Crystal structure of arabidopsis thaliana cyclophilin 38 (atcyp38 2

CHAPTER RESULTS AND DISCUSSION 4.1 EXPRESSION AND PURIFICATION OF NATIVE WILD TYPE ATCYP38 4.1.1 Expression Sufficient quantity of soluble AtCyP38 (83-437) was produced as a Glutathione-S-Transferase (GST) fusion protein at 25 oC, hrs (Fig. 19). M 97 kDa 66 kDa AtCyP38-GST 45 kDa 30 kDa 20.1 kDa 14.4 kDa Figure 19. SDS-PAGE showing the expression of soluble wt AtCyP38. M – Low Molecular Weight Marker, Lane – Cell lysate before induction, Lane – Whole cell lysate 4hrs after induction, Lane – Soluble protein hrs after induction 4.1.2 Affinity chromatographic purification The produced AtCyP38-GST fusion protein was purified using glutathione- sepharose beads (Amersham Biosciences), Fig. 20. Due to the slow kinetics of GST binding, considerable amount of the fusion protein came out along with the flow 79 through. Hence the flow through from the first step was subjected to a second round of purification process to retrieve the unbound fusion protein. 97 kDa M 66 kDa AtCyP38-GST 45 kDa 30 kDa 20.1 kDa 14.4 kDa Figure 20. SDS-PAGE showing the affinity chromatographic purification of the wt AtCyP38-GST fusion protein. M – Low Molecular Weight Marker, Lane – Soluble protein before affinity chromatography, Lane – Flow through from affinity chromatography column, Lane – Wash 1, Lane – Wash 2, Lane – Protein bound to glutathione sepharose beads. 4.1.3 Thrombin cleavage Removal of the GST tag was more efficient when cleaved on-column using the thrombin protease. The cleavage required over-night incubation at oC for the reaction to be complete. The cleaved protein had additional 14 residues, which belong to the linker that connects the tag and the protein, at its N-terminus. The presence of this linker with the sequence ‘GSPGISGGGGGILL’ is a peculiar feature of the pGEX-KG vector, which was used for protein expression. This additional glycine-rich region is expected to improve the thrombin cleavage and probably favored the complete cleavage. However, probably due to these extra residues from the linker, the 80 band of the tag-removed protein appears at a slightly higher molecular weight than its original 39 kDa weight in SDS-PAGE, Fig. 21. M 97 kDa 66 kDa 45 kDa 30 kDa AtCyP38 GST 20.1 kDa 14.4 kDa Figure 21. SDS-PAGE showing the thrombin cleavage of the wt AtCyP38-GST fusion protein. M – Low Molecular Weight Marker, Lane – Protein on resin before cleavage, Lane – Flow through from the column, after cleavage, Lane – Wash 1, Lane – Wash 2, Lane – GST, bound to the glutathione sepharose beads after cleavage. The cleaved AtCyP38 protein came out along with the flow through and the first wash. But it still had some amount of contaminant proteins. The two fractions were pooled together and subjected to another round of purification by size exclusion chromatography. 4.1.4 Size exclusion chromatography HiLoad 16/60 Superdex-75 column (Amersham Biosciences) was used for the size exclusion chromatographic experiment. Pure AtCyp38 protein was eluted out as a single peak, Fig. 22, and the corresponding fractions were pooled together. SDSPAGE of the pooled fractions, Fig. 23, had no other contaminant proteins. 81 Figure 22. Profile of size-exclusion chromatographic purification of wt AtCyP38 (83-437). The higher peak at around 60 ml corresponds to the protein. M 97 kDa 66 kDa 45 kDa Pure AtCyP38 30 kDa 20.1 kDa 14.4 kDa Figure 23. SDS-PAGE of size-exclusion chromatography fraction of purified AtCyP38 (83-437). M – Low Molecular Weight Marker, Lane – Protein before size-exclusion chromatography, Lane – Pooled fractions of the protein after size-exclusion chromatography. 4.1.5 Analyses for purity and homogeneity The position of the peak from size-exclusion chromatography indicated the protein to be a monomer of about 40 kDa size, Fig. 18. This was confirmed on 82 Native-PAGE, Fig. 24, and the protein appeared as a single band that corresponded to a monomer. M 232 kDa 140 kDa 66 kDa AtCyP38 Figure 24. Native-PAGE of purified wt AtCyP38 (83-437). M – High Molecular Weight Native Marker, Lane – Purified wt AtCyP38. The theoretical molecular weight of wt AtCyP38 (83-437) protein is 39.25 kDa. MALDI-TOF mass spectrometry of the recombinant wt AtCyP38, carried out using an Applied Biosystems 4700 Proteomics Analyzer 86, showed a mass of about 40389.69 + 1.217, Fig. 25. The additional stretch of 14 residues at the N-terminus of the protein makes its theoretical molecular weight 40.38 kDa. The molecular weight determined by mass spectrometry is quite close to this value. A Dynamic Light Scattering (DLS) experiment showed a dispersity index of 0.16 and this assured that the protein was monodispersed. The molecular weight predicted by DLS was 39.6 kDa, which again is quite close to the actual molecular weight and hence the protein was confirmed to be a monomer. 83 40389.69 Figure 25. Mass spectrometry for wt AtCyP38 (83-437). 4.2 EXPRESSION AND PURIFICATION OF SELENOMETHIONYLATED WILD TYPE ATCYP38 4.2.1 Expression Feedback inhibition of methionine biosynthesis (Doublie, 1997) at the protein expression stage was used for the incorporation of selenomethionine into the AtCyP38 protein. The expression of selenomethionylated protein in the minimal medium M9 resulted in slightly lower yield compared to the native protein which was expressed in nutrient-rich LB medium. 4.2.2 Purification mM DTT was maintained throughout in all the buffers to avoid the oxidation of selenomethionine during the purification process. The protocol that was used for native AtCyP38 purification was used for the purification of selenomethionylated 84 AtCyP38. MALDI-TOF analysis on the derivatized protein gave a mass of 40839.95 + 1.205. The mass difference between the native and selenomethionylated proteins indicates that out of the 10 methionine residues of AtCyp38 have been replaced by selenomethionine. 4.3 CRYSTALLIZATION AND DATA COLLECTION FOR WILD TYPE ATCYP38 4.3.1 Crystallization Initial crystallization screening of the native AtCyP38 protein by the hanging drop vapor diffusion method yielded crystals in two different conditions within days. Poly ethylene glycol (PEG) was the common precipitant in both the conditions and in addition to PEG, the conditions also had a volatile precipitant solution. One condition had 10% isopropanol and the other condition had 2.5% t-butanol. The presence of these volatile precipitants was indispensable for crystal formation. However, their volatile nature caused disintegration of the crystals as soon as the cover-slip was opened. Optimization of these hanging drop conditions did not show any improvement. The vapor batch method of crystallization was attempted in order to avoid the problem of excessive evaporation of volatile precipitants, as suggested by Mortuza and co-workers (2004). The volatile precipitant was provided in the reservoir and was allowed to diffuse slowly into the drops that were overlaid with paraffin oil. Crystal formation took slightly longer time than in the hanging drop method, probably due to the slow diffusion rate. Crystals were larger in size, Fig. 26, and fewer in number. The presence of oil prevented the fast evaporation rate of volatile precipitants out of the 85 drop when the cover was opened. The paraffin oil on the top of the drop also served as an additional cryo-protectant during the cryo-cooling of crystals for data collection. Figure 26. Crystal of AtCyP38 obtained by vapor batch method in a condition having PEG6000 and t-butanol as precipitants. Figure 27. Crystal of selenomethionylated AtCyP38 obtained by vapor batch method in a condition having PEG6000 and t-butanol as precipitants. Crystals of selenomethionylated AtCyP38 protein was obtained in the same condition as that of native AtCyP38 by the vapor batch method, Fig. 27. The native protein was required at an initial concentration of mg ml-1 for crystallization, whereas, the selenomethionylated protein was required only at mg ml-1, probably due to its lower solubility. 86 A few large crystals were picked-up, washed thoroughly in the crystallization buffer, dissolved in water and subjected to MALDI-TOF mass spectrometric analysis. The determined mass for the native and selenomethionylated crystals were the same as that of the corresponding purified protein, indicating that the proteins are intact in the crystal and no part of them has been cleaved off during the crystallization process. Crystals were tested at an in-house X-ray imageplate detector facility for diffraction quality and optimization of cryo-condition. Cryo-conditions with glycerol concentration less than 20% always resulted in the formation of ice-rings in diffraction images. Suitable cryo-protecting conditions were identified for crystals obtained from both the conditions. Essentially, the chosen cryo-conditions had 5% additional PEG in addition to 20-25% glycerol. Crystals from the vapor batch droplets were carefully picked-up with cryoloops and immediately transferred to the respective cryo-protectant. Crystals were either directly tested on the in-house system or flash-cooled in liquid nitrogen for later experiments. The native crystals from the condition containing PEG 6000 and tbutanol as precipitants diffracted up to Å whereas those from the condition containing PEG 4000 and isopropanol diffracted only up to Å. The diffraction quality of all selenomethionylated crystals was poor. But a few of these crystals from the condition containing PEG 6000 and t-butanol diffracted up to about Å. 4.3.2 Data collection and analysis Data collection was done at the X25 beamline, National Synchrotron Light Source, Brookhaven National Laboratory (Upton, NY, USA) with a Q315 chargecoupled device detector (Area Detector Systems Corporation). Prior to data collection, a fluorescence scan was carried out to identify the peak, remote and inflection 87 wavelengths for MAD dataset based on selenium absorption spectrum. 360 frames of the native and 360 frames for each of the MAD wavelengths were collected with an oscillation one degree for the respective crystals. The crystal parameters and data collection statistics are given in Table below. Table 1. Crystal parameters and data-collection statistics for wt AtCyP38. Values in parentheses are for the highest resolution shell (Native: 2.59-2.50 Å and Se-Met: 3.63-3.50 Å) Unit-cell parameters Space group C2221 Native Se-Met a (Å) 58.2 58.1 b (Å) 95.9 96.0 c (Å) 167.5 167.2 No. of molecules in ASU Data collection X-ray source and detector BNL (X-25) / ADSCQ-315 CCD Resolution (Å) 3.5 Inflection Peak Remote Wavelength (Å) 0.9500 0.9799 0.9798 0.9640 Total observations 109,448 38,377 38,181 37,311 Unique reflections 16,710 6,213 6,259 6,181 Completeness (%) 97.8 (99.8) 98.4 (93.3) 98.2 (91.9) 98.3 (92.3) Redundancy 6.7 (6.7) 6.3 (6.0) 6.2 (5.7) 0.071 (0.29) 0.059 (0.09) 0.065 (0.10) 0.050 (0.07) 21.3 (5.4) 30.2 (18.3) 30.7 (17.5) 20.5 (12.1) Rsym 〈I/σ(I)〉 2.5 6.1 (5.6) Rsym = ∑hkl∑i[|Ii(hkl) - | / ∑hkl∑i Ii(hkl)] 88 characteristic feature of the leucine zipper architecture. Also, some of the leucine residues fall within the loop region between the helices and that is quite unlikely for a leucine zipper. The four-helix bundle motif is relatively common in proteins. A DALI search (Holm & Sander, 1993) with the helical bundle of AtCyP38 covering residues 115217 showed that the closest structural homologues are spinach PsbQ (1nze; Z score, 10.1) and E. coli cytochrome b562 (256B; Z score, 10.0). PsbQ is a 16 KDa oxygen evolving subunit of Photosystem II and cytochrome b562 is a heme-binding protein involved in electron transport as well as DNA-dependant transcription regulation. These structures include a four-helix bundle with up-and-down topology. However, no region of the above proteins shows a strong sequence similarity to AtCyP38. PsbQ is only about 11% identical and aligns with the helical bundle of AtCyP38 with an R.M.S.D. of 1.71 over a stretch of 88 residues. Cytochrome b562 is about 10% identical and aligns with an R.M.S.D. of 1.73 for 83 residues, Fig. 32. A B Figure 32. Structure overlap for the helical bundle of AtCyP38 with (A) PsbQ and (B) cytochrome b562. AtCyP38 is drawn in red and green whereas PsbQ and cytochrome b562 are shown in cyan and pink. The overlap was generated with the MULTIPROT server and the figure was prepared using PYMOL. 99 The four helices of AtCyP38 helical bundle pack together mainly through hydrophobic interactions. The internal surface of the bundle is highly hydrophobic, being occupied mainly by Leu, Ile, Ala and Val residues. The helices are rich in leucine and isoleucine residues. The functional implication of this property is to be further verified. Also, the helices are rich in charged residues. All the potentially charged and hydrophilic residues point towards solvent. The region between Glu167 and Asp255 has clusters of charged residues with a net surplus of acidic residues (21 acidic, 16 basic). In other high molecular weight immunophilins, such as CyP40 and FKBP52, similar clusters of charged residues are involved in protein-protein interactions, such as interaction with Hsp90 (Ratajczak and Carrello, 1996). A short acidic linker region (~15 residues) connects the N-terminal domain with the C-terminal cyclophilin domain. It consists of aspartate and glutamate residues. Unlike bovine CyP40, there are no β-turns in this linker region. 4.7.2 Cyclophilin domain The cyclophilin domain of AtCyP38 (83-437) has the typical β-barrel structure, closed at both ends by α-helices. Eight anti-parallel β-strands make the barrel. Even though the AtCyP38 cyclophilin domain has quite extensive loops, they not have any conformation similar to the loops of other known divergent cyclophilins. Also, this domain does not have the conserved cysteine residues of other divergent cyclophilins. Fig. 33 shows the structural overlap of AtCyP38 CyP domain with hCyPA. A structure based sequence alignment of AtCyP38 with all these cyclophilins can be seen in Fig.34. 100 Figure 33. Structural overlap of cyclophilin domains of AtCyP38 (red) and hCyPA (green) in two different views. The N-terminal region of AtCyP38 that forms part of the CyP domain is shown in orange. The overlap was generated with the MULTIPROT server and the figure was prepared using PYMOL. 101 Several variations can be seen in the AtCyP38 (83-437) cyclophilin domain, when compared to other known cyclophilin structures. When the AtCyP38 sequence is aligned with that of other cyclophilins, it can be seen that the sequence in β5 and β6 region of other cyclophilins aligns well with the respective region of AtCyP38 and many of the active site residues can be seen in this region. But when structurally superimposed, it is the β1-β2 region of the current intermediate AtCyP38 that occupies the position of the β5-β6 region of other cyclophilins. This results in the displacement of a long stretch with the residues that must make the active site, in the form of a flexible loop. Strands β3 and β4 of AtCyP38 correspond to β1 and β2 of other cyclophilins. Strand β5 of AtCyP38 which corresponds to β3 of other cyclophilins, appears to be much longer. The current AtCyP38 structure lacks the βstrand, corresponding to the short β4 of other cyclophilins. Instead there is a β-strand (β6) in AtCyP38 in the later part of the structure. The corresponding region in other cyclophilins normally forms part of a loop. The β-strand β7 of AtCyP38 sits directly over the active site and does not form part of the barrel. In other cyclophilins the corresponding region forms a 310 helix, contributing to the active site. This β-strand of AtCyP38 is so oriented that it makes a cavity perpendicular to the β-barrel in the active sire region. β8 and β9 of AtCyP38 are slightly longer than the corresponding β7 and β8 of other cyclophilins. The helix α6 (between β4 and β5) of AtCyP38 caps one end of the β−barrel and this helix corresponds to α1 in other cyclophilins. α7 which caps the other end of the barrel is much shorter than the corresponding α−helix in other cyclophilins. The extreme C-terminus of AtCyP38 has a short 310 helix (α8), which is not seen in other cyclophilins. The current AtCyP38 intermediate structure has two long loops. The loop that connects β5 and β6 of AtCyP38 is about 102 26 residues longer than the corresponding loop in other cyclophilins. The other long loop in the structure between β6 and β7 is formed by the displacement due to the incorporation of the β1-β2 region into the β-barrel. AtCyP38 Bov CyP40 hCyPA hCyPB Eco CyPB 232 011 001 007 001 MPLLKGRASVDMKVKIKDNPNIEDCVFRIVLDGYNAPVTAGNFVDLVER----------SNPSNPRVFFDVDIGG-----ERVGRIVLELFADIVPKTAENFRALCTGEKGIGPTTGKP --MVNPTVFFDIAVDG-----EPLGRVSFELFADKVPKTAENFRALSTGEKGFG-----GPKVTVKVYFDLRIGD-----EDVGRVIFGLFGKTVPKTVDNFVALATGEKGFG-----------AKGDPHVLLTTSA-----GNIELELDKQKAPVSVQNFVDYVNSG---------- AtCyP38 Bov CyP40 hCyPA hCyPB Eco CyPB 281 HFYDGMEIQRS-DGFVVQTGDPEGPAEGFIDPSTEKTRTVPLEIMVTGEKTPFYGSTLEE 066 LHFKGCPFHRIIKKFMIQGGDFSNQNG--------------------------TGGESIY 048 --YKGSCFHRIIPGFMCQGGDFTRHNG--------------------------TGGKSIY 056 --YKNSKFHRVIKDFMIQGGDFTRGDG--------------------------TGGKSIY 040 -FYNNTTFHRVIPGFMIQGGGFTEQMQ--------------------------QKKPNPP AtCyP38 AtCyP38 Bov CyP40 hCyPA hCyPB Eco CyPB 078 GGILLVANPVIPDVSVLISGPP 099 340 LGLYKAQVVIPFNAF--GTMAMAREEFENDSGSSQVFWLL-KESELTPSNSNILDGRYAV 100 GEKFEDEN-FHYKHDKEGLLSMANAGSNT--NGSQFFITTVPTPHL--------DGKHVV 080 GEKFEDEN-FILKHTGPGILSMANAGPNT--NGSQFFICTAKTEWL--------DGKHVV 088 GERFPDEN-FKLKHYGPGWVSMANAGKDT--NGSQFFITTVKTAWL--------DGKHVV 073 IKNEADNG----LRNTRGTIAMARTADKD-SATSQFFINVADNAFLDH---GQRDFGYAV AtCyP38 Bov CyP40 hCyPA hCyPB Eco CyPB 397 FGYVTDN---EDFLADLKV-------------GDVIESIQVVSGLENLANPSYKIAG 149 FGQVIKGMGVAKILENVEVKG------EKPAKLCVIAECGELKEGDDWGIFPK---129 FGKVKEGMNIVEAMERFGSRN------GKTSKKITIADCGQLE-------------137 FGKVLEGMEVVRKVESTKTDS-----RDKPLKDVIIADCGKIEVEKPFAIAKE---125 FGKVVKGMDVADKISQVPTHDVG-PYQNVPSKPVVILSATVLP-------------- 437 195 165 184 166 Figure 34. Structure based sequence alignment of the cyclophilin domains of AtCyP38, bovine CyP40, human CyPA, human CyPB and E. coli CyPB. Residues corresponding to β-strands are underlined and those corresponding to helices are shown in bold. The conserved residues in all the sequences are highlighted in yellow. The residues that are known to be important for CsA binding are shown in red. The N-terminal region of AtCyP38, which enters into the cyclophilin domain, is shown in blue at the corresponding position. When compared with human CyPA, the cyclophilin domain of AtCyP38 shows the lowest degree of identity (17 %) to any known cyclophilin. This may be due to the functional role of this protein in plants. The cyclosporin A (CsA) binding of hCyPA involves 13 residues. In AtCyP38, only of these residues are conserved in the active site. This could be the reason for the inability of its spinach homolog 103 TLP40 to bind CsA in low concentrations. The conserved residues in AtCyP38 are: Arg290, Phe294, Gln297, Gly307, Ala358, Ala360, Gln372, Phe374 and Leu382, which correspond to Arg55, Phe60, Gln63, Gly72, Ala101, Ala103, Gln111, Phe113 and Leu122 of hCyPA. Of these residues, Ala101, Ala103, Gln111, and Phe113 of hCyPA are located in the β-barrel whereas, in the case of AtCyP38, the corresponding Figure 35. The cyclophilin domain of AtCyP38 (83-437). The region of the N-terminus that enters into the CyP domain is shown in cyan and the active site residues that are responsible for PPIase activity and CsA binding, as derived from the hCyPA structure, are shown in blue as stick model (figure prepared with PYMOL). residues Ala358, Ala360, Gln372 and Phe374 are in the loop regions that are far apart in the structure. The displacement of this region into a long loop could have happened due to the incorporation of two β-strands from the N-terminal domain and this is of great functional significance. The N-terminal region that replaces the active site provides two residues, Ala84 and Asn85, which are needed for CsA binding. As the active site residues are too scattered in the present unmatured structure of AtCyP38, CsA binding is abolished. 104 A shorter form of matured spinach TLP40 (123-449) does not bind to cyclosporin A, but still shows PPIase activity (Fulgosi et al., 1998). This region corresponds to AtCyP38 (111-437). The cyclophilin domain of AtCyP38, as seen from the crystal structure of AtCyP38 (83-437), explains this peculiar characteristic only to a small extent. The non-conserved position of the active site residues, when compared to other cyclophilins, could be the major reason for this property. However, the PPIase activity of mature TLP40 (123-449) seems to be unaffected. This is not surprising, even though this cannot be fully explained by the current structure. A few additional residues at the N-terminus make AtCyP38 (83-437) an intermediate form. The actual matured form of AtCyP38; i.e. residues 93-437 (or even a shorter stretch, 111-437), might have the loops rearranged, without much disturbance in the active site, making PPIase activity possible but still not conserved enough to elicit CsA binding characteristics when CsA is available only in very low concentrations. As described earlier, AtCyP38 (83-437) is an intermediate and not the mature protein. The incorporation of the two N-terminal β-strands into the C-terminal cyclophilin domain, there by causing dislocation of the active site residues, could be a strategic means to prevent the cyclophilin domain to be active during its transport to the thylakoid lumen of chloroplasts. This feature makes the present structure very important from the protein transportation and functional point of view. The structure of AtCyP38 in the mature form, when available, will confirm the complete mechanism of structural rearrangement and functional features such as CsA binding and PPIase activity. 4.8 SURFACE OF ATCYP38 105 The surface of the protein is more negatively charged, with intermittent positively charged patches. The inside of the β-barrel is filled with the side-chains of the residues that form the β-sheets, Fig 36A. However, the two long loops of the CyP domain are not heavily charged. Unlike other CyPs, the active site groove of the protein is not so well defined due to the over-hanging loops, Fig 36B. Fig 36C shows the rotated view of Fig 36B and it is quite obvious that this surface is highly negatively charged across the CyP domain, the adjacent helical bundle region as well as the sandwiched region in between. This is probably of great significance in its interaction with other proteins. . A B Helical bundle β-barrel Helical bundle C Helical bundle Active site Figure 36. Surface charge features (blue for positive and red for negative charge) of AtCyP38 (83-437). The dark red region indicates a potential of less than -10 kT/e, while dark blue indicates greater than 10 kT/e. (A) the view through the β barrel and the long loops; (B) the active site view for the CyP domain; (C) the rotated view of B. The figures were made with GRASP. 4.9 PRECURSOR, INTERMEDIATE AND MATURE ATCYP38 Many proteins, which are to be localized to specific organelles within the cell, are synthesized as precursors containing an N-terminal extension, which gets proteolytically removed during their translocation across the organelle membrane. In general, freshly synthesized precursors remain in a partially unfolded state for 106 efficient translocation and such unfolded states make structure analysis difficult. Some precursor proteins have more than one signal stretch, which aid the transportation to the interior location of the organelle. These intermediates are generally more stable than the precursor themselves. Understanding the mechanism of translocation of a protein requires knowledge of the structures of the intermediates in the pathway. The precursor AtCyP38 (residues 1-437) sequence starts with a hydrophilic tag region that is rich in serine, leucine and arginine residues. The precursor has a total length of 437 amino acids, spanning between Met1 and Gly437. This includes the bipartite signal sequences, which help the protein translocate to the chloroplast thylakoid lumen. The first signal that aids the transport across the chloroplast envelope corresponds to the region between Met1 and Arg36. The region between Cys37 and Ser92 forms the second signal, which helps the protein cross the thylakoid membrane and localize in the lumen. These signal regions get cleaved off once the transportation steps are over. Matured AtCyP38 encodes for the region Val93 to Gly437. The protein of this structural study is of an intermediate length with residues Val83 to Gly437. This includes a stretch of additional 10 residues compared to the mature protein. The complete sequence of AtCyP38 is given in Fig. 37. Our attempts to express the full length precursor AtCyP38 protein were not successful and the intermediate stretch from Cys37 to Gly437 turned out to be insoluble. In this study, we have expressed and solved the structure of the stretch, Val83 to Gly437. Currently, we have expressed and have been setting up the mature protein, Val93 to Gly437, for crystallization. When the structure of the mature protein is determined, we will be able to establish the actual mechanism of protein translocation and its functional features. 107 001 MAAAFASLPT FSVVNSSRFP RRRIGFSCSK KPLEVRCSSG NTRYTKQRGA FTSLKECAIS 061 LALSVGLMVS VPSIALPPNA HAVANPVIPD VSVLISGPPI KDPEALLRYA LPIDNKAIRE 121 VQKPLEDITD SLKIAGVKAL DSVERNVRQA SRTLQQGKSI IVAGFAESKK DHGNEMIEKL 181 EAGMQDMLKI VEDRKRDAVA PKQKEILKYV GGIEEDMVDG FPYEVPEEYR NMPLLKGRAS 241 VDMKVKIKDN PNIEDCVFRI VLDGYNAPVT AGNFVDLVER HFYDGMEIQR SDGFVVQTGD 301 PEGPAEGFID PSTEKTRTVP LEIMVTGEKT PFYGSTLEEL GLYKAQVVIP FNAFGTMAMA 361 REEFENDSGS SQVFWLLKES ELTPSNSNIL DGRYAVFGYV TDNEDFLADL KVGDVIESIQ 421 VVSGLENLAN PSYKIAG 437 Figure 37. The amino acid sequence of precursor AtCyP38 with its bipartite signals. The N-terminal region that is highlighted in yellow forms the first signal and the following region that is highlighted in grey forms the second signal. The protein used in the current study, amino acids 83-437, is shown in bold. 4.10 INSIGHTS FROM STUDIES ON SPINACH TLP40 AND PSBQ Protein phosphatases in the thylakoid membranes of plant chloroplasts are now known to be regulated by cyclophilins. Vener and co-workers (1999) used spinach TLP40 in their study to confirm this. However, not much study has been done on AtCyP38 in this regard. Since the sequences of these two proteins are very much conserved and both proteins get localized to the same region in the chloroplast, the TLP40 study results can be extrapolated to AtCyP38 as well. Similar to mammalian cyclophilins, TLP40 also binds to CsA, but only at nonphysiological concentrations. TLP40/CsA complex inhibits a protein phosphatase 2B (PP2B) type activity as seen from in vitro experiments. An investigation was carried out to understand the in vivo mode of action of TLP40 (Vener et al., 1999). Even though binding to an exogenous drug is not needed for the TLP40-phosphatase interaction, thylakoid protein dephosphorylation is activated by CsA treatment, 108 indicating a functional connection between the phosphatase and TLP40. PPIase activity of TLP40 is quite strong. Addition of prolyl-containing peptides and their subsequent binding to TLP40 could inhibit protein phosphatase activity. They proposed that thylakoid protein dephosphorylation is regulated by the lumenal TLP40 through a signaling pathway across the thylakoid membrane which is directed from the inside to the outside of the lipid bilayer. The phosphatase may be regulated by the reversible binding of TLP40 to a trans-membrane protein, which could be the phosphatase itself or an accessory trans-membrane protein, which anchors the phosphatase to TLP40 on the opposite side of the thylakoid membrane. TLP40 associates itself with the inner thylakoid membrane surface. It was also observed that membrane phosphatase activity is suppressed when TLP40 binds to a site in the inner thylakoid surface and is stimulated when TLP40 is released into the lumen. The activation of protein dephosphorylation by the addition of CsA is probably due to the formation of the CsA-TLP40 complex, which prevents TLP40 from associating with the membrane phosphatase. The regions of spinach TLP40 that correspond to the two short stretches 136 GVKALDSVERN146 and 162 VAGFAESKKDHG173 of AtCyP38 are about 60% similar to the sequence 33GKKFDSSRDRN43 of FKBP12, which binds to the protein phosphatase calcineurin (Aldape et al., 1992). However, analysis of these regions in AtCyP38 shows no common structural features, except the sequence similarity. These form part of the helices α3, α4 and the loop between them in the current structure. The charge distribution in the helical bundle for the protein PsbQ is similar to that of AtCyP38 in these two stretches. In the case of PsbQ, the charged cluster that is close to the N-terminal strand is thought to be involved in interaction with Photosystem II core and the second charged cluster is thought to be involved in interactions with 109 other extrinsic proteins or with a different site on the Photosystem II core (Calderone et al., 2003). Similarly, mature AtCyP38 might have an interaction mode wherein the stretch Gly136 to Asn146 gets involved with Photosystem II and the stretch Val162 to Gly173 takes part in phosphatase interaction or vise versa. But these two charged stretches not come anywhere close to CyP active site, to be affected by CsA binding. Biochemical data indicate that the N-terminal residues of spinach PsbQ are involved in binding to the Photosystem II complex, through an intermediary PsbP protein (Seidler, 1996). The structure of PsbQ and the helical bundle of AtCyP38 agree very well with each other as explained earlier. For AtCyP38, the region Nterminal to the helical bundle (i.e. Val93 to Asp114) could be involved in interaction with the thylakoid transmembrane phosphatase. The same region has the putative phosphatase-interacting sites 102 DPEALLR108 (Vener et al., 1999) which lies close to the CyP domain in the current structure in the form of a short helix. However the stretch which forms a short helix is only 57 % identical to the loop of regulatory subunit A that is responsible for binding the catalytic subunit of protein phosphatase 2A. At the same time, if CsA addition at high concentrations can inhibit phosphatase binding of AtCyP38, as seen for TLP40, then a region which lies close to the CyP active site might be involved in phosphatase binding. So chances of the phosphatase binding region being α1 of the current structure is more, compared to parts of and loops between α3 and α4. The intermediate form of AtCyP38 is not expected to perform any phosphatase activity as it is still in the transportation stage. The region that is responsible for this activity could be either protected or unfolded. Our current structure shows that the region remains protected from possible interactions with 110 phosphatases and from membrane binding by forming two of the β-strands of the cyclophilin domain. At the same time, the region 102 DPEALLR108 remains as a short helix, which gets sandwiched between the helical bundle and the cyclophilin domains, thus inaccessible for free interaction with a phosphatase. The cyclophilin domain is also unlikely to perform any PPIase activity during transportation. The displacement of the active site residues that are needed for binding to a prolyl peptide also supports this hypothesis. We speculate that once the transportation of AtCyp38 to the thylakoid lumen is over, the N-terminal region from signal gets cleaved by proteolytic activity. In the mature protein, the loop region of the cyclophilin domain that gets displaced by the signal residues during transport will take up their respective position and make the conserved β-barrel. In this context, the current structure indicates that the actual mature protein might start from residue Ile100 and not from Val93, as we expected before solving the structure. Otherwise, the signal region when cleaved will not remove only β1 and not β2. This won’t provide space for the displaced loop to reorganize and complete the barrel. This can be confirmed by N-terminal sequencing of the mature lumenal AtCyP38 of Arabidopsis, instead of a recombinant protein. However, this sort of reorganization is a very high energy requiring process and hence the whole barrel might even unfold and refold back to a stable conformation during maturation. Once folded, the cyclophilin domain should be able to perform PPIase activity, as the available nine active site residues will be in their preferred positions. These residues are enough to hold the prolyl peptide in place and elicit isomerization. The region Val93 to Asp114, along with the adjacent charged regions of the CyP domain and helical bundle will become accessible for interaction with the thylakoid trans-membrane phosphatase. Phosphatase binding of AtCyP38 inhibits the 111 dephosphorylation process by phosphatase. Binding of CsA to the cyclophilin domain of mature form of lumenal AtCyP38, by providing millimolar rates of CsA might cause changes in the domain’s interactions to allow phosphatase binding. The active site residues of CyP domain cannot hold the drug in place and probably the phosphatase binding region also gets affected in an attempt to bind the drug. In short, CsA binding induces phosphatase activity, simply because AtCyP38 can no longer bind to the phosphatase. The reduction in phosphatase activity, which happens when a prolyl-peptide binds to the cyclophilin domain, is also expected. The possible speculation from the current structure is that, some residues in the phosphatase binding region take part in stabilizing the prolyl peptide binding, similar to that of CsA-binding at high concentrations. The trans-membrane phosphatase that gets regulated by AtCyP38 is actually responsible for the dephosphorylation of protein subunits D1, D2 and CP43 of Photosystem II, as seen in spinach system (Rokka et al., 2000). So like TLP40 of spinach, CyP38 of Arabidopsis acts as a regulator to Photosystem II subunit dephosphorylation. The subunits D1, D2 and CP43 requires high turn over during temperature and light stress conditions. The damaged subunits have to be degraded fast and their dephosphorylation is required to initiate the degradation process. Also, new copies of the subunits should take up the position of degraded ones for the Photosystem to be functional. This makes it essential to have fast folding of the subunits within thylakoid lumen, so that the freshly folded proteins can get incorporated in to the Photosystem. Lumenal TLP40 / CyP38 along with other immunophilins are found to accumulate close to the region of incorporation of protein subunits into the Photosystem. The PPIase activity of AtCyP38 could be of great significance, wherein it performs the function of a foldase. Thus the thylakoid 112 phosphatase activity, protein degradation and protein folding processes are all connected to each other via CyP38. 4.11 FUTURE DIRECTIONS In this study, we have determined the structure of an intermediate form of AtCyP38, residues 83-437. We want to get the structure of the mature AtCyP38 (may be residues 100-437), to see how exactly the cyclophilin domain rearranges itself. Currently, we are attempting to crystallize the mature protein and we have not obtained any crystals for the mature protein so far. It is too early to conclude about the plausibility to crystallize the mature protein, due to the fact that the N-terminal helical domain of the mature protein is no longer in contact with the cyclophilin domain which makes the two domains more flexible. If the structure of the mature protein is obtained, we will have a better understanding of the differences between the two forms and how exactly that affects the functionality in binding to a phosphatase. Attempts are underway to crystallize the CyP domain; residues 232-437 as well. The structure of the domain alone can explain how exactly the loops are re-organized to complete the β-barrel structure. While structural analysis would provide valuable information about the function of AtCyP38, molecular genetic analyses; including mutational studies can provide considerable amount of information to complement it. Our collaborators are doing that part of the work. 4.12 CONCLUDING REMARKS This is the first structure of a plant cyclophilin. This structure is of much significance as it is a multi-domain cyclophilin that gets localized into the chloroplast lumen. The present AtCyP38 83-437 structure reveals significant details of the 113 structural features of a plant cyclophilin. The structure has two distinct domains, namely a helical bundle and the cyclophilin domain. A short stretch in the extreme Nterminus is expected to be involved in binding to phosphatase and thereby regulating the dephosphorylation process in plant Photosystem II. Apart from proposing a possible means of interaction with phosphatase and role as a PPIase in thylakoid lumen, the importance of the structure lies in the fact it shows how the active domain of a protein is protected during transport mechanism. This structure is of an intermediate form of a protein before the mature protein level. However, proper understanding of the exact mode of phosphatase interaction and its effect on binding prolyl-peptides require further investigations using the mature protein. 114 [...]... TEKTRTVPLE YKAQVVIPFN VFWLLKESEL NEDFLADLKV YKIAG 437 1 12 1 42 1 72 2 02 2 32 2 62 2 92 322 3 52 3 82 4 12 Figure 28 The sequence of AtCyP38 (83-437) The native methionine residues are highlighted in grey and the leucine residues that were mutated to methionine are highlighted in yellow 4.5 CRYSTALLOGRAPHY OF ATCYP38 MUTANTS 4.5.1 Expression, purification and crystallization The mutants and selenomethionine derivatives... in AtCyP38 are: Arg290, Phe294, Gln297, Gly307, Ala358, Ala360, Gln3 72, Phe374 and Leu3 82, which correspond to Arg55, Phe60, Gln63, Gly 72, Ala101, Ala103, Gln111, Phe113 and Leu 122 of hCyPA Of these residues, Ala101, Ala103, Gln111, and Phe113 of hCyPA are located in the β-barrel whereas, in the case of AtCyP38, the corresponding Figure 35 The cyclophilin domain of AtCyP38 (83-437) The region of the... active site, in the form of a flexible loop Strands β3 and β4 of AtCyP38 correspond to β1 and 2 of other cyclophilins Strand β5 of AtCyP38 which corresponds to β3 of other cyclophilins, appears to be much longer The current AtCyP38 structure lacks the βstrand, corresponding to the short β4 of other cyclophilins Instead there is a β-strand (β6) in AtCyP38 in the later part of the structure The corresponding... cyclophilin domain of AtCyP38 and other known cyclophilin structures (section 4.9 .2) The helical domain and the cyclophilin domain of the structure are connected β1 α1 2 2 GGILLVANPVIPDVSVLISGPPIKDPEALMRYAMPIDNKAIREVQKPME 90 80 110 100 2 α3 120 α3 α4 DITDSLKIAGVKAMDSVERNVRQASRTMQQGKSIIVAGFAESKKDHGNE 130 150 140 160 α4 170 α5 MIEKLEAGMQDMLKIVEDRKRDAVAPKQKEILKYVGGIEEDMVDGFPYE 180 190 21 0 20 0 β3 22 0... (X-12B) / ADSC Q210 CCD Resolution (Å) 2. 64 2. 46 Inflection Peak Remote Wavelength (Å) 0.9797 0.9794 0.9500 Total observations 23 0,354 94,780 91,174 96, 420 Unique reflections 14 ,20 9 17,556 17,609 19,441 Completeness (%) 97.8 (99.8) 98.1(93.3) 98 .2 (90.9) 98.6 (91.6) Redundancy 6 .2 (5.7) 6.3 (6.0) 6 .2 (5.7) 1 Rsym 0.069 (0 .29 ) 0.059 (0.30) 0.065 (0 .25 ) 0.067 (0 .23 ) 〈I/σ(I)〉 1 0.9500 21 .40 (5.4) 20 .2 (5.3)... similar to the loops of other known divergent cyclophilins Also, this domain does not have the conserved cysteine residues of other divergent cyclophilins Fig 33 shows the structural overlap of AtCyP38 CyP domain with hCyPA A structure based sequence alignment of AtCyP38 with all these cyclophilins can be seen in Fig.34 100 Figure 33 Structural overlap of cyclophilin domains of AtCyP38 (red) and hCyPA... (DeLano, 20 02) program A summary of the refinement statistics is shown in Table 3 Table 3 Structure refinement statistics for mutant AtCyP38 Mutant AtCyP38 1 Rcryst / Rfree 0 .22 /0 .28 Resolution range (Å) 8 - 2. 46 Reflections (working/test) 14317/1588 Final model: Non-hydrogen atoms 27 65 Waters 21 4 Average B-factors ( 2) : • Protein (all atoms) 64.36 • Protein (with two loops removed) 54.86 (21 86 atoms)... and aligns with the helical bundle of AtCyP38 with an R.M.S.D of 1.71 over a stretch of 88 residues Cytochrome b5 62 is about 10% identical and aligns with an R.M.S.D of 1.73 for 83 residues, Fig 32 A B Figure 32 Structure overlap for the helical bundle of AtCyP38 with (A) PsbQ and (B) cytochrome b5 62 AtCyP38 is drawn in red and green whereas PsbQ and cytochrome b5 62 are shown in cyan and pink The overlap... ∑hkl|Fo(hkl)| 95 4.7 THREE-DIMENSIONAL STRUCTURE OF ATCYP38 (83-437) The secondary structural organization of AtCyP38 is illustrated in Fig 28 AtCyP38 (83-437) has two distinct domains as seen from its crystal structure The Nterminus is a helical domain made up of 5 helices (α1 – α5) of varying lengths This domain is followed by a typical cyclophilin domain, made up of an eight-stranded βbarrel that is... β8 of other cyclophilins The helix α6 (between β4 and β5) of AtCyP38 caps one end of the β−barrel and this helix corresponds to α1 in other cyclophilins α7 which caps the other end of the barrel is much shorter than the corresponding α−helix in other cyclophilins The extreme C-terminus of AtCyP38 has a short 310 helix (α8), which is not seen in other cyclophilins The current AtCyP38 intermediate structure . α6 β 6 180 28 0 330 90 100 110 120 130 140 150 160 170 23 0 24 0 25 0 26 0 70 190 20 0 21 0 22 0 29 0 300 310 320 340 350 360 370 380 390 0 410 420 430 80 α 3 2 40 α 4 α 3 2. IEDCVFRIVL 26 2 26 3 DGYNAPVTAG NFVDLVERHF YDGMEIQRSD 29 2 29 3 GFVVQTGDPE GPAEGFIDPS TEKTRTVPLE 322 323 IMVTGEKTPF YGSTLEELGL YKAQVVIPFN 3 52 353 AFGTMAMARE EFENDSGSSQ VFWLLKESEL 3 82 383 TPSNSNILDG. 98.1(93.3) 98 .2 (90.9) 98.6 (91.6) Redundancy 6 .2 (5.7) 6.3 (6.0) 6 .2 (5.7) 6.3 (5.9) 1 R sym 0.069 (0 .29 ) 0.059 (0.30) 0.065 (0 .25 ) 0.067 (0 .23 ) 〈I/σ(I)〉 21 .40 (5.4) 20 .2 (5.3) 20 .7 (6.5) 19 .21 (5.1)