYOD1’s functional dimeric partner. It was clear from the EMSA that MYOD1 preferentially bound E47 than itself and confirmed MYOD1’s weak homodimerization capablities. Figure 24: EMSA 6% native gel showing the different ID-HLH binding affinities to 0.05 µM human E47. Residues for each human ID protein given in parentheses. “+” denoted presence of E47. All lanes contained 200nM DNA. Concentrations of each ID protein provided in the table above the gel. All ID proteins bound E47 to varying degrees. ! ! ! ! 66! Figure 25: EMSA 6% native gel showing the different ID-HLH binding affinities to 0.2 µM human MYOD1 (tagged with His-MBP). Residues for each human ID protein given in parentheses. “+” denoted presence of MYOD1. All lanes contained 100nM DNA. Concentrations of each ID protein provided in the table above the gel. ID1 and ID2 showed weak interactions with MYOD1 where a large fraction seemed to form an intermediate rather than complete inhibition. ID3 did not bind MYOD1. ! ! ! ! ! Figure 26: EMSA 6% native gel showing the different ID-HLH binding affinities to 0.2 µM human MYOD1 (tagged with His-MBP) heterodimerized with E47 (0.05µM). Residues for each human ID protein given in parentheses. “+” denotes presence of MYOD1 and/or E47. All lanes contained 200nM DNA. Concentrations of each ID protein provided in the table above the gel. MYOD1 had high propensity to bind E47. All IDs showed the same binding pattern as seen in Figures 24 and 25. ! 67! 5.3 ID helix-1 residues in binding specificity Since IDs shared an average 80% identity across the HLH domain, differences between ID2 and ID3 had to account for the differential binding to MYOD1. Langlands had proposed that three residues in helix-1 were important for binding specificity using a mammalian-2-hybrid system (Langlands, et al., 1997). In the ID2 structure, these three residues pointed away from the dimer interface (Figure 21). The only residue that could potentially be affected was the lysine at position 47 because of its interaction with the positive ion in the loop. To find out if these residues alone played a role in binding specificity, equivalent human positions from the Langlands paper were mutated in ID2 as follows Y37D (Y in ID1), D41H (G in ID1), K47R (K in ID1) as well as a double mutant Y37D-D41H. The expectation was that binding to E47 would not change but binding to MYOD1 (Appendix 3) would be affected. Using EMSAs and comparing binding of these mutants to E47 (Figure 27) and MYOD1 (Figure 28), it was found that there were no significant differences and they all bound like wild-type ID2. As with the previous EMSA for wild-type ID proteins, the mutants showed similar binding patterns for the MYOD1-E47 heterodimer as with MYOD1 and E47 homodimers (Figure 29). Hence, these residues were unlikely to confer binding specificity of the different ID proteins as was hypothesized from the ID2 and ID3 structures. ! 68! Figure 27: EMSA 6% native gel showing ID2 helix-1 mutants binding affinities to 0.2 µM human E47. “+” denotes presence of E47. All lanes contained 100nM DNA. Concentrations of each ID protein provided in the table above the gel. All mutants bound to E47. ! ! ! ! ! Figure 28: EMSA 6% native gel showing ID2 helix-1 mutants binding affinities to 0.2 µM human MYOD1 (HisMBP tagged). “+” denotes presence of MYOD1. All lanes contained 100nM DNA. Concentrations of each ID protein provided in the table above the gel. All ID2 helix-1 mutants bound to MYOD1 weakly just like wild-type ID2. ! ! 69! Figure 29: EMSA 6% native gel showing ID2 helix-1 mutants binding affinities to 0.2 µM human MYOD1 (HisMBP tagged) heterodimerized with 0.2 µM E47. “+” denotes presence of MYOD1 and/or E47. All lanes contained 100nM DNA. Concentrations of each ID protein provided in the table above the gel. IDs bound with similar affinities as with the E47 and MYOD1 homodimers. ! 70! 5.4 Exploring other differences in ID residues Other residues where ID1 and differed from ID3 were at ID2 positions Q55, N56, K58, K61 and H67. Of these, Q55 and K61 had associated interactions in the structure; Q55 to the ion and K61 as an intra-chain hydrogen bond to N40. The latter bond was also found in the E47 homodimer at N353-K375 and was not lost in the heterodimeric structure, E47-NeuroD1, at E47.N559-E47.K581. Mutations were made to these residues to change their size as well as mirror the opposing ID protein. ID2 mutations were Q55A, Q55R, K61A, K61Q and corresponding ID3 mutations were R60A, R60Q, Q66A and Q66K. Of these mutations, ID2.K61A, ID3.Q66K and ID3.Q66A did not produce soluble protein (Figure 17), suggesting a requirement for this bond to form a stable monomer. ID2.K61Q had expression similar to wild type and bound both E47 (Figure 30, top gel) and MYOD1 (Figure 30, bottom gel) that was similar to wt-ID2. Finally, a deletion at ID1.Q78 (ID2.Q55) caused partial binding loss to MYOD1 but not to E47 (Pesce, et al., 1993). It was found that ID2.Q55A and ID2.Q55R showed partial binding loss to both MYOD1 (Figure 31, bottom gel) and E47 (Figure 31, top gel). We postulate that the alanine causes a loss of one of the interactions to the positive ion and arginine would add a repelling force at that position. The reverse mutation in ID3 was striking. ID3.R60A and ID3.R60Q had higher protein solubility than wildtype, suggesting added stability. All previous experiments were done with the solubility tag, His-MBP attached to ID3. However, these mutants were soluble without the tag and EMSAs were performed with these untagged ID3 mutants. It was found that they bound better to E47 than wt-ID3, both with (Figure 32) and without (Figure 33) the solubility tag. R60A made no difference to MYOD1 binding. But R60Q showed partial binding to MYOD1. ! 71! Pesce and Benezra had shown that the loop of ID1 was not as flexible and was important for specificity (Pesce, et al., 1993). It is believed that this is also true for ID2. These results show that an ionic interaction in the loop makes for a more rigid loop that when disrupted, causes a conformational change that is not conducive to heterodimerization. The hypothesis is that ID1 and preferentially bind other bHLHcontaining proteins with a similar loop formation. Further, the arginine at position 60 in the loop of ID3 could have implications for its preference to different binding partners than ID1 and ID2. Figure 30: EMSA 6% native gels showing ID2 loop region mutants. wt = wild-type ID2, E47 concentration=100nM, DNA concentration=100nM, MYOD1 concentration=200nM. 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(2004) "Id4 regulates neural progenitor proliferation and differentiation in vivo", Development, 131, 5441-8 140. Zhu, W., Dahmen, J., Bulfone, A., Rigolet, M., Hernandez, M. C., Kuo, W. L., Puelles, L., Rubenstein, J. L. and Israel, M. A. (1995) "Id gene expression during development and molecular cloning of the human Id-1 gene", Brain Res Mol Brain Res, 30, 312-26 ! 96! ! LIST OF PUBLICATIONS 1. Wong, M. V., Jiang, S., Palasingam, P. and Kolatkar, P. R. (2012) "A Divalent Ion Is Crucial in the Structure and Dominant-Negative Function of ID Proteins, a Class of Helix-Loop-Helix Transcription Regulators", PLoS One, 7, e48591 2. Wong, M. V., Palasingam, P. and Kolatkar, P. R. (2012) "Cloning, purification and preliminary X-ray data analysis of the human ID2 homodimer", Acta Crystallogr Sect F Struct Biol Cryst Commun, 68, 1354-8 ! 97! Appendix 1: Protein Sequences (Human) ! ID2 sequence (residues 1-134) with methionines (that were substituted for selenium) in red, green G left over after TEV cleavage, HLH domain in bold: GMKAFSPVRSVRKNSLSDHSLGISRSKTPVDDPMSLLYNMNDCYSKLKELVPSIPQ NKKVSKMEILQHVIDYILDLQIALDSHPTIVSLHHQRPGQNQASRTPLTTLNTDISILS LQASEFPSELMSNDSKALCG MYOD1 sequence (residues 1-320) with HLH domain in bold: ! MELLSPPLRDVDLTAPDGSLCSFATTDDFYDDPCFDSPDLRFFEDLDPRLMHVGALL KPEEHSHFPAAVHPAPGAREDEHVRAPSGHHQAGRCLLWACKACKRKTTNADRR KAATMRERRRLSKVNEAFETLKRCTSSNPNQRLPKVEILRNAIRYIEGLQALLRDQ DAAPPGAAAAFYAPGPLPPGRGGEHYSGDSDASSPRSNCSDGMMDYSGPPSGAR RRNCYEGAYYNEAPSEPRPGKSAAVSSLDCLSSIVERISTESPAAPALLLADVPSES PPRRQEAAAPSEGESSGDPTQSPDAAPQCPAGANPNPIYQVL E47 sequence (residues 1-651) with HLH domain in bold: ! MNQPQRMAPVGTDKELSDLLDFSMMFPLPVTNGKGRPASLAGAQFGGSGLEDRP SSGSWGSGDQSSSSFDPSRTFSEGTHFTESHSSLSSSTFLGPGLGGKSGERGAYA SFGRDAGVGGLTQAGFLSGELALNSPGPLSPSGMKGTSQYYPSYSGSSRRRAADG SLDTQPKKVRKVPPGLPSSVYPPSSGEDYGRDATAYPSAKTPSSTYPAPFYVADGS LHPSAELWSPPGQAGFGPMLGGGSSPLPLPPGSGPVGSSGSSSTFGGLHQHERM GYQLHGAEVNGGLPSASSFSSAPGATYGGVSSHTPPVSGADSLLGSRGTTAGSSG DALGKALASIYSPDHSSNNFSSSPSTPVGSPQGLAGTSQWPRAGAPGALSPSYDGL HGLQSKIEDHLDEAIHVLRSHAVGTAGDMHTLLPGHGALASGFTGPMSLGGRHAGL VGGSHPEDGLAGSTSLMHNHAALPSQPGTLPDLSRPPDSYSGLGRAGATAAASEIK REEKEDEENTSAADHSEEEKKELKAPRARTSSTDEVLSLEEKDLRDRERRMANNA RERVRVRDINEAFRELGRMCQMHLKSDKAQTKLLILQQAVQVILGLEQQVRERNL NPKAACLKRREEEKVSGVVGDPQMVLSAPHPGLSEAHNPAGHM ID1 sequence (residues 1-155) with HLH domain in bold: MKVASGSTATAAAGPSCALKAGKTASGAGEVVRCLSEQSVAISRCAGGAGARLPAL LDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNSE SEVGTPGGRGLPVRAPLSTLNGEISALTAEAACVPADDRILCR ID3 sequence (residues 1-119) with HLH domain in bold: MKALSPVRGCYEAVCCLSERSLAIARGRGKGPAAEEPLSLLDDMNHCYSRLRELVP GVPRGTQLSQVEILQRVIDYILDLQVVLAEPAPGPPDGPHLPIQTAELTPELVISNDK RSFCH ! ! 98! Appendix 2: Purified proteins used in EMSA studies ! ! Figure 36: SDS-PAGE 4-12% gels showing proteins used in EMSA studies in Chapter 5. Marker in kDa (lane M), U = before induction. Gel A & B are the ID2 helix-1 mutants. Gel C is ID1-HLH, Gel D is His-MBP-ID3 fusion protein. Gel E is E47. Gel F has both the fusion MYOD1 as well as untagged MYOD1. ! ! ! ! 99! Appendix 3: E47 & MYOD1 cloning, expression and purification for EMSA studies ! Table 12: Constructs created for use in protein expression for E47 and MYOD1 human proteins showing their theoretical biochemical properties estimated by ProtParam(Wilkins, et al., 1999) Construct E47-bHLH MYOD1-bHLH cDNA (bp) 180 192 AA start 545 102 AA end 606 166 AA 62 64 Isoelectric Pt 11.10 11.36 MW (Da) 7430.6 7554.7 Ext coefficient 1490 ! ! A3.1 Cloning E47 HLH (residues 545-606) & MYOD1 HLH (residues 102-166) constructs were cloned from gene synthesized inserts (1st base) using Invitrogen’s Gateway cloning technology as per manufacturer’s instructions. Forward & reverse primers used for PCR amplification that included the attB sites for recombination into the entry clone as well as a Tobacco Etch Virus (TEV) protease cleavage site at the N-terminus were as follows 5’ GGGGACAAGT TTGTACAAAAAAGCAGGCTTCGAAAACCTGTATTTTCAGGGCCGGGAGAGGCGC ATGGCCAATAACG 3’, 5’ ggggaccactttgtacaagaaagctgggttTTATTACCGCTCTCGCACCTGCTG 3’, 5’GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAAACCTGTATTTTCAGGGCC GCAAGACCACCAACGCCGAC 3’, 5’ ggggaccactttgtacaagaaagctgggttTTATTACTGGTCGCGCAGCAGAGC 3’ respectively. BP reactions (Invitrogen) using the PCR products were recombined with vector pDONR221 & transformed into Top10 competent Escherichia coli cells (Invitrogen) to create an entry clone that was subsequently subcloned via the LR reaction (Invitrogen) into several expression vectors. In this case, the optimized expression vector was pDest-HisMBP which contains an N-terminal His-MBP tag. Inserts were confirmed by sequencing. ! 100! A3.2 Protein production The 6His-MBP-Tev-E47 & 6His-MBP-TEV-MYOD1 expression plasmids were transformed into OneShot competent Escherichia coli (DE3) cells (Invitrogen). The E.coli cells were cultured in 5L of Luria broth (LB) containing 100µg/mL Ampicillin in a shaker/incubator at 37°C until an OD600 of 0.7 was reached. The cultures were induced with 0.2mM IPTG and allowed to grow at 17°C for 18 hours. A3.3 Cells Harvesting Cells were harvested by ultracentrifugation in Nalgene plastic 50ml tubes at 11,952 g in a Sorvall SS-34 rotor for 10 at 4°C. The pellets were resuspended in cold lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM Imidazole) and ultrasonicated for mins at 30% amplitude (2 seconds on, seconds off) on ice. The supernatant was filtered through a 0.22 µm membrane after ultracentrifugation for hr at 36,603 g in a Sorvall SS-34 rotor, 4°C to remove any cell debris in preparation for purification. A3.4 Protein purification Protein purification was performed on the Akta Express system for all steps. The first step involved affinity chromatography using nickel beads (5mL HisTrap columns from GE Healthcare) equilibrated in lysis buffer to capture all 6His-tagged fusion proteins from crude lysate. The peak elutions (buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 300 mM Imidazole) were immediately desalted to 50 mM Tris-HCl pH 8.0,100 mM NaCl buffer by dilution with 50 mM Tris-HCl pH 8.0. To distinguish between the proteins, only E47 was subjected to protease cleavage by mixing with 1:100 (wt/wt) TEV protease at room temperature for hrs. ! 101! 6mL Resource-S (GE Healthcare) ion-exchange chromatography was performed in an increasing salt gradient up to 1M NaCl on the protein mixture to separate the fusion tag from the protein for E47 & to further purify the fusion protein for MYOD1. The eluted protein fractions were pooled and concentrated using a membranebased concentrator with a 3000 Da MW cutoff (Vivaspin, Sartorius). Buffer exchange whilst concentrating was done to ensure a salt content of 100mM NaCl or less. The Bradford assay was used to quantitate protein concentration as per manufacturer’s instructions. 20µL aliquots of each protein of 90% purity and higher were stored at 80°C for use in EMSAs (Electromobility shift assays). ! 102! Appendix 4: Summary of expression and purification protocols for ID mutants ! Expression and affinity chromatography followed the same protocol as native ID2 protein expression in Section 2: materials and methods. The following summary gives the differences in the expression vector used and the type of ion exchange chromatography used. ! Table 13: Changes to ID2 protocol for expression and purification of ID2 and ID3 mutants Construct ID2-D41H ID2-D41G ID2-Y37D ID2-Y37D_D41H ID2-Y37D_D41G ID2-Q55A ID2-Q55R ID2-Q55A_K61A ID3-R60A ID3-R60Q ID3-R60A ID3-R66K Expression vector pDest-565 pDest-565 pDest-565 pDest-565 pDest-565 pDest-565 pDest-565 pDest-565 pDest-HisMBP pDest-HisMBP pDest-HisMBP pDest-HisMBP Ion exchange column Resource-S cation exchanger Resource-S cation exchanger Resource-S cation exchanger Resource-S cation exchanger Resource-S cation exchanger Resource-S cation exchanger Resource-S cation exchanger Resource-S cation exchanger Resource-Q anion exchanger Resource-Q anion exchanger Resource-Q anion exchanger Resource-Q anion exchanger ! ! 103! Appendix 5: ID1 & ID3 cloning, expression and purification Table 14: ID1 & ID2 constructs and their theoretical biochemical properties estimated by ProtParam(Wilkins, et al., 1999) ID1-HLH cDNA (bp) 180 AA start 58 AA end 112 ID3-HLH 261 87 Construct 55 Isoelectric Pt 4.94 87 5.75 AA 6501.4 Ext coefficient 4470 9579.1 4470 MW (Da) Cloning primers for BP reaction (Invitrogen) were as follows: ID1 –HLH forward primer 5’ GGGGACAAGT TTGTACAAAA AAGCAGGCTT CGAAAACCTG TATTTTCAGG GC ATGGACGAGCAGCAGGTAAACGTG 3’, reverse primer 5’ ggggaccactttgtacaagaaagctgggttTTA TCATTCCGAGTTCAGCTCCAAC 3’. ID3-HLH forward primer 5’ GGGGACAAGT TTGTACAAAA AAGCAGGCTT CGAAAACCTG TATTTTCAGG GC ATGAAGGCGCTGAGCCCGGTGCGC 3’, reverse primer 5’ ggggaccactttgtacaagaaagctgggttTTA TCATGGCTCGGCCAGGACTACCTGCAG 3’. Differences in protein expression and purification are given here. All protocols follow the ID2 native protein expression and purification protocols set out in Chapter with the following exceptions in Table 15: Table 15: Changes to ID2 protocol for expression and purification of ID1 and ID3 HLH domains Construct ID1-HLH ID3-HLH ! Expression vector pDest-HisMBP pDest-HisMBP Ion exchange column Resource-Q anion exchanger Resource-Q anion exchanger 104! Appendix 6: ID2 as a dimer in solution. Gel filtration profile ! FPLC!machine:!Akta!Express! Column:!HiPrep™!16/60!Superdex0200! Injection!volume:!5!ml! Elution!buffer:!50!mM!TrisNHCl!pH8.0,!100!mM!NaCl! Flow!rate:!0.5!ml/min! Column!equilibrated!in!50!mM!TrisNHCl!pH8.0,!100!mM!NaCl,!150mLs! ! Calibration!markers:! ! Sample! Thyroglobulin! Ferritin! Catalase! Ovalbumin! Carbonic!anhydrase! Cytochrome!C! Aprotinin! M r! 669!000! 440!000! 67!000! 43!000! 29!000! 13!600! 6!512! Elution!peak!(ml)! 41! 49! 65! 71! 77! 81! 100! ! ! SDSNPAGE:!12%!SDS!gel,!200V,!40mins! ! ! ! ! ! Lanes:!ID2!HLH+14aa,!SN200.!Each!lane!is!a! sample!from!a!fraction!from!the!elution!profile! ! 1!=!D4! 2!=!E1! 3!=!E3! 4!=!E6! 5!=!E12! 6!=!F8! 7!=!F5! L!=!1kb!NEB!prestained!protein!ladder!P7708,! molecular!weight!labels!in!kDa! 105! Appendix 7: ID2 coordinates ! The coordinates for ID2 HLH crystal structure was deposited in the PDB with identification: 4AYA ! ! 106! [...]... O., Zhuang, Y., Manova, K and Benezra, R ( 199 9) "Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts", Nature, 401, 670-7 78 Ma, P C., Rould, M A., Weintraub, H and Pabo, C O ( 199 4) "Crystal structure of MyoD bHLH domain -DNA complex: perspectives on DNA recognition and implications for transcriptional activation", Cell, 77, 451 -9 79 Mantani, A., Hernandez,... 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B-cell factor (EBF) family of transcription factors reveals similarities to Rel DNA- binding proteins and a novel dimerization motif", J Biol Chem, 285, 25875 -9 120 Sun, X H ( 199 4) "Constitutive expression of the Id1 gene impairs mouse B cell development", Cell, 79, 893 -90 0 121 Sun, X H and Baltimore, D ( 199 1) "An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but . T. and Walker, M. D. ( 199 1) "Distribution and characterization of helix-loop-helix enhancer -binding proteins from pancreatic beta cells and lymphocytes", Nucleic Acids Res, 19, 3 893 -9. ID1, ID2 and ID3 all bound to E47 (Figure 24). ID1 and ID2 but not ID3 bound weakly to MYOD1 (Figure 25). This confirmed the reported observations by Langlands (Langlands, et al., 199 7). The. differential binding to MYOD1. Langlands had proposed that three residues in helix-1 were important for binding specificity using a mammalian-2-hybrid system (Langlands, et al., 199 7). In the