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• W2004152710 abstract "The human PPIL1 (peptidyl prolyl isomerase-like protein 1) is a specific component of human 35 S U5 small nuclear ribonucleoprotein particle and 45 S activated spliceosome. It is recruited by SKIP, another essential component of 45 S activated spliceosome, into spliceosome just before the catalytic step 1. It stably associates with SKIP, which also exists in 35 S and activated spliceosome as a nuclear matrix protein. We report here the solution structure of PPIL1 determined by NMR spectroscopy. The structure of PPIL1 resembles other members of the cyclophilin family and exhibits PPIase activity. To investigate its interaction with SKIP in vitro, we identified the SKIP contact region by GST pulldown experiments and surface plasmon resonance. We provide direct evidence of PPIL1 stably associated with SKIP. The dissociation constant is 1.25 × 10–7 M for the N-terminal peptide of SKIP-(59–129) with PPIL1. We also used chemical shift perturbation experiments to show the possible SKIP binding interface on PPIL1. These results illustrated that a novel cyclophilin-protein contact mode exists in the PPIL1-SKIP complex during activation of the spliceosome. The biological implication of this binding with spliceosome rearrangement during activation is discussed. The human PPIL1 (peptidyl prolyl isomerase-like protein 1) is a specific component of human 35 S U5 small nuclear ribonucleoprotein particle and 45 S activated spliceosome. It is recruited by SKIP, another essential component of 45 S activated spliceosome, into spliceosome just before the catalytic step 1. It stably associates with SKIP, which also exists in 35 S and activated spliceosome as a nuclear matrix protein. We report here the solution structure of PPIL1 determined by NMR spectroscopy. The structure of PPIL1 resembles other members of the cyclophilin family and exhibits PPIase activity. To investigate its interaction with SKIP in vitro, we identified the SKIP contact region by GST pulldown experiments and surface plasmon resonance. We provide direct evidence of PPIL1 stably associated with SKIP. The dissociation constant is 1.25 × 10–7 M for the N-terminal peptide of SKIP-(59–129) with PPIL1. We also used chemical shift perturbation experiments to show the possible SKIP binding interface on PPIL1. These results illustrated that a novel cyclophilin-protein contact mode exists in the PPIL1-SKIP complex during activation of the spliceosome. The biological implication of this binding with spliceosome rearrangement during activation is discussed. Pre-mRNA splicing, the removal of introns from mRNA precursors, is indispensable to the expression of most eukaryotic genes. The splicing of mRNA is catalyzed by spliceosome, a large machine formed by an ordered interaction of several small nuclear ribonucleoproteins (snRNPs), 3The abbreviations used are: snRNP, small nuclear ribonucleoprotein; PPIL1, peptidyl prolyl isomerase-like protein 1; SKIP, SKI-interacting protein; SKIP172, N-terminal fragment of SKIP-(1–172); nSKIP, N-terminal fragment of SKIP-(59–129); NOE, nuclear Overhauser effect; SPR, surface plasmon resonance; GST, glutathione S-transferase.3The abbreviations used are: snRNP, small nuclear ribonucleoprotein; PPIL1, peptidyl prolyl isomerase-like protein 1; SKIP, SKI-interacting protein; SKIP172, N-terminal fragment of SKIP-(1–172); nSKIP, N-terminal fragment of SKIP-(59–129); NOE, nuclear Overhauser effect; SPR, surface plasmon resonance; GST, glutathione S-transferase. U1, U2, U5, U4/U6, and numerous other less stably associated non-snRNP splicing factors (1Burge C.B. Tuschi T. Sharp P.A. Gesteland C. Cech T. Atkins F. The RNA World. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 525-560Google Scholar, 2Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). The formation of spliceosome goes through many intermediate stages. The stable intermediate complexes are the A, B, and C complexes. During the spliceosome maturation process, the most decisive step is the conversion from non-active complex B to the catalytically active spliceosome B*. The activated complex B* undergoes the first catalytic step of splicing and then forms complex C. Prior to the activation of the spliceosome and during the splicing process, a number of conformational rearrangements take place. Recently, human 45 S activated spliceosome (complex B*) and 35 S U5 snRNP have been isolated by immunoaffinity purification and characterized by mass spectrometry (3Makarov E.M. Makarova O.V. Urlaub H. Gentzel M. Will C.L. Wilm M. Lührmann R. Science. 2002; 298: 2205-2208Crossref PubMed Scopus (303) Google Scholar). Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition. More than 100 proteins were identified in the 45 S activated spliceosome, 80 of which are known splicing factors. The rest are non-snRNP proteins, including protein SKIP and one peptidyl prolyl isomerase-like protein 1 (PPIL1) (2Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, 3Makarov E.M. Makarova O.V. Urlaub H. Gentzel M. Will C.L. Wilm M. Lührmann R. Science. 2002; 298: 2205-2208Crossref PubMed Scopus (303) Google Scholar, 4Makarova O.V. Makarov E.M. Urlaub H. Will C.L. Gentzel M. Wilm M. Luhrmann R. EMBO J. 2004; 23: 2381-2391Crossref PubMed Scopus (150) Google Scholar). PPIL1 is a component in 45 S U5 snRNP in activated spliceosome (complex B*) and 35 S snRNP. It was believed to participate in the activation of spliceosome (5Folk P. Puta F. Skruzny M. Cell. Mol. Life. Sci. 2004; 61: 629-640Crossref PubMed Scopus (68) Google Scholar). The cDNA of PPIL1 was first cloned from human fetal brain, which encodes 166 amino acid residues. PPIL1 has 41.6% identity to human cyclophilin A (6Ozaki K. Fujiwara T. Kawai A. Shimizu F. Takami S. Okuno S. Takeda S. Shimada Y. Nagata M. Watanabe T. Takaichi A. Takahashi E. Nakamura Y. Shin S. Cytogenet. Cell Genet. 1996; 72: 242-245Crossref PubMed Scopus (16) Google Scholar). It belongs to a novel subfamily of cyclophilins, together with CypE in Dictyostelium discoideum and Cyp2 in Schizosaccharomyces pombe. SKIP (7Dahl R. Wani B. Hayman M.J. Oncogene. 1998; 16: 1579-1586Crossref PubMed Scopus (101) Google Scholar) is another essential component in 45 and 35 S U5 snRNP involved in the activation of spliceosome. Skip homologs have been identified from many diverse species, from yeast to human. It is a transcriptional coregulator that interacts with a variety of proteins (8Baudino T.A. Kraichely D.M. Jefcoat S.C. Winchester S.K. Partridge N.C. MacDonald P.N. J. Biol. Chem. 1998; 273: 16434-16441Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 9Leong G.M. Subramaniam N. Figueroa J. Flanagan J.L. Hayman M.J. Eisman J.A. Kouzmenko A.P. J. Biol. Chem. 2001; 276: 18243-18248Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 10Zhou S. Fujimuro M. Hsieh J.J. Chen L. Hayward S.D. J. Virol. 2000; 74: 1939-1947Crossref PubMed Scopus (98) Google Scholar). Since SKIP was found in 1998, more and more evidence has been accumulated for SKIP participation in pre-mRNA splicing (11Zhang C. Dowd D.R. Staal A. Gu C. Lian J.B. van Wijnen A.J. Stein G.S. MacDonald P.N. J. Biol. Chem. 2003; 278: 35325-35336Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 12Nagai K. Yamaguchi T. Takami T. Kawasumi A. Aizawa M. Masuda N. Shimizu M. Tominaga S. Ito T. Tsukamoto T. Osumi T. Biochem. Biophys. Res. Commun. 2004; 316: 512-517Crossref PubMed Scopus (12) Google Scholar). It has been demonstrated that Prp45p, ortholog of SKIP in Saccharomyces cerevisiae, is associated with spliceosome throughout the splicing process and is essential for pre-mRNA splicing (13Diehl B.E. Pringle J.R. Genetics. 1991; 127: 287-298Crossref PubMed Google Scholar). Neither SKIP nor PPIL1 is stably associated with U4/U6.U5 tri-snRNPs nor are they present in pre-spliceosomes; they are recruited before catalytic step 1 and might also function during activation of spliceosome (3Makarov E.M. Makarova O.V. Urlaub H. Gentzel M. Will C.L. Wilm M. Lührmann R. Science. 2002; 298: 2205-2208Crossref PubMed Scopus (303) Google Scholar, 4Makarova O.V. Makarov E.M. Urlaub H. Will C.L. Gentzel M. Wilm M. Luhrmann R. EMBO J. 2004; 23: 2381-2391Crossref PubMed Scopus (150) Google Scholar). In 2001, interaction was reported (14Skruzny M. Ambrozkova M. Fukova I. Martinkova K. Blahuskova A. Hamplova L. Puta F. Folk P. Biochim. Biophys. Acta. 2001; 1521: 146-151Crossref PubMed Scopus (22) Google Scholar) between SnwA and CypE, which are postulated as the orthologs of SKIP and PPIL1 in D. discoideum, respectively. Nevertheless, no direct evidence shows that PPIL1 can bind to SKIP directly, and the molecular mechanism of the interaction is still unknown. To investigate the interaction of PPIL1 with SKIP, we solved the solution structure of PPIL1 by NMR and have provided the kinetic constants by PPIase assay. GST pulldown experiments combined with surface plasmon resonance (SPR) measurements revealed the N-terminal of SKIP-(59–129) could associate with PPIL1 in vitro tightly. Furthermore, we provided experimental evidence for the formation of PPIL1-cyclosporin A-nSKIP ternary complex by NMR titration and chemical shift perturbation experiments. According to the results of perturbation, we mapped the interfaces of PPIL1 in binding to cyclosporin A and SKIP. PPIL1-nSKIP exhibits a novel interaction mode in contrast with other known cyclophilin-protein interactions. The biological implication of the association in spliceosome rearrangement during activation is discussed. Our results implicate more complicated roles that spliceosomal cyclophilin may play during the activation of spliceosome. Expression and Purification and Isotope Labeling of PPIL1—The hPPIL1 cDNA was obtained by PCR from human CD34+ hematopoietic stem/progenitor cell cDNA library cloned in-frame into the NdeI/XhoI sites of pET-22b (+) (Novagen). It was then transformed into the Escherichia coli expression strain BL21 (DE3). Bacteria were grown at 37 °C in Luria Bertani medium containing 100 mg/liter of ampicillin. Briefly, saturated Luria Bertani was diluted (1:300, v/v) in M9 minimal medium containing 0.05% 15NH4Cl and/or 0.25% [13C]glucose and incubated at 37 °C. Target protein expression was induced by addition of 0.8 mm isopropyl-1-thio-β-d-galactopyranoside to mid-log phase cultures (A600, 0.6–0.8). The culture was shaken at 20 °C overnight. After 20 h of additional growth, bacteria were harvested by centrifugation. The pellet was resuspended in a buffer containing 50 mm phosphate, pH 7.5, 500 mm NaCl. Cells were disrupted by sonication, and debris was spun down at 15,000 rpm, 4 °C for 0.5 h in a Beckman centrifuge using a JA-17 rotor. The supernatant was applied to a HiTrap chelating resin (Amersham Biosciences) charged with Ni2+ ions. The column was washed with 50 mm imidazole in 50 mm phosphate buffer, pH 7.5, 500 mm NaCl, and the protein was eluted using 200 mm imidazole in the same buffer. The purity of the protein was assessed by SDS-PAGE. The final yield for 15N-labeled protein was 30 mg/liter and for 13C/15N-labeled protein was 20 mg/liter. The purity of the protein was >95%. Characterization of Recombinant PPIL1—When recombinant PPIL1 was purified and concentrated to 50 mg/ml, only a few strong peaks could be observed in the 15N-1H HSQC due to severe aggregation. The peaks on HSQC were not well dispersed, and the number of the peaks did not increase significantly until the sample was diluted in the NMR buffer to the concentration of 10 mg/ml, estimated by BCA kits (Pierce). At that concentration, dynamic light scattering experiments indicated the protein was essentially monodisperse and stable with an estimated molecular mass of 20 kDa (data not shown). Therefore, the final concentration of PPIL1 sample applied in all NMR experiments was ∼10 mg/ml. The fractions of unlabeled protein were pooled and dialyzed against GST binding buffer. Construction, Expression of the N-terminal of Human SKIP Gene Fusions—The N-terminal 172 amino acids of the SKIP gene (SKIP172) and another three fragments (1–64, 59–129, and 126–172) were amplified by polymerase chain reaction from human brain cDNA library and cloned into the multiple cloning sites of pGEX-2T (Amersham Biosciences), resulting in a GST fusion gene. All GST fusion protein constructs were expressed in E. coli strain BL21 (DE3). The protein production was induced at A600 0.6–0.8, using 1 mm isopropyl-1-thio-β-d-galactopyranoside. The culture was then kept shaking at 30 °C for 5–6 h. After being harvested and centrifuged, cells were disrupted by sonication and debris spun down at 15,000 rpm, 4 °C for 0.5 h in a Beckman centrifuge using a JA-17 rotor. The expression of target protein was then analyzed by SDS-PAGE analysis of supernatant fluid. All four GST fusion proteins were purified as shown in protocols. GST was cleaved from GST fusion protein by incubation with thrombin at 4 °C for 20 h and removed by glutathione-agarose beads. Cleaved peptide was further purified to remove thrombin. PPIase Activity Assay and Cyclosporin A Inhibition Assays—Purified recombinant PPIL1 was assayed for PPIase activity essentially as described by Fischer et al. (15Fischer G. Wittmannliebold B. Lang K. Kiefhaber T. Schmid F.X. Nature. 1989; 337: 476-478Crossref PubMed Scopus (1204) Google Scholar) with the suggested substrate solvent application as described by Kofron et al. (16Kofron J. Kuzmic P. Kishore V. Colonbonilla E. Rich D.H. Biochemistry. 1991; 30: 6127-6134Crossref PubMed Scopus (483) Google Scholar). The peptide substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma) was dissolved in trifluroethanol with 470 mm LiCl to give 25 mm stock solution. The experimental setup was as follows: PPIL1 was diluted into 50 mm HEPES, 86 mm NaCl, pH 8.0 (PPIase buffer) to give 1 μm stock. In a 1-ml cuvette, 6 μl of PPIL1 (10 nm final concentration) was added to 535 μlof PPIase buffer and incubated on ice for 1 h. Chymotrypsin (60 μl of 6 mg/ml) was added to the cuvette, followed by transfer of the cuvette to a thermostated cell holder by which the reaction temperature was maintained at 5 °C. The reaction was started by the addition of 1.2 μl of peptide substrate (50 μm) followed by rapid mixing. The absorbance at 395 nm due to the release of p-nitroanilide was recorded with data collected every 0.1 s over a 2-min period. A number of samples were tested using the above method. Cyclosporin A inhibition assays were carried out essentially as described above except that the samples were preincubated for a minimum of 60 min with varying amounts of cyclosporin A (Calbiochem). GST Pulldown Experiments in Vitro—For interaction of His-tagged PPIL1 with GST-SKIP-(1–172), GST-SKIP-(1–64), GST-SKIP-(59–129), and GST-SKIP-(126–172), recombinant GST and GST fusion protein were immobilized on 300-μl bed volume of glutathione-agarose beads. After washing with 3 ml of GST binding buffer, the beads were incubated with an excessive amount of His-tagged PPIL1, which had been preincubated with cyclosporin A for 1 h, in 500-μl total volume at 4 °C overnight on a rotator. The beads were washed by GST binding buffer supplemented with different concentrations of NaCl (2 ml of each buffer). Finally, the beads were eluted by GST binding buffer supplemented with 10 mm glutathione. The bound proteins were detected by SDS-PAGE and stained with Coomassie Brilliant Blue. The bound PPIL1 were further affirmed by immunoblot using His tag antibody. Surface Plasmon Resonance Measurements—SPR experiments were carried out. nSKIP-(1–172) was coupled to a carboxymethyl-dextran CM5 sensor chip with an amine coupling kit. Binding was observed upon injection of different concentrations of PPIL1. The plateau values reached after completion of the association reactions were analyzed by a Langmuir binding isotherm. NMR Spectroscopy—The NMR sample of 15N/13C, 15N-PPIL1 was prepared and NMR experiments were recorded for assignments as described (17Xu C. Xu Y. Tang Y. Wu J. Shi Y. Huang Q. Zhang Q. J. Biomol. NMR. 2005; 31: 179-180Crossref PubMed Scopus (2) Google Scholar). NOE distance restraints were generated using 15N-edited NOESY and 13C-edited NOESY spectra employing 130-ms mixing times. NMR data were processed by NMRPipe and NMRDraw software (18Delagio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1596) Google Scholar) and assigned with Sparky (19Goddard, T. D., and Kneller, D. G. Sparky 3, University of California, San FranciscoGoogle Scholar). The CSI (20Wishart D.S. Sykes B.D. J. Biomol. NMR. 1994; 4: 171-180Crossref PubMed Scopus (1902) Google Scholar) program was used to obtain the backbone dihedral angles (ϕ and Ψ) in secondary structures on the basis of chemical shift information. The NOE-derived distance restraints were classified into four groups with the upper boundaries of 3.0, 4.0, 5.0, 6.0 Ä and lower boundary of 1.80 Ä on the basis of NOE intensity measurements. Hydrogen bond restraints were defined from slow exchanging amide protons identified after exchange of the H2O buffer to D2O. Structure Calculations—Structure calculation for PPIL1 was performed on the basis of proton-proton NOE restraints and dihedral angle restraints (ϕ and Ψ) with a simulated annealing protocol using the CNS v1.1 program (21Brunger 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. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar). High temperature torsion angle dynamics was performed at 50,000 K for 15 ps (1,000 steps) followed by a 15-ps cooling phase. Initial structure calculations included only hydrogen bonds in defined secondary structures from CSI. In the following refinement calculation, only hydrogen bonds whose donors could be identified unambiguously were added. In the final calculations, an ensemble of 100 structures (no distance violations >0.3 Ä and no dihedral angle violations >5°) was generated from unambiguous NOEs previously determined. 20 models were selected on the basis of energetic criteria (low total energy, using the accept.inp routine) to form a representative ensemble of the calculated structures. NMR Backbone Relaxation Experiments—15N relaxation experiments were carried out at 295 K on a Bruker DMX500 NMR spectrometer. 15N relaxation measurements were carried out using the published methods (22Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2002) Google Scholar). 15N T1 relaxation rates were measured with eight relaxation delays: 11, 62, 142, 243, 364, 525, 757, and 1150 ms. 15N T2 relaxation rates were measured with six relaxation delays: 17.6, 35.2, 52.8, 70.4, 105.6, and 140.8 ms. A recycle delay of 1 s was used for measurement of T1 and T2 relaxation rates. The spectra measuring 1H-15N NOE were acquired with a 2-s relaxation delay followed by a 3-s period of proton saturation. The spectra recorded in the absence of proton saturation employed a relaxation delay of 5 s. The exponential curve fitting and extract of T1 and T2 were processed by Sparky. The relaxation data R1/T1, R2/T2, and η were analyzed by Fast ModelFree v1.0. Chemical Shift Perturbation—For detecting the cyclosporin A and nSKIP binding sites on PPIL1, 1 mm 15N-labeled PPIL1 was used. After 1H, 15N-HSQC spectrum of free PPIL1 was recorded, and the sample was titrated with cyclosporin A in the method described by Weber et al. (23Weber C. Wider G. Freyberg B. Traber R. Braun W. Widmer H. Wüthrich K. Biochemistry. 1991; 30: 6563-6574Crossref PubMed Scopus (255) Google Scholar). After the 15N-1H HSQC of PPIL1-cyclosporin A was recorded, the sample was further titrated with 1 mm unlabeled SKIP until there was no change in the spectrum recorded. The final concentrations of both proteins at the end of the titration were ∼0.5 mm. All HSQC spectra for mapping the cyclosporin A and nSKIP binding interfaces on PPIL1 were performed on Bruker 600 MHz at 295 K. Exchange Experiments of PPIL1-nSKIP Complex in D2O—Freeze-dried 15N-PPIL1 mixed with nSKIP-(59–129) were dissolved in D2O. 15N-1H HSQC spectra were recorded to identify slow exchanging HNs of 15N-PPIL1 in the complex. Peptidyl Prolyl Cis-trans Isomerase Activity and Its Inhibition Activity by Cyclosporin A—PPIL1 clearly accelerates the rate of isomerization of the tetrapeptide substrate relative to the uncatalyzed thermal isomerization rate, and the catalysis is inhibited by addition of the cyclophilin binding drug cyclosporin A (in supplemental data). The enzyme reaction was found to follow Michaelis-Menten kinetics with the velocity of the reaction (v) increasing with substrate concentration [S] (Fig. 1). First-order rate kinetics were observed, and a double reciprocal Line-weaver-Burke plot of 1/v against 1/[S] gave values of kcat 960 s–1, Km 230 μm, which corresponds to a value of kcat/Km of 4.2 × 106 m–1 s–1. These values are similar to the published values for human CypA, which has kcat 12700 s–1, Km 870 μm and kcat/Km 14.6 × 106 m–1 s–1 (16Kofron J. Kuzmic P. Kishore V. Colonbonilla E. Rich D.H. Biochemistry. 1991; 30: 6127-6134Crossref PubMed Scopus (483) Google Scholar). Structure Determination—The solution structure of the recombinant protein PPIL1 was determined by multidimensional heteronuclear NMR spectroscopy. The last 8 amino acids as artifact from vector were not included in structure calculations. Fig. 2 shows an ensemble of 20 NMR structures and a ribbon representation of the energy-minimized average structure of human PPIL1 by MOLMOL (24Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6477) Google Scholar). The coordinates of these 20 NMR structures have been deposited into the Protein Data Bank (code 1XWN). Table 1 lists the structural statistics for the 20 deposited NMR structures. The root mean square deviation of the well defined secondary structure regions of the 20 structures to the average structure is 0.58 Ä for the backbone and 1.08 Ä for the heavy atoms. In contrast, the N-terminal residues (amino acids 1–11) are disordered because of few medium- and long-range NOEs.TABLE 1Summary of structure statisticsDistance restraints Intraresidue (i - j = 0)544 Sequential (|I - j| = 1)551 Medium range (2 < |i - j| <4)317 Long range (|i - j| >5)697 Hydrogen bonds96 Total2205Dihedral angle restraintsaThe Φ and Ψ angle restraints are generated from secondary structures by CSI. φ59 ψ59Mean r.m.s.d. from the experimental restraintsbR.m.s.d., root mean square deviation. Distance0.0037 ± 0.0009 Dihedral0.1010 ± 0.0193Mean r.m.s.d. from idealized covalent geometry Bond0.0011 ± 0.00005 Angle0.2752 ± 0.0027 Improper0.1084 ± 0.0057Mean energies (kcal mol-1) Etotal-855.79 ± 15.35 Evdw-566.42 ± 10.42 Enoe5.48 ± 1.30 Eangle83.16 ± 3.39 Ebond21.51 ± 2.20 Eimproper6.66 ± 0.58 EdihedralPROCHECK Ramachandran Plot analysis (%)cAll non-Gly residues, Φ/Ψ of most favored, and additional allowed regions are given by Procheck (23). Residues in most favored regions71.3% Residues in additionally allowed regions24.3% Residues in generously allowed regions3.6% Residues in disallowed regions0.8%Structural r.m.s.d. for secondary structures regionsdAtoms of well defined secondary structure regions: residues 13-64, 96-101, 110-145, and 157-164. (Ä) Backbone heavy atom (N, Cα, and C′)0.58 Heavy atom1.08a The Φ and Ψ angle restraints are generated from secondary structures by CSI.b R.m.s.d., root mean square deviation.c All non-Gly residues, Φ/Ψ of most favored, and additional allowed regions are given by Procheck (23Weber C. Wider G. Freyberg B. Traber R. Braun W. Widmer H. Wüthrich K. Biochemistry. 1991; 30: 6563-6574Crossref PubMed Scopus (255) Google Scholar).d Atoms of well defined secondary structure regions: residues 13-64, 96-101, 110-145, and 157-164. Open table in a new tab A PROCHECK (25Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4363) Google Scholar) analysis of the 20 NMR structures indicated that >95% of the residues lie in the most favored region and additional allowed region of the Ramachandran plot. The residues in the disallowed regions were those in the terminal part or in the loops because of the paucity of inter-residual NOEs. Description of PPIL1 Structure—Fig. 2A shows the superimposition of the backbone (N, Cα, C′) of the best fit of 20 structures with the lowest energy. The overall architecture of PPIL1 is similar to that of known x-ray and NMR cyclophilin structures (26Ke H. J. Mol. Biol. 1992; 228: 539-550Crossref PubMed Scopus (110) Google Scholar, 27Ottiger M. Zerbe O. Guntert P. Wuthrich K. J. Mol. Biol. 1997; 272: 64-81Crossref PubMed Scopus (68) Google Scholar, 28Mikol V. Kallen J. Walkinshaw M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5183-5186Crossref PubMed Scopus (98) Google Scholar) with an anti-parallel eight-stranded β-barrel (13–18, 22–27, 55–57, 61–63, 97–104, 107–115, 127–132, 160–163) capped by two α-helices (33–45, 136–144). One short 310 helix (120–122) lies within the loop connecting strands β6 and β7 (Fig. 2B). After 1H-2H exchange experiments, we found most observable HNs exist in regular secondary structures. However, it is interesting that no backbone amide protons in helix α2 show slow exchange, which indicates helix α2 may not be in very rigid conformation or it may be exposed to the solvent. However, the NδH2 of Asn-140 exchanged slow as observed in the recorded HSQC in D2O, which probably H-bonded to O′ of Gly-137. In the solution structure of PPIL1, helix α2 was further stabilized by a network of hydrophobic interactions between well conserved amino acids such as Met-20, Ile-56, Ile-62, Val-114, Leu-116, Val-139, Val-142, and Val-145 (in supplemental data). Residue 55–57 in the β3 strand showed fast amide proton exchange in contrast to the residues in the other β strands, which have been described as “structure breath” in the solution structure of CypA (27Ottiger M. Zerbe O. Guntert P. Wuthrich K. J. Mol. Biol. 1997; 272: 64-81Crossref PubMed Scopus (68) Google Scholar). This could be explained as the potential ability of adjusting the interior side chains to fit the binding of its substrates or inhibitors for two enzyme activity sites (Arg-55, Ile-57) located on β3. The Structure Comparison of PPIL1 and Cyclophilin A—In Fig. 3A, PPIL1 is superimposed upon the structure of human cyclophilin A. The root mean square deviation for most defined secondary structures of Cα atoms of PPIL1 and CypA is 1.2 Ä. PPIL1 is 41.6% identical to CypA (at the amino acid level). Fig. 4 shows the sequence alignment of PPIL1 with several human cyclophilins. The PPIL1 structure is highly conserved when compared with the structure of human cyclophilin A, the best characterized member of the family (26Ke H. J. Mol. Biol. 1992; 228: 539-550Crossref PubMed Scopus (110) Google Scholar, 27Ottiger M. Zerbe O. Guntert P. Wuthrich K. J. Mol. Biol. 1997; 272: 64-81Crossref PubMed Scopus (68) Google Scholar). The active sites of PPIL1 are also very similar to those found in human CypA; all 13 residues important in binding cyclosporin A are conserved. This is consistent with the facts that PPIL1 exhibits PPIase activity and the activity could be inhibited by cyclosporin A. By comparing the two structures, we found there were two highly conserved backbone-backbone hydrogen bonds located outside of regular secondary structures: Ala-32 NH-O′ Tyr-28 is in a helix-like turn, and Ser-51 NH-O′ Tyr-48 is a part of a β-turn (in supplemental data).FIGURE 4Sequence alignments. Sequence alignments of PPIL1 with cyclophilin A from Homo sapiens, accession code NP_066953 (CYPA_HUMAN); cyclophilin B from H. sapiens, accession code NP_000933 (CYPB_HUMAN); cyclophilin C from H. sapiens, accession code NP_000934; SnuCyp-20 from H. sapiens, accession code NP_006338 (CYPH_HUMAN). The sequences were aligned by CLUSTAL_W with dashes introduced for maximum alignment. Conserved residues involved in binding inhibitors and substrates in Cyp A were labeled with stars.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Despite the similarity in their overall structures, they are quite different at the C-terminal of helix α1 because of the loss of 3 amino acids in the loop linking α1 and β3 in PPIL1. In human cyclophilins, this feature is only present in high molecular mass cyclophilins Cyp-60 and Cyp-73 (29Galat A. Arch. Biochem. Biophys. 1999; 371: 149-162Crossref PubMed Scopus (95) Google Scholar). A special turn occurs following α1 due to the loss of inserted amino acids, and the conserved β-turn is linked to α1 by two hydrogen bonds, Tyr-48 NH-O′ Leu-42 and Tyr-47 NH-O′ Leu-42 (in supplemental data). The special hydrogen network makes the C-terminal of helix α1 of PPIL1 significantly different from that of hCypA and close to the loop G65-Y78 in structure comparison. The loop following α1 (65–74) is in poorly defined conformation because of the lack of inter-residual NOEs. We also note that there is one amino acid insert in the loop preceding β8, which causes the displacement of residue 146–151 with respect to the corresponding residues in hCypA; this leads to a significantly altered conformation of this loop with respect to hCypA." @default.
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