The Solution Structure of DNA-free Pax-8 Paired Box Domain Accounts for Redox Regulation of Transcriptional Activity in the Pax Protein Family

Pax-8 is a transcription factor belonging to the PAX genes superfamily and its crucial role has been proven both in embryo and in the adult organism. Pax-8 activity is regulated via a redoxbased mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys45 and Cys57). These residues belong to a highly evolutionary conserved DNA binding site: the Paired Box (Prd) domain. Crystallographic protein-DNA complexes of the homologues Pax-6 and Pax-5 showed a bipartite Prd domain consisting of two helix-turn-helix (HTH) motifs separated by an extended linker region. Here, by means of nuclear magnetic resonance, we show for the first time that the HTH motifs are largely defined in the unbound Pax-8 Prd domain. Our findings contrast with previous induced fit models, in which Pax-8 is supposed to largely fold upon DNA binding. Importantly, our data provide the structural basis for the enhanced chemical reactivity of residues Cys45 and Cys57 and explain clinical missense mutations that are not obviously related to the DNA binding interface of the paired box domain. Finally, sequence conservation suggests that our findings could be a general feature of the Pax family transcription factors. Pax-8 is a transcription factor belonging to the PAX genes superfamily and its crucial role has been proven both in embryo and in the adult organism. Pax-8 activity is regulated via a redoxbased mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys45 and Cys57). These residues belong to a highly evolutionary conserved DNA binding site: the Paired Box (Prd) domain. Crystallographic protein-DNA complexes of the homologues Pax-6 and Pax-5 showed a bipartite Prd domain consisting of two helix-turn-helix (HTH) motifs separated by an extended linker region. Here, by means of nuclear magnetic resonance, we show for the first time that the HTH motifs are largely defined in the unbound Pax-8 Prd domain. Our findings contrast with previous induced fit models, in which Pax-8 is supposed to largely fold upon DNA binding. Importantly, our data provide the structural basis for the enhanced chemical reactivity of residues Cys45 and Cys57 and explain clinical missense mutations that are not obviously related to the DNA binding interface of the paired box domain. Finally, sequence conservation suggests that our findings could be a general feature of the Pax family transcription factors. Pax-8 is an important eukaryotic transcription factor that is responsible, during embryogenesis, for the differentiation of several organs such as kidney, thyroid, and neural tube. Pax-8 is also essential in the adult organism, where it activates thyroid hormone production. During development, Pax-8 functionality is regulated by alternative splicing, generating isoforms that exhibit different transactivation properties but maintain unaltered the DNA binding domain (1Kozmik Z. Kurzbauer R. Dörfler P. Busslinger M. Mol. Cell. Biol. 1993; 13: 6024-6035Crossref PubMed Scopus (143) Google Scholar, 2Poleev A. Fickenscher H. Mundlos S. Winterpacht A. Zabel B. Fidler A. Gruss P. Plachov D. Development. 1992; 116: 611-623Crossref PubMed Google Scholar). In mature thyroid follicular cells, the complete Pax-8 splicing isoform regulates the expression of thyroglobulin, thyroperoxidase (3Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (275) Google Scholar, 4Espinoza C.R. Schmitt T.L. Loos U. J. Mol. Endocrinol. 2001; 27: 59-67Crossref PubMed Scopus (43) Google Scholar), sodium/iodide symporter, and thyrotropin receptor genes (5Ohno M. Zannini S. Levy O. Carrasco N. Di Lauro R. Mol. Cell. Biol. 1999; 19: 2051-2060Crossref PubMed Scopus (220) Google Scholar, 6Damante G. Di Lauro R. Biochim. Biophys. Acta. 1994; 1218: 255-266Crossref PubMed Scopus (195) Google Scholar, 7Santisteban P. Bernal J. Rev. Endocr. Metab. Disord. 2005; 6: 217-228Crossref PubMed Scopus (59) Google Scholar). Pax-8 activity is co-regulated by thyroid transcription factor-1, thyroid transcription factor-2, and Hex, indicating an active role in the early commitment and differentiation of thyrocytes (8Pasca di Magliano M. Di Lauro R. Zannini M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13144-13149Crossref PubMed Scopus (203) Google Scholar, 9Lang D. Powell S.K. Plummer R.S. Young K.P. Ruggeri B.A. Biochem. Pharmacol. 2007; 73: 1-14Crossref PubMed Scopus (225) Google Scholar). The Pax family shares a bipartite functionality consisting of a N-terminal binding region and a C-terminal transactivation region. The N-terminal region is usually comprised of three domains, namely a Paired Box (Prd) domain, a conserved octapeptide, and a further homeodomain (see Fig. 1). Differences in conservation and functionality in these domains have been used as evolution markers for the Pax family (10Balczarek K.A. Lai Z.C. Kumar S. Mol. Biol. Evol. 1997; 14: 829-842Crossref PubMed Scopus (77) Google Scholar), which has been subdivided into four groups according to the sequence homology in the N-terminal region. In particular, the N-terminal region of Pax-8 is composed by all three domains but presents an incomplete and inactive homeodomain. The Prd domain consists of a well conserved 128-residue-long region, formed by two distinct subdomains known in literature as PAI (N-terminal) and RED (C-terminal) (11Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar). DNA binding studies have demonstrated that the two subdomains of the Pax-8 Prd domain bind DNA independently (12Epstein J.A. Glaser T. Cai J. Jepeal L. Walton D.S. Maas R.L. Genes Dev. 1994; 8: 2022-2034Crossref PubMed Scopus (317) Google Scholar), are both required for proper promoter activation (13Pellizzari L. Tell G. Damante G. Biochem. J. 1999; 337: 253-262Crossref PubMed Google Scholar), and that binding is redox state dependent (14Kambe F. Nomura Y. Okamoto T. Seo H. Mol. Endocrinol. 1996; 10: 801-812PubMed Google Scholar, 15Tell G. Scaloni A. Pellizzari L. Formisano S. Pucillo C. Damante G. J. Biol. Chem. 1998; 273: 25062-25072Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Although both subdomains contain cysteine residues, it was demonstrated both in vivo and in vitro that PAI subdomain DNA-binding activity depends on a reducing environment, whereas RED subdomain binding activity does not (16Cao X. Kambe F. Lu X. Kobayashi N. Ohmori S. Seo H. J. Biol. Chem. 2005; 280: 25901-25906Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Recently, the conserved PAI cysteines (Cys45 and Cys57), which are predicted to be in the DNA binding interface, were shown to be sensitive to oxidation by glutathionylation, whereas reduction by APE1/Ref-1 restores Pax-8 functionality. The structures of three homologues of Pax-8 Prd domain have been characterized using x-ray diffraction techniques, in co-crystallization with their consensus DNA: the Drosophila (17Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (311) Google Scholar), Pax-6 (18Xu H.E. Rould M.A. Xu W. Epstein J.A. Maas R.L. Pabo C.O. Genes De. 1999; 13: 1263-1275Crossref PubMed Scopus (238) Google Scholar), and Pax-5 (19Garvie C.W. Hagman J. Wolberger C. Mol. Cell. 2001; 8: 1267-1276Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) Prd domains. For all the homologues, both subdomains are folded as helix-turn-helix (HTH) 2The abbreviations used are: HTHhelix-turn-helixPrdpaired boxCDcircular dichroismr.m.s.root mean squareNOEnuclear Overhauser effect. motifs connected by an extended linker region when bound to DNA. The N-terminal subdomain is preceded by an N-terminal β-hairpin that contacts DNA in the minor groove. With the exception of the Drosophila Prd domain, all the Prd homologues contact DNA both with the N- and C-terminal subdomains (12Epstein J.A. Glaser T. Cai J. Jepeal L. Walton D.S. Maas R.L. Genes Dev. 1994; 8: 2022-2034Crossref PubMed Scopus (317) Google Scholar). helix-turn-helix paired box circular dichroism root mean square nuclear Overhauser effect. Several missense mutations involving Pax-8 Prd are associated with thyroid dysgenesis, which leads to congenital hypothyroidism (20Macchia P.E. Lapi P. Krude H. Pirro M.T. Missero C. Chiovato L. Souabni A. Baserga M. Tassi V. Pinchera A. Fenzi G. Gruters A. Busslinger M. Di Lauro R. Nat. Genet. 1998; 19: 83-86Crossref PubMed Scopus (410) Google Scholar, 21Tell G. Pellizzari L. Esposito G. Pucillo C. Macchia P.E. Di Lauro R. Damante G. Biochem. J. 1999; 341: 89-93Crossref PubMed Google Scholar, 22Meeus L. Gilbert B. Rydlewski C. Parma J. Roussie A.L. Abramowicz M. Vilain C. Christophe D. Costagliola S. Vassart G. J. Clin. Endocrinol. Metab. 2004; 89: 4285-4291Crossref PubMed Scopus (104) Google Scholar). As a resulting phenotype, the thyroid is misplaced, severely reduced in size or totally absent. Most of these mutations are located in the N-terminal subdomain where a splicing variant has also been found (23Kozmik Z. Czerny T. Busslinger M. EMBO J. 1997; 16: 6793-6803Crossref PubMed Scopus (129) Google Scholar), although only one mutation in the RED subdomain (20Macchia P.E. Lapi P. Krude H. Pirro M.T. Missero C. Chiovato L. Souabni A. Baserga M. Tassi V. Pinchera A. Fenzi G. Gruters A. Busslinger M. Di Lauro R. Nat. Genet. 1998; 19: 83-86Crossref PubMed Scopus (410) Google Scholar) has been characterized. Mutations in the Pax-8 Prd domain are also associated to Wilms' tumor (1Kozmik Z. Kurzbauer R. Dörfler P. Busslinger M. Mol. Cell. Biol. 1993; 13: 6024-6035Crossref PubMed Scopus (143) Google Scholar). Interestingly, not all the affected residues are part of the predicted DNA binding surface (24Congdon T. Nguyen L.Q. Nogueira C.R. Habiby R.L. Medeiros-Neto G. Kopp P. J. Clin. Endocr. Metab. 2001; 86: 3962-3967Crossref PubMed Scopus (117) Google Scholar, 25Vilain C. Rydlewski C. Duprez L. Heinrichs C. Abramowicz M. Malvaux P. Renneboog B. Parma J. Costagliola S. Vassart G. J. Clin. Endocrinol. Metab. 2001; 86: 234-238Crossref PubMed Scopus (144) Google Scholar, 26Grasberger H. Ringkananont U. LeFrancois P. Abramowicz M. Vassart G. Refetoff S. Mol. Endocrinol. 2005; 19: 1779-1791Crossref PubMed Scopus (72) Google Scholar), suggesting that these mutants could affect folding of the Prd domain. In contrast to the DNA bound state of the Prd domains, the structure of the unbound state is still unknown. Early attempts using circular dichroism (CD) spectroscopy and classical homonuclear NMR approaches suggested that the free Prd domain is largely unstructured (27Epstein J. Cai J. Glaser T. Jepeal L. Maas R. J. Biol. Chem. 1994; 269: 8355-8361Abstract Full Text PDF PubMed Google Scholar). Using CD spectroscopy, studies of the structure of the Pax-8 Prd domain under oxidizing and reducing conditions concluded that the free domain has a low α-helical content (≈19% at 277 K) that increases upon DNA binding (13Pellizzari L. Tell G. Damante G. Biochem. J. 1999; 337: 253-262Crossref PubMed Google Scholar). Interestingly, the unbound and isolated PAI and RED subdomains also maintain a basal, low α-helical content. In summary, these experiments suggested a lack of secondary and tertiary organization of the free domain in solution. However, the C-terminal subdomain of the Drosophila Prd domain is not bound to the DNA, but is nonetheless structured (18Xu H.E. Rould M.A. Xu W. Epstein J.A. Maas R.L. Pabo C.O. Genes De. 1999; 13: 1263-1275Crossref PubMed Scopus (238) Google Scholar). This observation, together with the activity of Cys45 and Cys57 in the free protein, raises questions about the nature of the unbound state and justifies further research. Here, we investigate the unbound state of the Pax-8 Prd domain using high resolution heteronuclear NMR, providing the first detailed characterization of a free Prd domain. Based on NOE and chemical shift data, we show that the two subdomains have a well defined secondary structure and a defined tertiary HTH-fold. Our data indicate that the two subdomains behave as independent "beads" on an otherwise flexible "string." Our data show that the conserved cysteines in the N-terminal PAI subdomain have reduced pKa values, whereas the RED subdomain cysteine is largely buried, thereby providing a structural basis for the difference in glutathionylation susceptibility of the PAI and RED subdomains. Moreover, our findings help to explain the deleterious effects of mutations involving residues that do not belong to the DNA binding interface. Protein Expression and Purification—The DNA sequence encoding residues 1–146 of the human PAX-8 gene (SwissProt ID Q06710) cloned into a pIVEX2.3 MCS vector (Roche Molecular Biochemicals) was a kind donation of Prof. Cao. This construct has been thoroughly described elsewhere (16Cao X. Kambe F. Lu X. Kobayashi N. Ohmori S. Seo H. J. Biol. Chem. 2005; 280: 25901-25906Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and encodes a 159-residue fusion protein containing a 13-residue C-terminal linker ending with a His6 affinity tag. The 13C-15N doubly labeled fusion protein was expressed and purified by ASLA Biotech ltd. (Riga, Latvia) using an optimized ad hoc protocol described in the supplemental data. NMR Spectroscopy—NMR spectra were recorded on the in-house Bruker Avance 500 spectrometer and on a Bruker Avance II 750 spectrometer of the Large Scale NMR Facility, Utrecht, The Netherlands. All NMR experiments were performed at 298 K using a sample containing 0.9 mm U-13C/15N Pax-8 Prd domain in 50 mm sodium phosphate buffer at pH 6.2 in 90%, 10% H2O/D2O, supplemented with 10–15 mm d10-dithiothreitol and 0.1% NaN3. Backbone resonance assignment was accomplished using standard sensitivity enhanced (28Kay L.E. Keifer P. Saarinen T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2439) Google Scholar) decoupled 15N and 13C HSQC (29Bax A. Clore G.M. Gronenborn A.M. J. Magn. Reson. 1990; 88: 425-431Google Scholar, 30Vuister G.W. Bax A. J. Magn. Reson. 1992; 98: 428-435Google Scholar) experiments, three-dimensional HNCA, HNCO (31Kay L.E. Ikura M. Tschudin R. Bax A. J. Magn. Reson. 1990; 89: 496-514Google Scholar), HN(CO)CA, HN(CA)CO (32Ikura M. Bax A. J. Biomol. NMR. 1991; 1: 99-104Crossref PubMed Scopus (128) Google Scholar), CBCANH (33Wittekind M. Mueller L. J. Magn. Reson. Ser. B. 1993; 101: 201-205Crossref Scopus (856) Google Scholar, 34Grzesiek S. Bax A. J. Magn. Reson. 1992; 99: 201-207Google Scholar)–CBCACONH (35Grzesiek S. Bax A. J. Magn. Reson. 1992; 96: 432-440Google Scholar), H(CCO)NH (36Grzesiek S. Anglister J. Bax A. J. Magn. Reson. Ser. B. 1993; 101: 114-119Crossref Scopus (586) Google Scholar), and residue selective MUSIC experiments specific for glycine, serine, and valine-isoleucine-alanine residues (37Schubert M. Smalla M. Schmieder P. Oschkinat H. J. Magn. Reson. 1999; 141: 34-43Crossref PubMed Scopus (77) Google Scholar, 38Schubert M. Oschkinat H. Schmieder P. J. Magn. Reson. 2001; 148: 61-72Crossref PubMed Scopus (62) Google Scholar). Side chain resonances were assigned using 15N/13C-resolved HSQC-TOCSY (39Davis A.L. Keeler J.E. Laue D. Moskau D. J. Magn. Reson. 1992; 98: 207-216Google Scholar), HCCH-TOCSY (39Davis A.L. Keeler J.E. Laue D. Moskau D. J. Magn. Reson. 1992; 98: 207-216Google Scholar, 40Kay L.E. Xu G.Y. Singer A.U. Muhandiram D.R. Formankay J.D. J. Magn. Reson. Ser. B. 1993; 101: 333-337Crossref Scopus (562) Google Scholar), and 15N/13C-edited HSQC-NOESY (41Kay M.D. Sparks L.E. Torchia S.W. Bax A. J. Am Chem. Soc. 1988; 111: 1515-1517Google Scholar, 42Pascal S.M. Muhandiram D.R. Yamazaki T. Formankay J.D. Kay L.E. J. Magn. Reson. Ser B. 1994; 103: 197-201Crossref Scopus (280) Google Scholar) experiments. Interproton distance restraints were obtained from 15Nor 13C resolved three-dimensional HSQC-NOESY spectra, both recorded with a mixing time of 100 ms. Further details are described in supplemental data. Proton chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonic acid, whose resonance was set to 0.00 ppm whereas 13C and 15N chemical shifts were referenced indirectly to 2,2-dimethyl-2-silapentane-5-sulfonic acid, using the absolute frequency ratio (43Wishart D.S. Bigam C.G. Yao, J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2083) Google Scholar). The program CARA 3Keller, R. (2004) The Computer Aided Resonance Tutorial, First Edition, ISBN 3-85600-112-3. was used for spectral analysis. Collection of Conformational Restraints, Structure Calculation, and Refinement—Full backbone assignment of the Pax-8 Prd (residues 11–138 of the 159-amino acid protein construct) could be obtained with the exception of Pro44 and 85% of the nonexchangeable side chain resonances could be assigned. For the full construct residues 1–2 and 153–159 could not be assigned due to unfavorable water exchange or overlap. Chemical shifts values and NOE data for the N- and C-terminal cloning artifacts indicate that they are unstructured and do not interact with the Prd domain. In the following, the discussion will focus only on the Prd domain. NOE cross-peaks from the three-dimensional 15N- and 13C-resolved NOESY spectra were assigned and converted into distance restraints using a homemade script. Dihedral angle restraints for the φ and Ψ angles were derived from N, C′, Cα, Hα, and Cβ chemical shifts using TALOS (45Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar). The collected restraints were analyzed to remove redundant information and resulted in 1794 distance values, which, together with 123 dihedral angle restraints, formed the experimental data set for the restrained modeling. A trans-conformation was assumed for Pro44, supported by our experimental NOE restraints for the surrounding region. After applying standard pseudoatom corrections (46Wuthrich K. Billeter M. Braun W. J. Mol. Biol. 1983; 169: 949-961Crossref PubMed Scopus (1007) Google Scholar), 300 structures were calculated using CYANA 2.1 (47Güntert P. Methods Mol. Biol. 2004; 278: 353-378Crossref PubMed Scopus (1173) Google Scholar) starting from random conformations, simulated annealing details are described in supplemental data. The 20 conformers with the lowest CYANA target function were subjected to refinement in implicit solvent (dielectric constant = 4 × r) using restrained energy minimization with the AMBER force field (48Weiner S.J. Kollman P.A. Case D.A. Singh U.C. Ghio C. Alagona G. Profeta J.S. Weiner P. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4895) Google Scholar) in the program DISCOVER (Accelrys) to improve local structure quality and electrostatics (see details in supplemental data). The structural quality was evaluated with Procheck-NMR (49Laskowski R.A. Rullmann J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4474) Google Scholar) and WHATCHECK (50Hooft R.W.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1818) Google Scholar). The programs MOLMOL (51Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar) and PyMol (52DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA2002Google Scholar) (DeLano Scientific, Palo Alto, CA) were used to visualize and evaluate r.m.s. deviations between the structures. WHAT-IF (53Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3377) Google Scholar) server has been used to evaluate salt bridges. A homemade script has been written to evaluate hydrophobic interactions, following the rules of Israelachvili and Pashley (55Israelachvili J. Pashley R. Nature. 1982; 300: 341-342Crossref PubMed Scopus (977) Google Scholar). Complete NMR restraints and structure calculation statistics are given in Table 1 for residues 11–138 of the fusion protein corresponding to the Prd and excluding the N- and C-terminal cloning artifacts.TABLE 1Structural statistics and restraint information for the Pax-8 Prd domainStructural statistics and restraint informationaStatistics were calculated for residues 11–138 corresponding to the Prd domain using the 20 lowest energy structuresRestraint information Number of structures20 Number of distance restraints1,794 Intra/short/medium/long rangebDistance restraints between hydrogens of residues i and j are classified as intra (intraresidue, i.e. i – j = 0), short (short-range, i.e. i – j = 1), medium (medium range, i.e. 1 < [i – j] <5), and long (long range, i.e. i – j ≥ 5)946/588/185/75 Restraints per residue (overall)11.3 Restraints per residue (N-terminal subdomain)16.3 Restraints per residue (C-terminal subdomain)16.6 TALOS derived dihedral angle restraints123Average r.m.s. deviation from experimental restraints Distance upper limits (Å)0.028 ± 0.050 [Range][0.01, 0.57] Distance lower limits0.035 ± 0.040 [Range][–0.01, –0.57] Dihedral angle restraints (°)Ramachandran quality parameters in selected subdomains (%) Residues in most favored regions90.20% Residues in allowed regions8.20% Residues in additionally allowed regions1.40% Residues in disallowed regions0.10%Consistency check over individual structures (mean value [ranges]) Abnormally short interatomic distances6 [2, 9] Unsatisfied H bond acceptors (buried)1 [0, 2] Unsatisfied H bond donors (buried)13 [6, 19]Average G factors (53Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3377) Google Scholar) Phi-psi0.140 Chi1-chi20.360 Chi1-only0.040 Overall0.190Coordinate pairwise root mean square deviation (Å) Global backbone atoms11.1 ± 4.5 Global side chain atoms11.6 ± 4.5N-terminal subdomaincThe N-terminal subdomain encompasses fragment 29 – 62, whereas the C-terminal, fragment 87–135 Backbone1.61 ± 0.54 Heavy atoms2.77 ± 0.56C-terminal subdomaincThe N-terminal subdomain encompasses fragment 29 – 62, whereas the C-terminal, fragment 87–135 Backbone1.61 ± 0.32 Heavy atoms2.75 ± 0.29a Statistics were calculated for residues 11–138 corresponding to the Prd domain using the 20 lowest energy structuresb Distance restraints between hydrogens of residues i and j are classified as intra (intraresidue, i.e. i – j = 0), short (short-range, i.e. i – j = 1), medium (medium range, i.e. 1 < [i – j] <5), and long (long range, i.e. i – j ≥ 5)c The N-terminal subdomain encompasses fragment 29 – 62, whereas the C-terminal, fragment 87–135 Open table in a new tab Molecular Dynamics Simulations—Molecular dynamics simulation has been performed starting from the crystal structure of human Pax-6 Prd domain (Protein Data Bank code 6PAX) with the cognate DNA removed and chloride ions added to neutralize the overall charge (see details in supplemental data). The system was simulated for 10 ns. In the simulation the temperature was kept constant through a simple velocity rescaling procedure, whereas the pressure was controlled through a Berendsen bath (57Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Di Nola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23947) Google Scholar) using a relaxation time of 100 fs. The volume of the box was fluctuating about 540 nm3 with a standard deviation of less than 0.002 of its value. All structural analyses, in particular r.m.s. deviations, secondary structure, and angular order parameter analyses, have been performed using the program MOLMOL. Cysteine pKa Calculation—pKa values for cysteines were calculated, based on the Poisson-Boltzmann equation, essentially according to the method of Antosiewicz et al. (58Antosiewicz J. McCammon J.A. Gilson M.K. J. Mol. Biol. 1994; 238: 415-436Crossref PubMed Scopus (760) Google Scholar) with minor modifications (59Fogolari F. Ragona L. Licciardi S. Romagnoli S. Michelutti R. Ugolini R. Molinari H. Struct. Funct. Genet. 2000; 39: 317-330Crossref PubMed Scopus (76) Google Scholar). The starting structures were a model built by homology with Pax-6 (PDB code 6PAX) as template, and the 20 NMR structures. pKa calculations have been also performed on Pax-5 and Pax-6 structures. Circular Dichroism—The purified Pax-8 Prd domain was used for CD spectroscopy at a concentration of 15 μm using a Jasco J-600 CD/ORD spectropolarimeter interfaced to a computer for data collection. Standard conditions were 50 mm Na2HPO4 (pH 6.2), 250 μm dithiothreitol, 277 K, 0.2-cm path-length cuvette. Spectra are presented in terms of mean residue molecular ellipticity ([θ]; deg cm2 dmol-1), based on a mean residue weight of 110.4 Da. Data Deposition—Chemical shifts have been deposited at the BMRB data base, with access code 15693, whereas the protein coordinates were deposited in the protein data bank (PDB code 2K27). NMR Secondary Structure Identification—The 1H-15N HSQC spectrum is a sensitive indicator of the structural and dynamical properties of a protein. The spectrum of free Pax-8-Prd shows a chemical shift dispersion typical of an all α-helical folded protein (supplemental Fig. S1). Secondary structure assessment based on diagnostic sequential and medium-range NOEs and chemical shift deviation from random coil values (60Wang Y. Jardetzky O. J. Am. Chem. Soc. 2002; 124: 14075-14084Crossref PubMed Scopus (94) Google Scholar, 61Wishart D.S. Sykes B.D. Methods Enzymol. 1994; 239: 363-392Crossref PubMed Scopus (939) Google Scholar) revealed the presence of six α-helices, consistent with the Prd domain crystal structures described in literature (Fig. 2 and supplemental S2). The helices are named starting from the N-terminal by ordinal numbering. The total helix content of the free Pax-8 Prd amounts to ∼42%. This value is in agreement with control CD experiments (supplemental Fig. S3), which show an α-helical content that, depending on the analysis method adopted, varies from ∼28 to ∼39% (62Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32-44Crossref PubMed Scopus (946) Google Scholar, 63Chen Y.H. Yang J.T. Chau K.H. Biochemistry. 1974; 13: 3350-3359Crossref PubMed Scopus (1971) Google Scholar). CD experimental conditions were the same as those of the NMR with exception of protein and dithiothreitol concentrations. The reduced state of the Prd domain in solution is confirmed by α and β carbon chemical shifts of the conserved cysteines, which are in agreement with the values reported by Sharma and Rajarathnam (64Sharma D. Rajarathnam K. J. Biomol. NMR. 2000; 18: 165-171Crossref PubMed Scopus (250) Google Scholar). Pax-8 Prd Domain Conformation—Analysis of the NOESY cross-peaks resulted in the identification of 76 long range NOEs that reflect crucial interactions within each subdomain and are typical of a tertiary organization (supplemental Fig. S4). The backbone traces of the lowest energy structure and the ensemble of structures of the free Pax-8 Prd domain are shown in Fig. 3. Structural statistics are reported in Table 1. The tertiary organization of the free Pax-8 DNA binding domain shows the characteristics of a canonical Prd domain: two HTH motifs are connected by an unstructured linker region. NOE data and chemical shift differences from random coil values (60Wang Y. Jardetzky O. J. Am. Chem. Soc. 2002; 124: 14075-14084Crossref PubMed Scopus (94) Google Scholar) support the random coil nature of the linker (Figs. 2 and supplemental S4). Furthermore, the backbone chemical shifts of the linker are consistent with a highly flexible backbone, according to the random coil index (65Berjanskii M.V. Wishart D.S. J. Am. Chem. Soc. 2005; 127: 14970-14971Crossref PubMed Scopus (331) Google Scholar) (supplemental Fig. S5). Additionally, the random coil index indicates a random coil-like structure and high backbone flexibility for the C- and N-terminal regions flanking the two HTH domains. The flexibility and lack of structure of the linker and the absence of NOEs between the two subdomains indicate that the two HTH subdomains are independent "beads on a string" and explain the high global coordinate r.m.s. deviation. The individual N- and C-terminal subdomains have considerably lower pairwise r.m.s. deviations values for both backbone and heavy atoms (∼1.6 and 2.7 Å, respectively) in comparison with the whole Prd domain; these values are consistent with a defined tertiary fold for both subdomains but are somewhat higher than typical for a well defined rigid structure. Closer inspections reveals that, whereas the individual helices are well defined with pairwise r.m.s. deviation of ∼0.6 Å, the interhelical segments and the interhelical angles have considerable lower definition, resulting in an increased r.m.s. deviation. We suggest that the lack of a precise definition of the tertiary structure is a signature of protein dynamics. This is also reflected in the increased random coil index and predicted flexibility for the interhelical segments. Thus, whereas the free domain has a well defined secondary structure and a defined tertiary fold, it is most likely a dynamic structure. Both the PAI and RED subdomains contain three helices consistent with the HTH motif (see Table 2 for an analysis).TABLE 2Inter-helix tilt (degrees) comparisons in helix-turn-helix motifsPax-8Pax-5Pax-6Helix I–II114 ± 14143141Helix I–III108 ± 97877Helix II–III103 ± 18124117Helix IV–V112 ± 7120122Helix IV–VI73 ± 87577Helix V–VI121 ± 6103104 Open table in a new tab Helix I and II in the PAI subdomain pack against each other in an antiparallel arrangement and are almost perpendicular to helix III, which is the DNA recognition helix. Helices IV, V, and VI of the RED subdomain are packed analogously. A striking difference between the two domains is the length of the DNA recognition helix. Although helix III spans on average only 1.5 helical turns, helix VI is 2.5 turns lo