High Resolution Structure of Deinococcus Bacteriophytochrome Yields New Insights into Phytochrome Architecture and Evolution
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m611824200
ISSN1083-351X
AutoresJeremiah R. Wagner, Junrui Zhang, J.S. Brunzelle, Richard D. Vierstra, Katrina T. Forest,
Tópico(s)Plant and Biological Electrophysiology Studies
ResumoPhytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IXα. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45Å resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C32 carbon of biliverdin to Cys24, the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors. Phytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IXα. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45Å resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C32 carbon of biliverdin to Cys24, the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors. Phytochromes (Phys) 3The abbreviations used are: Phy or PHY, phytochrome; BV, biliverdin IXα; CBD, chromophore-binding domain; DrCBD, D. radiodurans CBD; GAF, cGMP phosphodiesterase/adenyl cyclase/FhlA; HK, histidine kinase; PAS, Per/Arndt/Sim; PCB, phycocyanobilin; PΦB, phytochromobilin; Pfr, far red light-absorbing form of Phy; Pr, red-light absorbing form of Phy; R, red light; FR, far red light; BphP, bacteriophytochrome; DrBphP, D. radiodurans BphP.3The abbreviations used are: Phy or PHY, phytochrome; BV, biliverdin IXα; CBD, chromophore-binding domain; DrCBD, D. radiodurans CBD; GAF, cGMP phosphodiesterase/adenyl cyclase/FhlA; HK, histidine kinase; PAS, Per/Arndt/Sim; PCB, phycocyanobilin; PΦB, phytochromobilin; Pfr, far red light-absorbing form of Phy; Pr, red-light absorbing form of Phy; R, red light; FR, far red light; BphP, bacteriophytochrome; DrBphP, D. radiodurans BphP. comprise a ubiquitous superfamily of photoreceptors present in the plant, fungal, and bacterial kingdoms. They play critical roles in various light-regulated processes, ranging from phototaxis and pigmentation in bacteria to seed germination, chloroplast development, shade avoidance, and flowering in higher plants (1Vierstra R.D. Karniol B. Briggs W.R. Spudich J.L. Handbook of Photosensory Receptors. Wiley-VCH Press, Weinheim, Germany2005: 171-196Crossref Scopus (23) Google Scholar, 2Quail P.H. Nat. Rev. Mol. Cell Biol. 2002; 3: 85-93Crossref PubMed Scopus (574) Google Scholar). Phys are homodimeric complexes with each polypeptide containing an N-terminal chromophore-binding domain (CBD) that autocatalytically attaches via a thioether linkage a single linear tetrapyrrole (or bilin) chromophore, a PHY domain that is important for spectral integrity, and a C-terminal domain that promotes dimerization and often signal transmission (3Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (800) Google Scholar). Through unique interactions among the bilin and the CBD and PHY domains, Phys can exist as two metastable conformers, a red light (R)-absorbing Pr form and a far red light (FR)-absorbing Pfr form. By photoconverting between Pr and Pfr, Phys act as unique light-regulated switches in various signal transduction cascades. In bacteria and fungi, a canonical histidine kinase (HK) domain is typically present downstream of the PHY domain, thus allowing these Phys to participate in various two-component phosphorelays (1Vierstra R.D. Karniol B. Briggs W.R. Spudich J.L. Handbook of Photosensory Receptors. Wiley-VCH Press, Weinheim, Germany2005: 171-196Crossref Scopus (23) Google Scholar, 4Karniol B. Wagner J.R. Walker J.M. Vierstra R.D. Biochem. J. 2005; 392: 103-116Crossref PubMed Scopus (172) Google Scholar, 5Karniol B. Vierstra R.D. J. Bacteriol. 2004; 186: 445-453Crossref PubMed Scopus (77) Google Scholar). Although higher plant Phys contain a C-terminal HK-related sequence that promotes dimerization (2Quail P.H. Nat. Rev. Mol. Cell Biol. 2002; 3: 85-93Crossref PubMed Scopus (574) Google Scholar, 3Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (800) Google Scholar), it remains unclear if this domain has phosphotransferase activity (6Yeh K.C. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13976-13981Crossref PubMed Scopus (351) Google Scholar) or is even involved in signal transmission (7Matsushita T. Mochizuki N. Nagatani A. Nature. 2003; 424: 571-574Crossref PubMed Scopus (241) Google Scholar, 8Krall L. Reed J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8169-8174Crossref PubMed Scopus (80) Google Scholar). Despite intensive physico-chemical analysis of various Phys, we do not yet understand how contacts between the polypeptide and the bilin enable photointerconversion between Pr and Pfr, how this transformation reversibly alters the activity of the photoreceptor, or how the holoprotein dimerizes (3Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (800) Google Scholar). Important insights were made recently with our determination of the first three-dimensional structure of the CBD domain derived from the sole bacteriophytochrome (BphP) present in the proteobacterium Deinococcus radiodurans (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar). This ground state Pr structure confirmed sequence predictions that the CBD is composed of Per/Arndt/Sim (PAS) and cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and revealed that the GAF domain contains a deep pocket that cradles the bilin in a ZZZsyn, syn, anti conformation in the Pr form. The most unexpected feature of the DrCBD structure is the presence of a deep knot, where a conserved insertion within the GAF domain lassos the N-terminal 34 residues upstream of the PAS domain (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar, 10Virnau P. L A.M. Kardar M. PLoS Comput. Biol. 2006; 2: 1-9Crossref Scopus (261) Google Scholar). This knot bridges the PAS and GAF domains and contacts the chromophore, suggesting that it stabilizes the CBD and possibly participates in signal transmission. The high degree of conservation for residues that form the PAS and GAF domains, contact the chromophore, and create the knot strongly suggests that the unique topology of the DrCBD is present in most, if not all, members of the Phy superfamily and probably helps generate the unique R/FR photochromic spectral properties of these photoreceptors (4Karniol B. Wagner J.R. Walker J.M. Vierstra R.D. Biochem. J. 2005; 392: 103-116Crossref PubMed Scopus (172) Google Scholar, 9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar). Despite this similarity, there are differences in bilin preference and in the bilin attachment site between the bacterial and fungal Phys and their cyanobacterial and higher plant counterparts. In bacterial and fungal Phys, the chromophore is biliverdin IXα (BV) (11Froehlich A.C. Noh B. Vierstra R.D. Loros J. Dunlap J.C. Eukaryot. Cell. 2005; 4: 2140-2152Crossref PubMed Scopus (125) Google Scholar, 12Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (252) Google Scholar), which is synthesized by oxidative cleavage of heme by a heme oxygenase. BV is attached to the CBD via a thioether linkage between a conserved cysteine upstream of the PAS domain and the A-ring C3 vinyl side chain of BV (4Karniol B. Wagner J.R. Walker J.M. Vierstra R.D. Biochem. J. 2005; 392: 103-116Crossref PubMed Scopus (172) Google Scholar, 13Lamparter T. Michael N. Caspani O. Miyata T. Shirai K. Inomata K. J. Biol. Chem. 2003; 278: 33786-33792Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In contrast, the cyanobacterial and higher plant Phys use phycocyanobilin (PCB) or phytochromobilin (PΦB), respectively (14Yeh K.C. Wu S.H. Murphy J.T. Lagarias J.C. Science. 1997; 277: 1505-1508Crossref PubMed Scopus (448) Google Scholar, 15Lagarias J.C. Rapoport H. J. Am. Chem. Soc. 1980; 102: 4821-4828Crossref Scopus (259) Google Scholar, 16Lamparter T. Mittmann F. Gartner W. Borner T. Hartmann E. Hughes J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11792-11797Crossref PubMed Scopus (156) Google Scholar). Production of PCB and PΦB includes enzymatic reduction of the A-ring in BV to generate an ethylidene side chain at the C3 position. The conserved cysteine attachment site for these Phys is within the GAF domain (3Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (800) Google Scholar, 15Lagarias J.C. Rapoport H. J. Am. Chem. Soc. 1980; 102: 4821-4828Crossref Scopus (259) Google Scholar, 17Park C.M. Shim J.Y. Yang S.S. Kang J.G. Kim J.I. Luka Z. Song P.S. Biochemistry. 2000; 39: 6349-6356Crossref PubMed Scopus (48) Google Scholar, 18Wu S.H. Lagarias J.C. Biochemistry. 2000; 39: 13487-13495Crossref PubMed Scopus (158) Google Scholar). Although these differences in bilin chemistry and ligation site do not appreciably alter the ability of cyanobacterial and plant Phys to photointerconvert between Pr and Pfr, they may have important adaptive consequences for maximizing light perception. Most noticeably, they blue shift the absorption spectrum of Pr to better coincide with those of chlorophylls and thus to be more efficient in detecting shade from other photosynthetic organisms (19Otwinowski Z. Minor W. Macromol. Crystallogr. A. 1997; 276: 307-326Crossref Scopus (38526) Google Scholar, 20Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-167Crossref Scopus (5028) Google Scholar). In an attempt to better define the structure of Phys, we have engineered the surface of DrCBD to optimize crystal packing and have obtained substantially higher resolution diffraction. The new 1.45 Å resolution structure supports conclusions drawn from the previous 2.5 Å model (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar) and unequivocally confirms that BV is linked to Cys24 via the C32 carbon of the A-ring side chain. Comparisons of the crystal contacts in the 2.5 and 1.45 Å resolution structures identified an extensive buried surface between symmetry mates that we speculate represents a biologically relevant dimerization site in the photoreceptor. The 1.45 Å resolution structure also revealed that upon ligation, BV assumes a chiral center at C2 in the A-ring and is converted to 2(R),3(E)-PΦB, a structure similar to that proposed for bound PCB and PΦB. Given this similarity of the bound bilins, we then tested the hypothesis that one key step in the evolution of higher plant Phys from bacterial BphPs involved swapping the attachment site from the N-terminal region to the GAF domain to favor PCB/PΦB ligation. Collectively, our results provide a more accurate description of the bilin-binding pocket of a Phy and offer a partial mechanism for how plant Phys evolved from prokaryotic progenitors. Protein Production and Crystallization—DrCBD encompassing the first 321 amino acids of DrBphP and containing an N-terminal T7 tag and a C-terminal His6 tag was as described (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar). DrCBD-Y307S was generated by correcting the inadvertent point mutation that substituted Pro-240 for a Thr and converting the Tyr-307 codon to that for a serine with the QuikChange method (Stratagene, La Jolla, CA). The DrCBD-Y307S apoprotein was expressed in Escherichia coli strain Rosetta (DE3) (Novagen, San Diego, CA) with or without simultaneous expression of the heme oxygenase from Synechocystis PCC6803 (12Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (252) Google Scholar). The crude lysates were incubated for 30 min with a >10-fold molar excess of BV (Porphyrin Products, Logan, UT), and the resulting holoproteins were purified by sequential nickel-chelate affinity and anion exchange chromatography as previously described (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar). Bilin binding was assayed by zinc-induced fluorescence of the chromoprotein following SDS-PAGE (12Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (252) Google Scholar). Optimal crystallization conditions were identified by the hanging drop vapor diffusion method using the sparse matrix Cryoscreen (Nextal Biotechnologies, Montreal, Canada). Each trial included 2 μl of well solution and 2 μl of 20 mg/ml DrCBD-Y307S in 30 mm Tris-HCl (pH 8.0). Large crystals (0.2 × 0.18 × 0.08 mm) were generated with 0.095 m sodium citrate (pH 5.6), 19% (v/v) isopropyl alcohol, 19% (v/v) polyethylene glycol 4000, and 5% (v/v) glycerol. The crystals were briefly washed in mother liquor and then flash-frozen in liquid nitrogen. Structure Determination—Two x-ray diffraction data sets were collected on DrCBD-Y307S crystals. One data set was collected at the University of Wisconsin (Madison, WI) with a Proteum CCD detector with x-rays generated by a Microstar rotating anode (Bruker AXS, Madison, WI). One degree images were captured for 360 degrees with 30-s exposure times and processed with HKL2000 and Scalepack (19Otwinowski Z. Minor W. Macromol. Crystallogr. A. 1997; 276: 307-326Crossref Scopus (38526) Google Scholar) (Table 1). The second data set was collected at the Advanced Photonic Source (APS, Argonne, IL) on beamline 51DB using a MAR225 detector. One degree images were captured for 180 degrees with 1.2-s exposure times. These images were integrated and scaled to the edge of the detector to a maximum resolution of 1.45 Å (Table 1).TABLE 1Data collection and refinement statisticsData setRotating anodeSynchrotronData collectionWavelength (Å)1.540.972No. of unique reflections17,484 (1,401)aThe value for the highest resolution shell is shown in parentheses.56,868 (4,813)Resolution (Å)24-2.15 (2.23-2.15)20-1.45 (1.5-1.45)Completeness (%)96.4 (78.8)97.5 (83.3)Redundancy6.2 (2.6)3.7 (2.9)Rsymm (%)10.4 (33.3)3.9 (27.6)I/σI10.0 (2.8)12.6 (3.0)Space groupC2C2Cell dimensionsa, b, c (Å)89.5, 51.6, 80.989.3, 51.8, 80.4α, β, γ (degrees)90.0, 116.4, 90.090.0, 116.3, 90.0Refinement statisticsResolution range (Å)24.0-2.15 (2.2-2.15)20-1.45 (1.49-1.45)R-factor (%)18.9 (21.8)16.5 (21.3)Free R-factor (%)23.7 (25.1)19.2 (27.3)Protein atoms2,4452,531Solvent molecules111301Heteroatoms4343Dual conformers1025Isotropic average temperature factors (Å2)Overall32.424.2Protein atoms32.623.2Heteroatoms22.921Water molecules31.832.1Root mean square deviationsBond lengths (Å)0.0140.012Bond angles (degrees)1.81.8Estimated coordinate error (Å2)0.140.044a The value for the highest resolution shell is shown in parentheses. Open table in a new tab Phases for both x-ray data sets were determined by molecular replacement with the program AmoRe (20Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-167Crossref Scopus (5028) Google Scholar), using the previously described DrCBD structure (Protein Data Bank accession code 1ZTU (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar)) without the chromophore or His6 tag as the search model. Unambiguous solutions for both DrCBD-Y307S data sets were nearly identical with correlation coefficients of 68.2 and 67.0 and R-factors of 42.6 and 38.5% for the low resolution and high resolution data sets, respectively. After an initial round of positional refinement with Refmac5 (21Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13854) Google Scholar), electron density maps were constructed; nearly all of the residues from the search model correctly fit into a 2Fo - Fc electron density map. The protein-chromophore linkage was modeled by creating a modified amino acid consisting of a cysteine bonded via a thioether linkage to the C32 carbon of the A-ring vinyl group of BV. To account for the rotation of the D-ring, the energy penalty for rotation about the C15=C16 double bond in this adduct was removed. This adduct was introduced into the structure using the Fo - Fc difference electron density maps, and the model was again refined with Refmac5 (24Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D. 2001; 57: 122-133Crossref PubMed Scopus (1651) Google Scholar). Based on interpretation of both the 2Fo - Fc and Fo - Fc electron density maps generated from data to 1.45 Å, the double bond in the A-ring was removed from between the C2 and C3 carbons and introduced between the C3 and C31 carbons, and the restraints forcing the A-ring to be planar and parallel with the B- and C-rings were released. Both models were refined by iterative rounds of model building with Xfit (22McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2021) Google Scholar) or COOT (23Emsley P. Cowtan K. Acta Crystallogr. Sect. D. 2004; 60: 2126-2132Crossref PubMed Scopus (23226) Google Scholar) and TLS and positional refinement with Refmac5 (24Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D. 2001; 57: 122-133Crossref PubMed Scopus (1651) Google Scholar). The final models have excellent geometry and appropriate R-factors (Table 1). In both models, the 16 N-terminal residues (13 of which comprise the T7 tag) and the 5 C-terminal histidines of the His6 tag were not modeled because of disorder. Although the model constructed from the 1.45 Å data set exhibited no gaps in the polypeptide chain, several residues forming the linker between the PAS and GAF domains were omitted in the 2.15 Å model. Data collection and refinement statistics are presented in Table 1. Structure figures were generated with PyMOL (Delano Scientific, Palo Alto, CA). Atomic coordinates and structure factors for the 2.15 and 1.45 Å resolution models have been deposited in the Protein Data Bank (25Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27325) Google Scholar) under accession codes 2O9B and 2O9C, respectively. Native Size Determinations—The coding sequence for DrCBD-PHY (amino acids 1-501) was PCR-amplified from the full-length DrBphP construction by using primers designed to introduce BamHI and XhoI sites to the 5′ and 3′ ends, respectively. The BamHI-XhoI-digested PCR product was cloned into pET21b(+) (Novagen), which was similarly digested, to direct the expression of DrCBD-PHY bearing an N-terminal T7 tag and a C-terminal His6 tag. Nickel-chelate affinity-purified fulllength DrBphP, DrCBD-PHY, DrCBD, and DrCBD-Y307S were concentrated by ammonium sulfate precipitation and resuspended in 150 mm NaCl and 50 mm Tris-HCl (pH 7.5) to a final concentration of ∼5 mg/ml. Dynamic light scattering was performed using a DynaPro (Protein Solutions/Wyatt Technology Corp., Santa Barbara, CA) multiangle light scattering instrument with a 5 mg/ml concentration of each DrBphP dissolved in 30 mm Tris-HCl (pH 8.0). Approximate molecular radii assuming spherical proteins were calculated by the Dynamics software package (Wyatt Technology Corp.). Size exclusion fast protein liquid chromatography was performed using a Superdex 200 (Amersham Biosciences) column equilibrated with 150 mm NaCl and 50 mm Tris-HCl (pH 7.5) and a flow rate of 200 μl/min. The column was calibrated using the gel filtration standards from Bio-Rad. Nondenaturing PAGE was performed as described (26Book A.J. Yang P. Scalf M. Smith L.M. Vierstra R.D. Plant Physiol. 2005; 138: 1046-1057Crossref PubMed Scopus (53) Google Scholar) using bovine serum albumin (U. S. Biochemical Corp.) and ovalbumin (Sigma) as standards (16Lamparter T. Mittmann F. Gartner W. Borner T. Hartmann E. Hughes J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11792-11797Crossref PubMed Scopus (156) Google Scholar). Analysis of Site-directed Mutants—The PAS and GAF cysteine codon mutations were generated by the QuikChange method using the full-length DrBphP construction bearing a C-terminal His6 tag (12Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (252) Google Scholar). To assess chromophore preference, the apoproteins were expressed as above without co-expression of Synechocystis heme oxygenase (12Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (252) Google Scholar) and assembled with the chromophore in vitro, using a >10-fold molar excess of either BV, PCB purified from Spirulina platensis (27Terry M.J. Maines M.D. Lagarias J.C. J. Biol. Chem. 1993; 268: 26099-26106Abstract Full Text PDF PubMed Google Scholar), or various ratios of BV and PCB. The assembled proteins were purified from the crude lysates by nickel-chelate affinity chromatography. Absorption and difference spectra were obtained with a PerkinElmer Life Sciences Lambda 650 spectrophotometer following saturating irradiations with R (690 or 660 nm) and FR (770 nm) enriched by interference filters. To quantitate binding efficiency of BV relative to PCB, the absorbance maxima of BV- and PCB-bound proteins were identified in the Soret and Q bands for the Pr spectrum generated by each BV/PCB mixture. By incorporating these absorbance values in the ratio (QBV/QPCB)/(SoretBV/SoretPCB), the relative contributions of BV-bound and PCB-bound holoproteins in the mixtures were calculated. These ratios were normalized by subtracting the value calculated for the 100% PCB sample (100% PCB-bound sample now represented 0% BV incorporation) from all other values and scaling all of the adjusted values so that the values from samples generated solely with BV equaled 100%. Crystal Lattice Engineering Leads to a High Resolution Structure of DrCBD—To better define the structure of BV and its interactions with the CBD and to generate crystals suitable for time-resolved x-ray analysis of the Pr to Pfr phototransformation, we sought to improve the diffraction resolution of DrCBD crystals. We noticed a chemically impossible overlap of symmetry-related Tyr-307 side chains in the 1ZTU structure (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar) that could create local disorder detrimental to crystal formation (Fig. 1A). To reduce this conflict, we replaced Tyr-307 with Ser (Fig. 1B), reasoning that a substitution of residue 307 should not appreciably affect DrCBD folding or photochemical activity, given its surface location and considerable distance from the chromophore (Fig. 1C). Indeed, the DrCBD-Y307S apoprotein was stable and soluble and readily assembled with BV to generate a chromoprotein spectrophotometrically indistinguishable from DrCBD following R or FR irradiation. 4J. R. Wagner, J. Zhang, R. D. Vierstra, and K. T. Forest, unpublished data. DrCBD-Y307S holoprotein did not crystallize under the previously described condition (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar), but in subsequent unbiased screens, we identified new conditions that readily generated bright blue crystals in the space group C2. Importantly, the DrCBD-Y307S crystals yielded data sets to maximum resolutions of 2.15 and 1.45 Å using x-rays generated from a rotating anode and synchrotron radiation, respectively (Table 1). From this pair of data sets, two models for DrCBD-Y307S were created by molecular replacement and subsequent refinement (Table 1). Whereas the prior 2.5 Å model lacked several residues in two disordered loops (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar), the 1.45 Å model for DrCBD-Y307S includes all 318 residues of the CBD with no breaks in the polypeptide chain from amino acid 4 to 322 (Fig. 1D). Alternate conformations were modeled for 25 mostly solvent-exposed residues; however, even in this high resolution structure, seven disordered side chains were truncated to alanine. Although DrCBD-Y307S crystallized in a different space group than DrCBD, the 1ZTU model and the new structure were remarkably coherent, with a least squares alignment of all protein backbone atoms yielding a root mean square deviation of 0.49 Å2. Even the structure of α8 surrounding residue 307 was not substantially altered, indicating that the Tyr to Ser substitution in DrCBD-Y307S had minimal impact on CBD folding. The adjacent α4 was slightly distorted, presumably to accommodate the altered crystal packing (Fig. 1, A-C). In contrast to the clash at Tyr-307 with its symmetry mate in the DrCBD structure, the Ser at this position in the DrCBD-Y307S structure pointed away from its symmetry mate, which we presume improved crystal packing (Fig. 1B). The higher resolution model confirmed the presence of the knot between the GAF and PAS domains (Fig. 1D), along with the positioning of Ile35 and Gln36 at its center (9Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (435) Google Scholar). As recently noted by Virnau et al. (10Virnau P. L A.M. Kardar M. PLoS Comput. Biol. 2006; 2: 1-9Crossref Scopus (261) Google Scholar) in a large scale survey of knotted protein structures, the knot in DrCBD has four crossover points and is thus defined as a figure-of-eight knot (Fig. 1, D and E). Although we have not explicitly addressed the mechanism of protein folding in our current work, we note parenthetically that this accurately refined CBD structure revealed a cis peptide bond at Asp235-Pro236 within the extended GAF insertion that forms the knot crossover (Fig. 1D). Cis-trans isomerization of prolines is well known to be a rate-limiting step in protein folding in general (28Kiefhaber T. Schmid F.X. J. Mol. Biol. 1992; 224: 231-240Crossref PubMed Scopus (100) Google Scholar), and is important for the folding of at least one knotted protein (29Mallam A.L. Jackson S.E. J. Mol. Biol. 2005; 346: 1409-1421Crossref PubMed Scopus (107) Google Scholar). For this reason, we speculate that the proline-rich nature of the GAF insertion (5 prolines within 15 residues) and in particular the cis conformation of Pro236 could be critical in stabilizing intermediate folded states until the knot is threaded and the hydrophobic amino acids in the GAF domain insertion condense around the invariant Ile35 within the N-terminal extension. Dimerization of DrCBD—Our previous 2.5 Å resolution structure of DrCBD contained a large contact surface between 2-fold symmetry mates. Although we altered a residue in this crystallographic interface and obtained a different space group (C2 versus P21212), the same overall di
Referência(s)