PAS Domains
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m301701200
ISSN1083-351X
AutoresJocelyne Vreede, Michael Horst, Klaas J. Hellingwerf, Wim Crielaard, Daan M. F. van Aalten,
Tópico(s)bioluminescence and chemiluminescence research
ResumoPAS (PER-ARNT-SIM) domains are a family of sensor protein domains involved in signal transduction in a wide range of organisms. Recent structural studies have revealed that these domains contain a structurally conserved α/β-fold, whereas almost no conservation is observed at the amino acid sequence level. The photoactive yellow protein, a bacterial light sensor, has been proposed as the PAS structural prototype yet contains an N-terminal helix-turn-helix motif not found in other PAS domains. Here we describe the atomic resolution structure of a photoactive yellow protein deletion mutant lacking this motif, revealing that the PAS domain is indeed able to fold independently and is not affected by the removal of these residues. Computer simulations of currently known PAS domain structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities. The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins. PAS (PER-ARNT-SIM) domains are a family of sensor protein domains involved in signal transduction in a wide range of organisms. Recent structural studies have revealed that these domains contain a structurally conserved α/β-fold, whereas almost no conservation is observed at the amino acid sequence level. The photoactive yellow protein, a bacterial light sensor, has been proposed as the PAS structural prototype yet contains an N-terminal helix-turn-helix motif not found in other PAS domains. Here we describe the atomic resolution structure of a photoactive yellow protein deletion mutant lacking this motif, revealing that the PAS domain is indeed able to fold independently and is not affected by the removal of these residues. Computer simulations of currently known PAS domain structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities. The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins. PER-ARNT-SIM periodic clock pattern light oxygen voltage photoactive yellow protein ground state 4-morpholineethanesulfonic acid wild type PAS1 domains are structural modules that can be found in proteins in all kingdoms of life (1Taylor B.L. Zhulin I.B. Microbiol. Mol. Biol. Rev. 1999; 63: 479-506Crossref PubMed Google Scholar, 2Zhulin I.B. Taylor B.L. Trends Biochem. Sci. 1997; 22: 331-333Abstract Full Text PDF PubMed Scopus (345) Google Scholar). The PAS module was first identified in theDrosophila clock protein PER and the basic helix-loop-helix containing transcription factors ARNT (aryl-hydrocarbon receptor nuclear translocator) in mammals and SIM (single-minded protein) iinsects (3Nambu J.R. Lewis J.O. Wharton K.A. Crews S.T. Cell. 1991; 67: 1157-1167Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Most PAS domains are sensory modules, typically sensing oxygen tension, redox potential, or light intensity (1Taylor B.L. Zhulin I.B. Microbiol. Mol. Biol. Rev. 1999; 63: 479-506Crossref PubMed Google Scholar, 4Pellequer J.-L. Wager-Smith K.A. Kay S.A. Getzoff E.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5884-5890Crossref PubMed Scopus (218) Google Scholar). Alternatively, they mediate protein-protein interactions or bind small ligands (5Anantharaman V. Koonin E. Aravind L. J. Mol. Biol. 2001; 307: 1271-1292Crossref PubMed Scopus (216) Google Scholar). Although the amino acid sequences of the different PAS domains show little similarity, their three-dimensional structures appear to be conserved. All of the PAS domains resemble the structure of photoactive yellow protein (PYP) (4Pellequer J.-L. Wager-Smith K.A. Kay S.A. Getzoff E.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5884-5890Crossref PubMed Scopus (218) Google Scholar), a photoreceptor presumed to be involved in a phototactic response of the bacterium Ectothiorhodospira halophila to intense blue light (6Sprenger W.W. Hoff W.D. Armitage J.P. Hellingwerf K.J. J. Bacteriol. 1993; 175: 3096-3104Crossref PubMed Scopus (350) Google Scholar). Its structure reveals an α/β-fold with the light-sensitive chromophore p-coumaric acid bound to the protein via a thioester linkage (7Borgstahl G.E.O. Williams D.R. Getzoff E.D. Biochemistry. 1995; 34: 6278-6287Crossref PubMed Scopus (439) Google Scholar). It is the only PAS domain of which the catalytic function, i.e. signal generation and transduction, has been studied in great detail. The protein has been shown to undergo a photocycle linked to isomerization of the chromophore (8Xie A.H. Hoff W.D. Kroon A.R. Hellingwerf K.J. Biochemistry. 1996; 35: 14671-14678Crossref PubMed Scopus (183) Google Scholar, 9Perman B. Srajer V. Ren Z. Teng T. Pradervand C. Ursby T. Bourgeois D. Schotte F. Wulff M. Kort R. Hellingwerf K. Moffat K. Science. 1998; 279: 1946-1950Crossref PubMed Scopus (284) Google Scholar, 10Kort R. Vonk H. Xu X. Hoff W.D. Crielaard W. Hellingwerf K.J. FEBS Lett. 1996; 32: 73-78Crossref Scopus (204) Google Scholar, 11Genick U.K. Borgstahl G.E.O. Kingman N. Ren Z. Pradervand C. Burke P. Srajer V. Teng T. Schildkamp W. McRee D.E. Moffat K. Getzoff E.D. Science. 1997; 275: 1471-1475Crossref PubMed Scopus (388) Google Scholar, 12Genick U.K. Soltis S.M. Kuhn P. Canestrelli I.L. Getzoff E.D. 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Biophys. J. 1998; 75: 406-412Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). A more moderately red-shifted intermediate absorbing maximally at 465 nm is formed on the nanosecond time scale (18Hoff W.D. van Stokkum I.H.M. van Ramesdonk H.J. van Brederode M.E. Brouwer A.M. Fitch J.C. Meyer T.E. van Grondelle R. Hellingwerf K.J. Biophys. J. 1994; 67: 1691-1705Abstract Full Text PDF PubMed Scopus (252) Google Scholar). The red-shifted intermediate spontaneously converts into a blue-shifted intermediate absorbing maximally at 355 nm at the sub-millisecond time scale (18Hoff W.D. van Stokkum I.H.M. van Ramesdonk H.J. van Brederode M.E. Brouwer A.M. Fitch J.C. Meyer T.E. van Grondelle R. Hellingwerf K.J. Biophys. J. 1994; 67: 1691-1705Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 19Meyer T.E. Yakali E. Cusanovich M.A. Tollin G. Biochemistry. 1987; 26: 418-423Crossref PubMed Scopus (286) Google Scholar). The blue-shifted intermediate subsequently relaxes back to pG on a sub-second time scale (15Meyer T.E. Tollin G. Causgrove T.P. Cheng P. Blankenship R.E. Biophys. J. 1991; 59: 988-991Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 18Hoff W.D. van Stokkum I.H.M. van Ramesdonk H.J. van Brederode M.E. Brouwer A.M. Fitch J.C. Meyer T.E. van Grondelle R. Hellingwerf K.J. Biophys. J. 1994; 67: 1691-1705Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 19Meyer T.E. Yakali E. Cusanovich M.A. Tollin G. Biochemistry. 1987; 26: 418-423Crossref PubMed Scopus (286) Google Scholar, 20Meyer T.E. Tollin G. Hazzard J.H. Cusanovich M.A. Biophys. J. 1989; 56: 559-564Abstract Full Text PDF PubMed Scopus (167) Google Scholar) or faster in a light-dependent reaction (21Miller A. Leigeber H. Hoff W.D. Hellingwerf K.J. Biochim. Biophys. Acta. 1998; 1141: 190-196Crossref Scopus (38) Google Scholar, 22Hendriks J. Hoff W.D. Crielaard W. Hellingwerf K.J. J. Biol. Chem. 1999; 274: 17655-17660Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Several detailed studies, including Laue diffraction and cryo-crystallography (9Perman B. Srajer V. Ren Z. Teng T. Pradervand C. Ursby T. Bourgeois D. Schotte F. Wulff M. Kort R. Hellingwerf K. Moffat K. Science. 1998; 279: 1946-1950Crossref PubMed Scopus (284) Google Scholar, 11Genick U.K. Borgstahl G.E.O. Kingman N. Ren Z. Pradervand C. Burke P. Srajer V. Teng T. Schildkamp W. McRee D.E. Moffat K. Getzoff E.D. Science. 1997; 275: 1471-1475Crossref PubMed Scopus (388) Google Scholar,12Genick U.K. Soltis S.M. Kuhn P. Canestrelli I.L. Getzoff E.D. Nature. 1998; 392: 206-209Crossref PubMed Scopus (327) Google Scholar), NMR spectroscopy (23Rubinstenn G. Vuister G.W. Mulder F.A.A. Dux P.E. Boelens R. Hellingwerf K.J. Kaptein R. Nat. Struct. Biol. 1998; 5: 568-570Crossref PubMed Scopus (177) Google Scholar), small angle x-ray scattering (24Sasaki J. Kumauchi M. Hamada N. Oka T. Tokunaga F. Biochemistry. 2002; 41: 1915-1922Crossref PubMed Scopus (40) Google Scholar, 25Imamoto Y. Kamikubo H. Harigai M. Shimizu N. Kataoka M. Biochemistry. 2002; 41: 13595-135601Crossref PubMed Scopus (64) Google Scholar), biochemical experiments (26van Brederode M.E. Hoff W.D. van Stokkum I.H.M. Groot M.L. Hellingwerf K.J. Biophys. J. 1996; 71: 365-380Abstract Full Text PDF PubMed Scopus (115) Google Scholar), and Fourier transform infrared spectroscopy (27Hoff W.D. Xie A. van Stokkum I.H.M. Tang X.-J. Gural J. Kroon A.R. Hellingwerf K.J. Biochemistry. 1999; 38: 1009-1017Crossref PubMed Scopus (121) Google Scholar, 28Xie A.H. Kelemen L. Hendriks J. White B.J. Hellingwerf K.J. Hoff W.D. Biochemistry. 2001; 40: 1510-1517Crossref PubMed Scopus (211) Google Scholar) and computer simulations (29van Aalten D.M.F. Hoff W.D. Findlay J.B.C. Crielaard W. Hellingwerf K.J. Protein Eng. 1998; 11: 873-879Crossref PubMed Scopus (28) Google Scholar, 30van Aalten D.M.F. Crielaard W. Hellingwerf K.J. Joshua-Tor L. Protein Sci. 2000; 9: 64-72Crossref PubMed Scopus (43) Google Scholar), have revealed that during the PYP photocycle distinct significant conformational changes occur. It is these conformational changes that are thought to translate the photon signal into a cellular response via subsequent protein/protein interactions. To study the possible protein motions involved in the photocycle, PYP dynamics have been investigated by computer simulation (29van Aalten D.M.F. Hoff W.D. Findlay J.B.C. Crielaard W. Hellingwerf K.J. Protein Eng. 1998; 11: 873-879Crossref PubMed Scopus (28) Google Scholar). This study suggested that chromophore-linked concerted motions may be present in pG and that these motions might be amplified upon isomerization of the chromophore. The simulations, later supported by x-ray crystallographic studies (30van Aalten D.M.F. Crielaard W. Hellingwerf K.J. Joshua-Tor L. Protein Sci. 2000; 9: 64-72Crossref PubMed Scopus (43) Google Scholar), also suggest that conserved glycines were serving as hinge points, allowing substructures in the protein to fluctuate relative to each other. In a subsequent study where the rigidity of the PYP backbone was altered by mutation of these glycines, the role of these hinge points in the signal transduction process was further confirmed (31van Aalten D.M.F. Haker A. Hendriks J. Hellingwerf K. Joshua-Tor L. Crielaard W. J. Biol. Chem. 2002; 277: 6463-6468Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The glycines that were investigated in this study fall within the PAS-fold (4Pellequer J.-L. Wager-Smith K.A. Kay S.A. Getzoff E.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5884-5890Crossref PubMed Scopus (218) Google Scholar) and show a large degree of conservation throughout the PAS family. This has led to the speculation that apart from a conserved structure the PAS domains may have similar conformational freedom and associated signal transduction mechanism (30van Aalten D.M.F. Crielaard W. Hellingwerf K.J. Joshua-Tor L. Protein Sci. 2000; 9: 64-72Crossref PubMed Scopus (43) Google Scholar). Here we investigate whether the dynamic properties of PAS domains are intrinsic properties associated with their conserved fold. First, we have mutated the PYP from E. halophila into a minimal PAS domain by the removal of the N-terminal cap (see also Ref. 32van der Horst M.A. van Stokkum I.H. Crielaard W. Hellingwerf K.J. FEBS Lett. 2001; 497: 26-30Crossref PubMed Scopus (69) Google Scholar). To be able to tackle the dynamic properties of this minimal PAS domain, its three-dimensional structure was refined against 1.14-Å synchrotron diffraction data. Second, this structure was used in a comparative computational study on the conformational flexibility of all of the PAS domains for which crystals structures are available: HERG, the N-terminal domain of a human potassium channel (33Cabral J.H.M. Lee A. Cohen S.L. Chait B.T. Li M. MacKinnon R. Cell. 1998; 95: 649-655Abstract Full Text Full Text PDF PubMed Google Scholar); LOV2, a photoreceptor domain from plants (34Crosson S. Moffat K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2995-3000Crossref PubMed Scopus (419) Google Scholar); and FixL, a bacterial oxygen sensor (35Gong W. Hao B. Mansy S.S. Gonzalez G. Gilles-Gonzalez M.A. Chan M.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15177-15182Crossref PubMed Scopus (343) Google Scholar). Essential dynamics analyses on the sampled configurational space of all of these PAS domains reveal conserved concerted motions. This supports the hypothesis that the common structure of PAS domains implies common flexibility and that it is this conserved property that is fundamental for PAS domain function in signal transduction. Δ25PYP encompassing residues 26–125 of PYP was expressed and purified as described previously (32van der Horst M.A. van Stokkum I.H. Crielaard W. Hellingwerf K.J. FEBS Lett. 2001; 497: 26-30Crossref PubMed Scopus (69) Google Scholar). Crystals were grown by equilibration of 1 μl of 30 mg/ml protein with 1 μl of mother liquor (1.8 m ammonium sulfate, 10 mmCoCl2, 100 mm MES, pH 6.5) against a 1-ml reservoir of mother liquor. Crystals appeared after 2–3 days with a largest dimension of 0.4 mm. Diffraction data were collected at beamline ID14-EH1 (European Synchrotron Radiation Facility, Grenoble, France) and processed with the HKL package (Table I) (36Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). The structure of Δ25PYP was solved by molecular replacement with AMoRe (37Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar) using the native PYP structure (Protein Data Bank code 2PHY) (7Borgstahl G.E.O. Williams D.R. Getzoff E.D. Biochemistry. 1995; 34: 6278-6287Crossref PubMed Scopus (439) Google Scholar) as a search model (excluding the chromophore) against 8–4-Å data. A solution was found (r = 0.479, correlation coefficient = 0.282) with two molecules in the asymmetric unit. Initial refinement was carried out with CNS (38Brunger A.T. Adams P.D. Clore G.M. 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. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar) interspersed with model building in O (39Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). The chromophore was not included in the refinement until it was well defined by an unbiased Fo − Fc ,φcalc map (Fig. 1). Further rounds of refinement with SHELX97 (40Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1886) Google Scholar) allowed the placement of water molecules and the assignment of some alternate side chain conformations. In the last stages of the refinement, hydrogen atoms were included (Table I).Table IDetails of data collection and structure refinementResolution range (Å)17-1.14 (1.18-1.14)No. observed reflections299780 (23225)No. unique reflections75208 (7010)Redundancy4.0 (3.3)I/ςI13.5 (4.3)Completeness (%)94.4 (89.2)Rmerge0.060 (0.217)Rcryst, Rfree0.147, 0.177No. groups200 residues, 406 H2Oroot mean square deviation from ideal geometry Bonds (Å)0.010 Angles (°)2.0B-factor root mean square deviation (Å2) all bonds2.6B (Å2)15.8 (protein), 33.5 (water)Values between brackets are for the highest resolution shell. Crystals were of space group P43212 (a =b = 82.57 Å, c = 63.45 Å) and were cryo-cooled to 100 K. All measured data were included in structure refinement. Open table in a new tab Values between brackets are for the highest resolution shell. Crystals were of space group P43212 (a =b = 82.57 Å, c = 63.45 Å) and were cryo-cooled to 100 K. All measured data were included in structure refinement. Residues 113 (leucine) and 114 (serine) in one monomer and residue 116 (aspartic acid) in the other monomer were disordered, although some evidence for several possible conformations was visible in the map. The building of these regions was attempted, but their conformations could not be determined with confidence. Similar observations were made in previous crystallographic studies of PYP in the P65 space group (30van Aalten D.M.F. Crielaard W. Hellingwerf K.J. Joshua-Tor L. Protein Sci. 2000; 9: 64-72Crossref PubMed Scopus (43) Google Scholar, 31van Aalten D.M.F. Haker A. Hendriks J. Hellingwerf K. Joshua-Tor L. Crielaard W. J. Biol. Chem. 2002; 277: 6463-6468Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 41van Aalten D.M.F. Crielaard W. Hellingwerf K. Joshua-Tor L. Acta Crystallogr. Sec. D. 2002; 58: 585-590Crossref PubMed Scopus (12) Google Scholar). At the N terminus, the well defined electron density was present for Ala-27 at the early stages of refinement. Subsequent maps also defined the conformation of Leu-26. Sampling of conformational space by the computer simulation method CONCOORD (42de Groot B.L. van Aalten D.M.F. Scheek R.M. Amadei A. Vriend G. Berendsen H.J.C. Proteins. 1997; 29: 240-251Crossref PubMed Scopus (227) Google Scholar) was performed for crystal structures of the PAS domains depicted in Fig. 2. Besides these existing PAS domain structures, the Δ25PYP crystal structure described here was also simulated. As a negative control for the subsequent comparisons, a CONCOORD ensemble starting from the crystal structure of turkey lysozyme (Protein Data Bank code135L) bearing no structural resemblance to PAS domains was also calculated. During the CONCOORD runs, 1000 structures were generated and a damping factor of 0.25 was applied to avoid unreasonable side chain geometries. Essential dynamics (43Amadei A. Linssen A.B.M. Berendsen H.J.C. Proteins. 1993; 17: 412-425Crossref PubMed Scopus (2636) Google Scholar) determines concerted motions of atoms from an ensemble of structures, for example, a set of crystal structures (44van Aalten D.M.F. Conn D.A. de Groot B.L. Berendsen H.J.C. Findlay J.B.C. Amadei A. Biophys. J. 1997; 73: 2891-2896Abstract Full Text PDF PubMed Scopus (72) Google Scholar, 45de Groot B.L. Hayward S. van Aalten D.M.F. Amadei A. Berendsen H.J.C. Proteins. 1998; 31: 116-127Crossref PubMed Scopus (156) Google Scholar, 46van Aalten D.M.F. Chong C.R. Joshua-Tor L. Biochemistry. 2000; 39: 10082-10089Crossref PubMed Scopus (21) Google Scholar, 47Biondi R.M. Komander D. Thomas C.C. Deak J.M.L.M. Alessi D.R. van Aalten D.M.F. EMBO J. 2002; 21: 4219-4228Crossref PubMed Scopus (166) Google Scholar) or a trajectory from a computer simulation (43Amadei A. Linssen A.B.M. Berendsen H.J.C. Proteins. 1993; 17: 412-425Crossref PubMed Scopus (2636) Google Scholar, 48van Aalten D.M.F. Amadei A. Linssen A.B.M. Eijsink V.G.H. Vriend G. Berendsen H.J.C. Proteins. 1995; 22: 45-54Crossref PubMed Scopus (181) Google Scholar, 49van Aalten D.M.F. Amadei A. Bywater R. Findlay J.B.C. Berendsen H.J.C. Sander C. Stouten P.F.W. Biophys. J. 1996; 70: 684-692Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 50van Aalten D.M.F. Findlay J.B.C. Amadei A. Berendsen H.J.C. Protein Eng. 1995; 8: 1129-1135Crossref PubMed Scopus (168) Google Scholar, 51Mello L.V. de Groot B.L. Li S.L. Jedrzejas M.J. J. Biol. Chem. 2002; 277: 36678-36688Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Here the CONCOORD ensembles were used as input. A covariance matrix is constructed that describes the correlation of the positional shifts of one atom with those of another atom as shown in Equation 1, Cij=((xi−xi,0)(xj−xj,0))Equation 1 where xi and xj represent the coordinates of atoms i and j in a conformation, whereas xi,0 andxj,0 represent the average coordinates of the atoms over the ensemble. The average is calculated over all structures after they are superimposed on a reference structure to remove overall translational and rotational motion. Diagonalizing this matrix yields a set of eigenvectors and eigenvalues. The eigenvectors are directions in a 3N-dimensional space (where Nis the number of atoms), and motion along a single eigenvector corresponds to concerted displacements of groups of atoms in Cartesian space. The eigenvalues are a measure of the mean square fluctuation of the system along the corresponding eigenvectors. The eigenvectors are sorted according to their eigenvalue, the first eigenvector having the largest eigenvalue. To allow direct comparison of concerted motions for different proteins, an equal number of atoms must be used in the essential dynamics calculations. A first simplification is that only Cαatoms are taken into account, which sufficiently represent the large motions of the protein backbone (48van Aalten D.M.F. Amadei A. Linssen A.B.M. Eijsink V.G.H. Vriend G. Berendsen H.J.C. Proteins. 1995; 22: 45-54Crossref PubMed Scopus (181) Google Scholar, 52van Aalten D.M.F. de Groot B.L. Findlay J.B.C. Berendsen H.J.C. Amadei A. J. Comp. Chem. 1997; 18: 169-181Crossref Scopus (147) Google Scholar). When the structures also contain insertions and deletions such as in the PAS domains (Fig. 3), further simplifications will need to be applied to reduce all of the structures to a common core (44van Aalten D.M.F. Conn D.A. de Groot B.L. Berendsen H.J.C. Findlay J.B.C. Amadei A. Biophys. J. 1997; 73: 2891-2896Abstract Full Text PDF PubMed Scopus (72) Google Scholar). Residues in the PAS domains that overlapped structurally were selected by the DALI server (53Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3565) Google Scholar), which performs a pairwise comparison of secondary structure elements. The results of the pairwise alignment on secondary structure were compared to yield the common structural elements present in the PAS domains (Fig. 3). For lysozyme, the negative control, an equal number of residues was selected starting from the N terminus. The structure of Δ25PYP was solved by molecular replacement and refined to a 1.14-Å resolution (R-factor = 0.147,Rfree = 0.177) (Fig. 1 and TableI). The asymmetric unit contains two protein molecules related by a non-crystallographic 2-fold rotation axis (Fig. 1). The molecules have a similar conformation with a root mean square deviation of 0.77 Å on Cα atoms. Compared with the wtPYP structure, the two molecules superimpose with root mean square deviations of 0.99 and 0.76 Å, respectively. From these superpositions, positional shifts of the Cα atoms of the mutant structures with respect to the positions of the Cαatoms in wild type PYP are given in Fig.2. The N terminus and the loops consisting of residues 84–88, 98–101, and 111–117 in Δ25PYP have a different conformation than those in wild type PYP. The different conformation of the Δ25PYP N terminus compared with the equivalent residues in wtPYP is most probably caused by the deletion of the first 25 residues (Fig.3). When the first two residues at the N terminus of Δ25PYP are excluded from the superposition, the root mean square deviation is reduced by ∼0.2 Å. From the NMR structure and the comparison of two crystal forms of wild type PYP, the loop around residue Met-100 is observed to be flexible (7Borgstahl G.E.O. Williams D.R. Getzoff E.D. Biochemistry. 1995; 34: 6278-6287Crossref PubMed Scopus (439) Google Scholar, 30van Aalten D.M.F. Crielaard W. Hellingwerf K.J. Joshua-Tor L. Protein Sci. 2000; 9: 64-72Crossref PubMed Scopus (43) Google Scholar, 54Dux P. Rubinstenn G. Vuister G.W. Boelens R. Mulder F.A.A. Hard K. Hoff W.D. Kroon A. Crielaard W. Hellingwerf K.J. Kaptein R. Biochemistry. 1998; 37: 12689-12699Crossref PubMed Scopus (128) Google Scholar). Close contacts between the two monomers in the asymmetric unit cell affect the conformation of the "100 loop" (Fig. 1). The distance between the backbone atoms of the two Met-100 residues is 95%) of the motion is covered by the first 5% (12Genick U.K. Soltis S.M. Kuhn P. Canestrelli I.L. Getzoff E.D. Nature. 1998; 392: 206-209Crossref PubMed Scopus (327) Google Scholar) of the eigenvectors. With this condensed description of flexibility in the individual PAS domains, comparisons are facilitated. Sets of eigenvectors can be projected onto each other yielding a cumulative square inner product, indicating the degree of similarity of the motions described by the eigenvectors. Here we have focused on the first 12 eigenvectors (5% 3N = 234 total eigenvectors), because these together describe approximately 95% of the total motion in the ensembles. TableII shows that the eigenvectors from the different PAS domains are very similar, suggesting that the cores of the PAS domains share common motions, which are not present in lysozyme (the negative control). This is further confirmed by projection of the PAS domain eigenvectors onto the first three eigenvectors calculated from the wtPYP ensemble (Fig. 5). Whereas the other PAS domains reproduce these largest wtPYP motions for up to 90% within the first 12 eigenvectors, they are almost absent in the lysozyme ensemble. Thus, the PAS domains not only share a common structure but also share a common conformational flexibility.Table IIComparison of essential subspacesPYPFixLHERGLOV2Δ25PYPLysozymePYP1.00FixL0.701.00HERG0.660.721.00LOV20.690.710.731.00Δ25PYP0.780.680.690.701.00Lysozyme0.240.240.220.240.231.00The first twelve (i.e. 5% of the total dimension of the system) eigenvectors are pairwise compared through calculation of a cumulative square inner product (57de Groot B.L. van Aalten D.M.F. Amadei A. Berendsen H.J.C. Biophys. J. 1996; 71: 1707-1713Abstract Full Text PDF PubMed Scopus (82) Google Scholar). Open table in a new tab The first twelve (i.e. 5% of the total dimension of the system) eigenvectors are pairwise compared through calculation of a cumulative square inner product (57de Groot B.L. van Aalten D.M.F. Amadei A. Berendsen H.J.C. Biophys. J. 1996; 71: 1707-1713Abstract Full Text PDF PubMed Scopus (82) Google Scholar). To understand the motions described by the eigenvectors on a molecular level, the minimum and maximum projections onto an eigenvector can be translated back to Cartesian space and compared as Cαtraces. In Fig. 6, the minimum and maximum projections of the first 3 eigenvectors of Δ25PYP are compared. The central β-sheet appears to be relatively static, whereas the loops, most notably the αA/αB segment, show the largest fluctuations. In the PAS domains, this segment is generally important for the binding of the ligand (7Borgstahl G.E.O. Williams D.R. Getzoff E.D. Biochemistry. 1995; 34: 6278-6287Crossref PubMed Scopus (439) Google Scholar, 34Crosson S. Moffat K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2995-3000Crossref PubMed Scopus (419) Google Scholar, 35Gong W. Hao B. Mansy S.S. Gonzalez G. Gilles-Gonzalez M.A. Chan M.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15177-15182Crossref PubMed Scopus (343) Google Scholar). For instance, in PYP, residue Arg-52 on this segment is known to undergo a conformational change (9Perman B. Srajer V. Ren Z. Teng T. Pradervand C. Ursby T. Bourgeois D. Schotte F. Wulff M. Kort R. Hellingwerf K. Moffat K. Science. 1998; 279: 1946-1950Crossref PubMed Scopus (284) Google Scholar, 11Genick U.K. Borgstahl G.E.O. Kingman N. Ren Z. Pradervand C. Burke P. Srajer V. Teng T. Schildkamp W. McRee D.E. Moffat K. Getzoff E.D. Science. 1997; 275: 1471-1475Crossref PubMed Scopus (388) Google Scholar, 29van Aalten D.M.F. Hoff W.D. Findlay J.B.C. Crielaard W. Hellingwerf K.J. Protein Eng. 1998; 11: 873-879Crossref PubMed Scopus (28) Google Scholar, 30van Aalten D.M.F. Crielaard W. Hellingwerf K.J. Joshua-Tor L. Protein Sci. 2000; 9: 64-72Crossref PubMed Scopus (43) Google Scholar) upon isomerization of the chromophore. Glu-46, which shares a proton with the chromophore, is also located in this region (Fig. 4). Similarly, the αA/αB segment is involved in binding the heme in FixL (35Gong W. Hao B. Mansy S.S. Gonzalez G. Gilles-Gonzalez M.A. Chan M.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15177-15182Crossref PubMed Scopus (343) Google Scholar) and the FMN in LOV (34Crosson S. Moffat K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2995-3000Crossref PubMed Scopus (419) Google Scholar) both via interaction with a phenylalanine, which lies at the equivalent position of Glu-46 in PYP. In addition, a recent analysis of LOV domains has revealed that the αA/αB region participates in a conserved salt bridge, which is also observed in FixL and HERG and has been proposed to be involved in signal transduction (55Crosson S. Rajagopal S. Moffat K. Biochemistry. 2003; 42: 2-10Crossref PubMed Scopus (350) Google Scholar). It is noteworthy that despite these similar interactions and conservation of conformational flexibility, there is almost no sequence conservation in the αA/αB segment. The data presented here show that in the absence of the N-terminal domain, PYP maintains its PAS-fold despite the exposure of several hydrophobic residues to solvent. The Δ25PYP structure together with the recently determined of the LOV domain in complex with FMN (34Crosson S. Moffat K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2995-3000Crossref PubMed Scopus (419) Google Scholar) allowed further structural comparisons of the PAS family. Although these proteins have almost entirely dissimilar sequences, their structures are remarkably similar with the conserved parts, the β-sheet and the αA/B helices, making up the PAS core. This finding suggests that although these proteins bind different ligands, their signaling states are reached through similar conformational changes. We investigated this by simulating the complete PAS domain proteins that have been structurally defined to date and extracting from that the structurally conserved core. An analysis of the data shows that in particular the αA/B segment moves in a concerted fashion. Thus, we propose that despite the absence of any sequence conservation, the PAS domains are not only structurally conserved but also share a common conformational flexibility that may have evolved to (i) accommodate the various input signals from different ligands/co-factors located at different positions in the domain and (ii) transmit the sensing event to downstream transducer proteins.
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