Structural Basis for the Conformational Integrity of the Arabidopsis thaliana HY5 Leucine Zipper Homodimer
2007; Elsevier BV; Volume: 282; Issue: 17 Linguagem: Inglês
10.1074/jbc.m611465200
ISSN1083-351X
AutoresMi‐Kyung Yoon, Ho-Min Kim, Giltsu Choi, Jie‐Oh Lee, Byong‐Seok Choi,
Tópico(s)Plant Molecular Biology Research
ResumoThe leucine zipper (LZ) domain of the HY5 transcription factor from Arabidopsis thaliana has unique primary structural properties, including major occupation by the Leu residues as well as two buried polar residues in the a positions and a localized distribution of charged and polar residues in the first three heptad repeats. In this study, we solved the crystal structure of the HY5 LZ domain and show that the peculiarities in the primary sequence yield unusual structural characteristics. For example, the HY5 LZ domain exhibits a bipartite charge distribution characterized by a highly negative electrostatic surface potential in its N-terminal half and a nearly neutral potential in its C-terminal half. The LZ N-terminal region also contains two consecutive putative trigger sites for dimerization of the coiled coils. In addition, two buried asparagines at a positions 19 and 33 in the HY5 LZ domain display distinct modes of polar interaction. Whereas Asn19 shows a conformational flip-flop, Asn33 is engaged in a permanent hydrogen bond network. CD spectropolarimetry and analytical ultracentrifugation experiments performed with versions of the HY5 LZ domain containing mutations in the a positions yielded further evidence that position a amino acid residues are crucial for achieving an oligomeric state and maintaining stability. However, a low correlation between position a amino acid preference, core packing geometry, and rotamer conformations suggests that the oligomeric state of the LZ domain is not governed entirely by known structural properties. Taken together, our results suggest structural factors conferring conformational integrity of the HY5 LZ homodimer that are more complicated than proposed previously. The leucine zipper (LZ) domain of the HY5 transcription factor from Arabidopsis thaliana has unique primary structural properties, including major occupation by the Leu residues as well as two buried polar residues in the a positions and a localized distribution of charged and polar residues in the first three heptad repeats. In this study, we solved the crystal structure of the HY5 LZ domain and show that the peculiarities in the primary sequence yield unusual structural characteristics. For example, the HY5 LZ domain exhibits a bipartite charge distribution characterized by a highly negative electrostatic surface potential in its N-terminal half and a nearly neutral potential in its C-terminal half. The LZ N-terminal region also contains two consecutive putative trigger sites for dimerization of the coiled coils. In addition, two buried asparagines at a positions 19 and 33 in the HY5 LZ domain display distinct modes of polar interaction. Whereas Asn19 shows a conformational flip-flop, Asn33 is engaged in a permanent hydrogen bond network. CD spectropolarimetry and analytical ultracentrifugation experiments performed with versions of the HY5 LZ domain containing mutations in the a positions yielded further evidence that position a amino acid residues are crucial for achieving an oligomeric state and maintaining stability. However, a low correlation between position a amino acid preference, core packing geometry, and rotamer conformations suggests that the oligomeric state of the LZ domain is not governed entirely by known structural properties. Taken together, our results suggest structural factors conferring conformational integrity of the HY5 LZ homodimer that are more complicated than proposed previously. A major challenge that faces protein chemists is the ability to predict the three-dimensional structures of proteins from their primary amino acid sequences. Because of its simplicity and periodicity, the coiled-coil motif is an ideal model system with which to investigate properties of the protein folding process. In terms of biological function, the coiled coil is one of the most common structural motifs and participates in many cellular processes through the formation of protein-protein interactions.Among coiled-coil proteins, the basic leucine zipper (bZIP) 3The abbreviations used are: bZIP, basic leucine zipper; LZ, leucine zipper; WT, wild-type; GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SeMet, selenomethionine; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CLZ, heterogeneous nuclear ribonucleoprotein Cleucine zipper-like. 3The abbreviations used are: bZIP, basic leucine zipper; LZ, leucine zipper; WT, wild-type; GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SeMet, selenomethionine; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CLZ, heterogeneous nuclear ribonucleoprotein Cleucine zipper-like. proteins play crucial roles in the regulation of transcription. These proteins bind to their cognate DNA elements through their basic regions as they dimerize via their leucine zipper (LZ) domains. The LZ domain consists of an α-helix that contains a series of leucine residues on one face. Two LZ α-helices intertwine to produce a dimeric structure with a slight superhelical twist (1Cohen C. Parry D.A. Proteins. 1990; 7: 1-15Crossref PubMed Scopus (665) Google Scholar). The LZ domain consists of n heptad sequence repeats, designated (abcdefg)n (2McLachlan A.D. Stewart M. J. Mol. Biol. 1975; 98: 293-304Crossref PubMed Scopus (567) Google Scholar), where positions a and d create the interface between two LZ strands by engaging in hydrophobic interactions, and positions e and g flank the dimer interface by forming electrostatic interactions. The various amino acid positions of the LZ heptad repeat have been shown to play unique roles in determining the specificity (homo- or heterodimer) (1Cohen C. Parry D.A. Proteins. 1990; 7: 1-15Crossref PubMed Scopus (665) Google Scholar, 3John M. Briand J.P. Granger-Schnarr M. Schnarr M. J. Biol. Chem. 1994; 269: 16247-16253Abstract Full Text PDF PubMed Google Scholar, 4O'Shea E.K. Rutkowski R. Kim P.S. Cell. 1992; 68: 699-708Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 5O'Shea E.K. Lumb K.J. Kim P.S. Curr. Biol. 1993; 3: 658-667Abstract Full Text PDF PubMed Scopus (403) Google Scholar), orientation (parallel or antiparallel) (6Oakley M.G. Kim P.S. Biochemistry. 1998; 37: 12603-12610Crossref PubMed Scopus (176) Google Scholar), oligomeric state (7O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1273) Google Scholar, 8Harbury P.B. Zhang T. Kim P.S. Alber T. Science. 1993; 262: 1401-1407Crossref PubMed Scopus (1332) Google Scholar, 9Junius F.K. Mackay J.P. Bubb W.A. Jensen S.A. Weiss A.S. King G.F. Biochemistry. 1995; 34: 6164-6174Crossref PubMed Scopus (69) Google Scholar, 10Lumb K.J. Kim P.S. Biochemistry. 1995; 34: 8642-8648Crossref PubMed Scopus (300) Google Scholar), and stability of the LZ domain (3John M. Briand J.P. Granger-Schnarr M. Schnarr M. J. Biol. Chem. 1994; 269: 16247-16253Abstract Full Text PDF PubMed Google Scholar, 7O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1273) Google Scholar).The recently sequenced Arabidopsis thaliana genome (11The Arabidopsis Genome InitiativeNature. 2000; 408: 796-815Crossref PubMed Scopus (7059) Google Scholar) contains 75 genes that encode members of the bZIP transcription factor family (12Jakoby M. Weisshaar B. Droge-Laser W. Vicente-Carbajosa J. Tiedemann J. Kroj T. Parcy F. Trends Plant Sci. 2002; 7: 106-111Abstract Full Text Full Text PDF PubMed Scopus (1247) Google Scholar). Compared with those of human bZIP proteins, the primary structures of A. thaliana LZ domains show unique characteristics, including longer heptad repeats and abundant Asn residues in position a (13Deppmann C.D. Acharya A. Rishi V. Wobbes B. Smeekens S. Taparowsky E.J. Vinson C. Nucleic Acids Res. 2004; 32: 3435-3445Crossref PubMed Scopus (86) Google Scholar). It has been suggested that these unique properties might specify LZ dimers in ways that differ from mammalian bZIP proteins. Dimerization of A. thaliana LZ domains was predicted to be specified by the unique distribution of Asn residues in position a, not by electrostatic interactions between positions e and g in human LZ motifs (13Deppmann C.D. Acharya A. Rishi V. Wobbes B. Smeekens S. Taparowsky E.J. Vinson C. Nucleic Acids Res. 2004; 32: 3435-3445Crossref PubMed Scopus (86) Google Scholar).The Arabidopsis bZIP protein HY5 is a positive regulator of photomorphogenesis (14Oyama T. Shimura Y. Okada K. Genes Dev. 1997; 11: 2983-2995Crossref PubMed Scopus (571) Google Scholar). HY5 is constitutively nuclear localized to be involved in light regulation of the transcriptional activity of promoters containing the G-box light-responsive element (15Chattopadhyay S. Ang L.H. Puente P. Deng X.W. Wei N. Plant Cell. 1998; 10: 673-683Crossref PubMed Scopus (333) Google Scholar). Interaction of HY5 with G-box elements is expected to be mediated by the C-terminal region of the protein, which contains a bZIP domain. The HY5 LZ domain shares common structural features with other Arabidopsis LZ motifs such as the presence of two Asn residues in position a (13Deppmann C.D. Acharya A. Rishi V. Wobbes B. Smeekens S. Taparowsky E.J. Vinson C. Nucleic Acids Res. 2004; 32: 3435-3445Crossref PubMed Scopus (86) Google Scholar). In addition, HY5 displays some primary structural features that differ from those of other Arabidopsis bZIP proteins: major occupation by leucines of the hydrophobic residues (three of four) of position a and localized distribution of the charged and polar residues in the first three heptad repeats. Thus, we hypothesized that classical notions of dimerization specificity and stability might not apply to HY5 LZ dimers. To investigate the structural basis for the conformational integrity of the HY5 LZ homodimer, we determined its crystal structure at 2.0 Å and characterized the functions of position a amino acids using CD spectropolarimetry and analytical ultracentrifugation. This is the first LZ structure among the Arabidopsis LZ motifs.EXPERIMENTAL PROCEDURESPeptide Synthesis—N-terminally acetylated synthetic peptides were purchased from Peptron, Inc. (Daejeon, South Korea). The molecular mass and purity of each peptide were verified by mass spectrometry and analytical high performance liquid chromatography.Cloning and Mutagenesis—To obtain the wild-type (WT) LZ domain of HY5 (amino acids 111-150), PCR was performed using, as a template, the A. thaliana gene that encodes fulllength HY5. PCR fragments generated from the HY5 gene were inserted into the pGEX4T3 vector (Amersham Biosciences) between the BamHI and XhoI restriction sites.For the production of mutant LZ domains, mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were introduced by PCR using, as a template, the WT LZ domain-containing pGEX4T3-based vector described above. PCR was followed by digestion with DpnI to digest the methylated parental plasmid. The resulting DNA was transformed into XL10 competent cells. All mutant plasmid constructs were verified by DNA sequencing.Peptide Expression and Purification—The WT and mutant versions of the HY5 LZ domain were overexpressed as glutathione S-transferase (GST) fusions in Escherichia coli strain BL21 and grown in LB medium. When the cells reached A600 = 0.8 - 1.0, recombinant protein expression was induced by the addition of 0.4 mm isopropyl β-d-thiogalactopyranoside. After further incubation for 4 h, the cells were harvested by centrifugation. The cell pellets were resuspended in buffer containing 50 mm sodium phosphate (pH 7.4) and 0.1 m NaCl and frozen at -70 °C. When the cell suspensions were thawed, 1 mm phenylmethylsulfonyl fluoride was added, and the cells were disrupted by sonication. The resulting cell lysates were subjected to centrifugation at 15,000 rpm for 1 h; the clarified supernatants were filtered using 0.45-μm filters (Sartorius AG); and the HY5 LZ domain-GST fusion proteins were purified using a GST affinity column (Amersham Biosciences). After removal of the GST protein by thrombin digestion, the resulting proteins were further purified by gel filtration on a Superdex-75 fast protein liquid chromatography system (Amersham Biosciences) in 5 mm sodium phosphate (pH 7.4). The elution flow rate was 1 ml/min. The resulting eluents were concentrated using an Amicon Ultra centrifugal filter (Millipore Corp.). The final protein preparations used in this study were shown to be pure by Tricine/SDS-PAGE. Protein concentrations were determined by UV absorbance at 280 nm using an extinction coefficient of 1280 cm-1m-1 for each HY5 LZ peptide.The selenomethionine (SeMet)-containing WT HY5 LZ domain was prepared from E. coli strain BL21 grown in M9 medium (16Jansson M. Li Y.C. Jendeberg L. Anderson S. Montelione B.T. Nilsson B. J. Biomol. NMR. 1996; 7: 131-141Crossref PubMed Scopus (148) Google Scholar) supplemented with 2 mm MgSO4, 0.4% (w/v) glucose, and 0.1 mm CaCl2. When this culture reached A600 ∼ 0.8 at 37 °C, 100 mg each of Leu, Ile, Val, Thr, Phe, and Lys and 50 mg of l-(+)-SeMet (Sigma) were added per liter of growth medium. The culture was incubated for an additional 20 min at 37 °C, and the cells were then induced to express the recombinant proteins by the addition of 1 mm isopropyl β-d-thiogalactopyranoside. Protein production was allowed to proceed for 4 h at 37 °C, after which the proteins were purified as described above. When the SeMet-containing version of the WT HY5 LZ domain was purified, 5 mm dithiothreitol was added to the buffers used for a native sample preparation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated >95% substitution of one Met residue with SeMet.CD Spectropolarimetry—CD measurements were performed on a Jasco J-715 spectrometer with ∼100 μm protein samples in 5mm sodium phosphate (pH 7.4) or 5 mm sodium acetate (pH 4.0) at the Mokpo National University Central Laboratory (Mokpo, South Korea) or at the Korea Basic Science Institute (Ochang, South Korea). A cuvette with a 1-mm path length was used. Each spectrum is the result of the averaging of five consecutive scans. A separate spectrum was generated for the buffer alone, and this spectrum was subtracted from spectra taken in the presence of protein. For thermal denaturation profiles, ellipticity at 222 nm (which allowed us to inspect the peptide backbone region) was measured for each protein construct over a linear temperature gradient of 5-90 °C at a constant scan rate of 1 °C/min. The reversibility of the thermal denaturation was determined by monitoring the return of the CD signal at 222 nm upon cooling from 90 to 5 °C immediately after the thermal unfolding experiments. The thermal transitions were >95% reversible for all recombinant proteins except HM12 and HM13 in 5 mm sodium acetate (pH 4.0). The helicity was estimated with the following equation: % α-helicity = (-([θMRW]222 + 2340)/30,300) × 100 (where [θMRW]222 is the mean residue ellipticity at 222 nm) (17Yang J.T. Wu C.S. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Crossref PubMed Scopus (1731) Google Scholar).Sedimentation Equilibrium—Sedimentation equilibrium experiments with the WT and mutant versions of the HY5 LZ domain was performed at 20 °C in a ProteomeLab XL-A analytical ultracentrifuge (Beckman Coulter, Inc.) using a An-60 Ti rotor and 6-channel centerpieces in the Mokpo National University Central Laboratory. All peptides were dissolved in buffer containing 5 mm sodium phosphate (pH 7.4) or 5 mm sodium acetate (pH 4.0). Samples with an initial A280 of ∼0.13 (∼100 μm) were centrifuged at 30,000 and/or 35,000 rpm. Radial absorbance scans for 10 replicates were collected in continuous scan mode at 280 or 275 nm with a step size of 0.001 cm and a time interval of 4 h. Global fitting of data sets was performed with the program UltraScan Version 8.0 (18Demeler B. Scott D.J. Harding S.E. Rowe A.J. Modern Analytical Ultracentrifugation: Techniques and Methods. Royal Society of Chemistry, London2005: 210-229Google Scholar).Crystallization—WT HY5 LZ crystals were grown at 23 °C by the hanging-drop vapor-diffusion method, mixing 7.5 mg/ml peptide with an equal volume of reservoir solution containing 0.1 m BisTris (pH 6.0), 0.1 m MgCl2, and 25% (w/v) polyethylene glycol 3350. Crystals were equilibrated in cryoprotectant buffer containing reservoir buffer plus 20% (v/v) glycerol and flash-frozen in liquid nitrogen. The crystals belong to space group C2, with a = 48.4 Å, b = 24.5 Å, c = 76.3 Å, α = γ = 90°, and β = 100°.Data Collection and Structure Determination—The diffraction data were collected at the 4A and 6B beam lines of the Pohang Accelerator Laboratory and processed using the HKL package (see Table 1). The three-dimensional structure was determined by combining the three-wavelength anomalous dispersion data set from an SeMet-containing crystal and by single isomorphous replacement analysis using the SeMet crystal as a derivative. Two ordered selenium sites were identified from remote and native data sets using the program SAPI (19Hao Q. Gu Y.X. Yao J.X. Zheng C.D. Fan H.F. J. Appl. Crystallogr. 2003; 36: 1274-1276Crossref Scopus (9) Google Scholar). Phasing and density modifications were carried out using MLPHARE (20Otwinowski Z. Wolf W. Evans P.R. Leslie A.G.W. Isomorphous Replacement and Anomalous Scattering. Science and Engineering Research Council, Daresbury, United Kingdom1991: 80-86Google Scholar) and RESOLVE (21Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1632) Google Scholar). Ninety percent of the amino acid residues were placed by the automatic modeling procedure of RESOLVE. Iterative cycles of manual model building and refinement were carried out using the programs O (22Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar) and CNS (23Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar), respectively. All non-glycine amino acid residues were located in the most favored region of the Ramachandran plot calculated with the refined model.TABLE 1Data collection, phasing, and refinement statistics for the SeMet-containing HY5 LZ domainNativeSeleniumData collectionSpace groupC2C2Cell dimensionsa, b, c (Å)48.3, 24.5, 76.548.4, 24.5, 76.3α, β, γ90.0°, 100.0°, 90.0°90.0°, 100.0°, 90.0°PeakInflectionRemoteWavelength1.127140.978800.978950.97114Resolution (Å)2.02.12.12.1RsymaThe highest resolution shell is shown in parentheses.9.5 (39.2)5.6 (11.1)5.7 (11.0)5.8 (11.6)I/σI12.845.546.045.0Completeness (%)aThe highest resolution shell is shown in parentheses.97.7 (87.8)97.5 (95.4)97.5 (96.5)97.4 (98.1)Redundancy2.94.34.64.8RefinementResolution (Å)20.0-2.0No. reflections (works/test)5059/621Rwork/Rfree24.3/29.6No. atomsProtein694Water102B-factorsProtein36.6Water47.9r.m.s.bRoot mean square. deviationsBond lengths (Å)0.007Bond angles1.24°a The highest resolution shell is shown in parentheses.b Root mean square. Open table in a new tab RESULTSOverall Structure of the HY5 LZ Homodimer—A peptide corresponding to the LZ fragment (residues 111-150) of Arabidopsis HY5 with additional N-terminal Gly and Ser residues was crystallized using the hanging-drop vapor-diffusion method. The HY5 LZ crystals belong to space group C2 and contain two monomers in the asymmetrical unit with a dimer formed around the crystallographic 2-fold symmetry axis. The HY5 LZ crystal structure was determined by multiwavelength anomalous dispersion methods using a peptide containing SeMet. Diffraction data were collected to 2.0 Å from crystals of both the SeMet-containing and native peptides. The structure was refined to a crystallographic R-factor of 24.3% with Rfree = 29.6% over a resolution range of 20.0 to 2.0 Å (Table 1). The current structure includes 42 residues (Fig. 1A) as well as 102 water molecules. The root mean square deviations of bond lengths and bond angles from the idea values were 0.007 Å and 1.24°, respectively (Table 1).The HY5 LZ domain consists of a parallel, two-stranded, α-helical coiled coil (Fig. 1B) with a diameter of ∼18 Å and a length of ∼60 Å. The two α-helices wrapped around one another with a left-handed superhelical twist. Residues 3-41 form an α-helix that makes a 23.9° crossing angle. Because there are just 3.5 residues/α-helical turn, each strand contained 11 turns. The average backbone dihedral angles for the helical region (residues 3-41) are -66.5 ± 8.6° for φ and -40.0 ± 10.0° for ψ. These values are similar to but differ slightly from those measured for the yeast Gcn4-p1 (-63 ± 7° for φ and -42 ± 7° for ψ) (7O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1273) Google Scholar) and human c-Jun (-63 ± 10° for φ and -42 ± 13° for ψ) homodimers (24Junius F.K. O'Donoghue S.I. Nilges M. Weiss A.S. King G.F. J. Biol. Chem. 1996; 271: 13663-13667Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). As predicted by "knobs-into-holes" packing (25Crick F.H.C. Acta Crystallogr. 1953; 6: 689-697Crossref Google Scholar), the dimer interface is formed by the side chains of residues at positions a and d of one monomer and those at positions a′ and d′ of the other (Fig. 1C). This classical intertwined packing has been observed in the Gcn4-p1 (7O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1273) Google Scholar) and c-Jun homodimers (24Junius F.K. O'Donoghue S.I. Nilges M. Weiss A.S. King G.F. J. Biol. Chem. 1996; 271: 13663-13667Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).Contributions of Electrostatic Interactions to the Stability and Orientation of the HY5 LZ Domain—Positions e and g of the heptad repeat flank the dimer interface of the helices and are generally occupied by charged or polar residues (2McLachlan A.D. Stewart M. J. Mol. Biol. 1975; 98: 293-304Crossref PubMed Scopus (567) Google Scholar, 26Hurst H.C. Protein Profile. 1994; 1: 123-168PubMed Google Scholar, 27Hu J.C. O'Shea E.K. Kim P.S. Sauer R.T. Science. 1990; 250: 1400-1403Crossref PubMed Scopus (317) Google Scholar, 28Vinson C.R. Hai T. Boyd S.M. Genes Dev. 1993; 7: 1047-1058Crossref PubMed Scopus (288) Google Scholar). Interhelical electrostatic interactions are formed between an amino acid in position g of a heptad repeat in one helix (gi) and the following amino acid in position e of a heptad repeat in the other helix (ei′+5) of an LZ domain (29Krylov D. Mikhailenko I. Vinson C. EMBO J. 1994; 13: 2849-2861Crossref PubMed Scopus (226) Google Scholar). These positions have been suggested to be involved in stabilizing or destabilizing the coiled coil (3John M. Briand J.P. Granger-Schnarr M. Schnarr M. J. Biol. Chem. 1994; 269: 16247-16253Abstract Full Text PDF PubMed Google Scholar, 7O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1273) Google Scholar) and in the dimerization specificity of an LZ domain (1Cohen C. Parry D.A. Proteins. 1990; 7: 1-15Crossref PubMed Scopus (665) Google Scholar, 3John M. Briand J.P. Granger-Schnarr M. Schnarr M. J. Biol. Chem. 1994; 269: 16247-16253Abstract Full Text PDF PubMed Google Scholar, 4O'Shea E.K. Rutkowski R. Kim P.S. Cell. 1992; 68: 699-708Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 5O'Shea E.K. Lumb K.J. Kim P.S. Curr. Biol. 1993; 3: 658-667Abstract Full Text PDF PubMed Scopus (403) Google Scholar) depending on whether the interhelical electrostatic interactions are attractive or repulsive. Although they contribute to the global stability of the coiled coil far less than do the energetic contributions of van der Waals packing effects of residues at positions a and d (30Hu J.C. Newell N.E. Tidor B. Sauer R.T. Protein Sci. 1993; 2: 1072-1084Crossref PubMed Scopus (84) Google Scholar, 31Spek E.J. Bui A.H. Lu M. Kallenbach N.R. Protein Sci. 1998; 7: 2431-2437Crossref PubMed Scopus (92) Google Scholar, 32Mittl P.R. Deillon C. Sargent D. Liu N. Klauser S. Thomas R.M. Gutte B. Grutter M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2562-2566Crossref PubMed Scopus (61) Google Scholar), salt bridges formed between the residues at positions e and g are still believed to be important for modulating both interhelical (29Krylov D. Mikhailenko I. Vinson C. EMBO J. 1994; 13: 2849-2861Crossref PubMed Scopus (226) Google Scholar, 30Hu J.C. Newell N.E. Tidor B. Sauer R.T. Protein Sci. 1993; 2: 1072-1084Crossref PubMed Scopus (84) Google Scholar, 33Zhou N.E. Kay C.M. Hodges R.S. Protein Eng. 1994; 7: 1365-1372Crossref PubMed Scopus (132) Google Scholar, 34Krylov D. Barchi J. Vinson C. J. Mol. Biol. 1998; 279: 959-972Crossref PubMed Scopus (105) Google Scholar) and intrahelical (30Hu J.C. Newell N.E. Tidor B. Sauer R.T. Protein Sci. 1993; 2: 1072-1084Crossref PubMed Scopus (84) Google Scholar, 31Spek E.J. Bui A.H. Lu M. Kallenbach N.R. Protein Sci. 1998; 7: 2431-2437Crossref PubMed Scopus (92) Google Scholar, 35Kammerer R.A. Jaravine V.A. Frank S. Schulthess T. Landwehr R. Lustig A. Garcia-Echeverria C. Alexandrescu A.T. Engel J. Steinmetz M.O. J. Biol. Chem. 2001; 276: 13685-13688Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) stability.On the basis of the interatomic distances in the final HY5 LZ structure, we predicted that five interhelical salt bridges occur at the dimer interface: Arg11(A)-Glu16(B), Glu16(A)-Arg11(B), Glu23(A)-Lys18(B), Glu32(A)-Arg37(B), and Arg37(A)-Glu32(B) (where A and B in parentheses refer to strands A and B) (Fig. 2A). In addition to the expected interhelical salt bridges between residues in positions e and g, intrahelical salt bridges were also predicted to form between the residues in positions c and g: Glu7-Arg11, Glu21-Lys18, and Glu21-Arg25 for strand A and Glu21-Lys18 and Glu21-Arg25 for strand B (Fig. 2A).FIGURE 2Electrostatic interactions of the HY5 LZ homodimer. A, helical wheel diagrams of the HY5 LZ homodimer. The view is from the N terminus. Heptad positions are labeled a-g. Solid and dashed gray lines indicate electrostatic attractions between oppositely charged residues and hydrogen bonds, respectively. Boxed amino acid residues are within the putative trigger sites. The intrahelical hydrogen bond between Leu8 and Glu9 in strand B was omitted for simplicity. B, front (upper) and back (lower) views of electrostatic surface potential representations of the HY5 LZ dimer. All positively charged residues and some negatively charged residues that interact with positive charges are labeled. Surface potentials were calculated using GRASP (59Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5313) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ninety-one percent of positions b, c, e, and g of the HY5 LZ domain have either charged or polar residues across all of the heptad repeats. Specifically, in the first three repeats, all of the residues in positions c and e are negatively charged, whereas all of the residues in positions b and g are either positively charged or polar. Because of the localized charge distribution along each helix, 8 of 10 intra/interhelical ion pairs occur in the N-terminal half of the HY5 LZ domain. Accordingly, the occurrence of a negative charge-rich region in the N-terminal half of the helix gives rise to a highly negative electrostatic surface potential, whereas the C-terminal half shows low electrostatic potential (Fig. 2B). Electrostatic attractions between oppositely charged residues provide local charge compensation in three different regions around the positively charged residues Arg11, Lys18/Arg25, and Arg37, which correspond to the N-terminal, central, and C-terminal domains of the helix, respectively. Of six positively charged residues in one strand, two are not involved in electrostatic interactions because of their positions in the helix and the orientation of their side chains. Specifically, the side chain of Lys13 stretches out to the exterior of the helix, thus avoiding the formation of a salt bridge with neighboring negatively charged residues, whereas Lys41 is not positioned near any negatively charged residues, thus rendering Lys41 incapable of forming an ion pair. Despite the net charge of +1 for the complete HY5 LZ domain, localized charge distributions allow the negatively charged N-terminal half of the helix to be distinguishable from the C-terminal half. This is the first LZ domain that shows such a localized charge distribution and thus a bipartite electrostatic surface potential.In general, charges are sufficiently delocalized such that repulsive interactions do not interfere with the conformation (folding and orientation) of LZ domains (7O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1273) Google Scholar, 32Mittl P.R. Deillon C. Sargent D. Liu N. Klauser S. Thomas R.M. Gutte B. Grutter M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2562-2566Crossref PubMed Scopus (61) Google Scholar, 36Whitson S.R. LeStourgeon W.M. Krezel A.M. J. Mol. Biol. 2005; 350: 319-337Crossref PubMed Scopus (32) Google Scholar). In the case of the HY5 LZ domain, however, a highly localized negative electrostatic potential seems to lead to unfavorable electrostatic repulsion in its N-terminal half. It is noteworthy that the negative charge-rich region in the N-terminal ha
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