Crystal structure of arabidopsis thaliana cyclophilin 38 (atcyp38 1

CRYSTAL STRUCTURE OF ARABIDOPSIS THALIANA CYCLOPHILIN 38 (ATCYP38) DILEEP VASUDEVAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 CRYSTAL STRUCTURE OF ARABIDOPSIS THALIANA CYCLOPHILIN 38 (ATCYP38) DILEEP VASUDEVAN (M. Fisheries Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS This thesis is the most significant scientific accomplishment in my life so far and it is my pleasure to thank those who made this possible and who supported me in one way or other. I am grateful to my PhD supervisor, Dr. Kunchithapadam Swaminathan. With his enthusiasm, inspiration and great efforts to explain things clearly and simply, he helped me understand what crystallography is. Without his patience and help, the structure of this thesis would not have been possible. The support of our collaborator Prof. Sheng Luan and his group at University of California, Berkeley, USA is deeply acknowledged. I also thank Dr. Leslie Haire of National Institute of Medical Research, London, UK for her suggestion to use vapor batch plates for my crystallization experiments. I would like to thank the beamline staff at National Synchrotron Light Source, USA for all their help during my data collection. I am indebted to my peers at NUS for providing a stimulating and fun environment in which I could learn and grow. I am especially grateful to my DBS labmates Asha, Gayathri and Tien Chye, as well as my friends Dileep, Thakur and Jobi. I convey my heartfelt thanks to Dr. Curt Davey of School of Biological Sciences (SBS), Nanyang Technological University (NTU) for accommodating me in his lab as a Research Associate even before the completion of my PhD and letting me finish my PhD thesis, along with my job. I also thank my friends at NTU. Words are not enough to thank my dearest wife Anuradha for all her support at both lab and back home. Had she not been there, my PhD project would not have i reached this point. I wish to dedicate this thesis to her. Also, I offer my special thanks to my mother and our entire family for providing a loving environment. The NUS research scholarship, which supported my research and stay in Singapore, is greatly acknowledged. ii PUBLICATION Parts of this thesis have already been or will be published in due course: Vasudevan D, Gopalan G, He Z, Luan S, Swaminathan K. 2005. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38). ACTA Cryst. F 61: 1087-1089. Vasudevan D, Radhakrishnan A, Luan S, Swaminathan K. 2007. Crystal structure of an intermediate form of Arabidopsis thaliana cyclophilin 38 (AtCyP38) (to be submitted). iii TABLE OF CONTENTS Acknowledgements Page i Publication iii Table of contents iv Summary ix List of abbreviations xi List of figures xiii List of tables xv CHAPTER MACROMOLECULAR X-RAY CRYSTALLOGRAPHY 1.1 PROTEIN STRUCTURE DETERMINATION 1.2 PROTEIN CRYSTALLIZATION 1.3 BASIC CONCEPTS IN PROTEIN CRYSTALLOGRAPHY 1.3.1 Unit-cell, lattices and Miller indices 1.3.2 Symmetry, point groups and space groups 1.3.3 Crystals and X-rays 1.3.4 X-ray diffraction 1.3.5 Bragg’s law 10 1.3.6 Reciprocal space 11 1.3.7 The Ewald sphere 13 1.3.8 Fourier transform and structure factor 14 1.3.9 Phase problem 15 1.4 GEOMETRIC DATA COLLECTION 16 1.4.1 Data reduction 17 iv 1.5 STRUCTURE DETERMINATION 18 1.5.1 Phasing methods 18 1.5.1.1 The Multi-wavelength anomalous dispersion (MAD) method 21 1.5.1.2 Principle of anomalous scattering 23 1.5.2 Phase improvement 25 1.5.3 Model building 26 1.5.4 Refinement 28 1.5.5 Validation 31 1.5.6 Presentation 34 CHAPTER BIOLOGICAL BACKGROUND 2.1 ORGAN TRANSPLANTATION & IMMUNOSUPPRESSIVE DRUGS 35 2.1.1 Cyclosporin 36 2.1.2 FK506 37 2.1.3 Rapamycin 37 2.2 IMMUNOPHILINS 38 2.2.1 PPIase activity of immunophilins and protein folding 40 2.2.2 Immunosuppressive activity of immunophilins 41 2.3 DIVERSITY OF IMMUNOPHILINS 42 2.3.1 Archaeal cyclophilins 42 2.3.2 Bacterial cyclophilins 43 2.3.3 Fungal cyclophilins 43 2.3.4 Animal cyclophilins 44 2.3.5 Plant cyclophilins 47 2.3.5.1 Domain organization & phylogenetic analysis in Arabidopsis cyclophilins 49 v 2.3.5.2 Evolutionary dynamics of the lumenal cyclophilins 56 2.4 CYLOPHILIN STRUCTURES 57 2.4.1 Ligand-free cyclophilin structures 57 2.4.2 Cyclophilin-ligand complex structures 63 2.4.2.1 Structural features of cyclosporin A binding 65 2.4.2.2 Structural features of prolyl-peptide binding 67 2.5 HOMOLOGS OF ATCYP38 68 2.6 PURPOSE OF THIS STUDY 69 CHAPTER MATERIALS AND METHODS 3.1 EXPRESSION AND PURIFICATION OF RECOMBINANT ATCYP38 71 3.1.1 Expression 71 3.1.2 Affinity chromatographic purification 73 3.1.3 Thrombin cleavage 73 3.1.4 Size exclusion chromatography 73 3.1.5 Analyses for purity and homogeneity 3.2 74 EXPRESSION AND PURIFICATION OF SELENOMETHIONYLATED ATCYP38 74 3.2.1 Expression ` 74 3.2.2 Purification 75 3.3 CRYSTALLIZATION AND TESTING OF CRYSTAL QUALITY 75 3.3.1 Hanging drop vapor diffusion method 75 3.3.2 Vapor batch method 75 3.3.3 Cryo-protection of the crystals and testing of diffraction quality 77 3.4 77 DATA COLLECTION vi 3.5 DATA ANALYSIS AND STRUCTURE DETERMINATION 78 CHAPTER RESULTS AND DISCUSSION 4.1 EXPRESSION AND PURIFICATION OF NATIVE WILD TYPE ATCYP38 79 4.1.1 Expression 79 4.1.2 Affinity chromatographic purification 79 4.1.3 Thrombin cleavage 80 4.1.4 Size exclusion chromatography 81 4.1.5 Analyses for purity and homogeneity 82 4.2 EXPRESSION AND PURIFICATION OF SELENOMETHIONYLATED WILD TYPE ATCYP38 84 Expression 84 4.2.2 Purification 84 4.2.1 4.3 CRYSTALLIZATION, DATA COLLECTION AND ANALYSIS FOR WILD TYPE ATCYP38 85 4.3.1 Crystallization 85 4.3.2 Data collection and analysis 87 4.4 SELECTIVE MUTATION OF ATCYP38 RESIDUES TO AID PHASING 90 4.5 CRYSTALLOGRAPHY OF ATCYP38 MUTANTS 91 4.5.1 Expression, purification and crystallization 91 4.5.2 Data collection and analysis 92 4.6 STRUCTURE DETERMINATION OF MUTANT ATCYP38 93 4.7 THREE-DIMENSIONAL STRUCTURE OF ATCYP38 (83-437) 96 vii 4.7.1 N-terminal domain 98 4.7.2 Cyclophilin domain 100 4.8 SURFACE OF ATCYP38 105 4.9 PRECURSOR, INTERMEDIATE AND MATURE ATCYP38 106 4.10 INSIGHTS FROM STUDIES ON SPINACH TLP40 and PSBQ 108 4.11 FUTURE DIRECTIONS 113 4.12 CONCLUDING REMARKS 113 REFERENCES 115 viii 2.4.2 Cyclophilin-ligand complex structures Several cyclophilin structures are available in complex with a ligand. More than 20 different X-ray structures of native and CsA analogue structures have been determined. All structures of native and CsA analogues show very similar conformations and share the same binding mode. The binding is enhanced by conserved hydrogen bonds between CsA and CyPA. The different CsA analogues can have a significant effect on binding and on calcineurin inhibition (Kallen et al., 1998), even though there are few significant changes in their 3D structures. Even chemically, different inhibitors like peptolide 214103 adopt the same bound conformation as CsA (Mikol et al., 1998). A series of non-immunosuppressant CsA analogues have also been developed which can bind strongly to CyPA but have small chemical differences in the effector loops. However, the binding of the complex to calcineurin is dramatically reduced and there is no measurable immunosuppressant effect. Such modified cyclosporins are of interest in anti-HIV therapy, where inhibition of CyPA alone prevents replication of the HIV virion (Thali et al., 1994; Franke and Luban, 1996). Another non-immunosuppressive CsA derivative is the drug Valspodar (PSC833), which exhibits the capacity to chemosensitise tumor cells that show multidrug resistance associated with MDR1-glycoprotein over-expression. The changes in Valspodar, as compared to CsA, significantly reduce the binding to CyPA and there is a very significant drop in immunosuppressive activity (Keller et al., 1992). Three families of small molecular ligands that can bind to the active site of cyclophilins have also been discovered. The piperidine family has been designed, based on the chemical similarity with dipeptide structures. The X-ray structures of complexes with 1-acetyl-3-methyl piperidine and ethyl-piperidine glyoxylate have been characterised (Kontopidis et al., 2004). The structure of a complex of hCyPA 63 with dimethylsulfoxide (DMSO) bound in the active site has led to the design of a series of related inhibitors. When crystallized in the presence of DMSO, two DMSO molecules were found in close proximity to the active site of human CyPD (Schlatter et al., 2005). A number of reports have appeared in the literature describing the isolation, characterization and immunosuppressive activity of a novel cyclophilin binding compound, Sanglifehrin A (Zenke et al., 2001). A number of anti-arthritic gold (I) drugs are in clinical use. Their critical target sites are thought to be thiolate sulphurs (cysteine residues). However, there is little structural data on the adducts of anti-arthritic Au (I) complexes with proteins. The crystal structure of a medically relevant gold compound [Au (I) phosphine complex (AuPEt3Cl)] in complex with CyP3 from C. elegans has been reported (Zou et al., 2000). The gold is bound to the active site histidine and it is shown to be inhibitory to PPIase activity. Calcineurin (protein phosphatase 2B, Cn) is activated by calcium-loaded calmodulin (CaM) and is inhibited by both the complexes, FKBP12-FK506 and CyPA-CsA. It is a heterodimer of chains CnA (60 kDa) and CnB (19 kDa). The crystal structures of the ternary complex between CyPA, CsA, truncated CnA and full length CnB have been reported (Huai et al., 2002; Jin and Harrison, 2002). The protein-drug complex binds at the interface between the catalytic and regulatory subunits of Cn. The conformation of both CyPA and CsA are largely unaltered from those seen in the binary complex alone. Cyclophilin complexes with proline containing dipeptides, tripeptides and tetrapeptides have been determined (Kallen et al., 1991; Kallen and Walkinshaw, 1992; Ke et al., 1993; Konno et al., 1996; Zhao and Ke, 1996). In all these cases, 64 binding of the peptide to a cyclophilin is similar and always the proline residue in the peptide adopts a cis-conformation. Cyclophilins are potential drug targets for anti-HIV therapy as the interaction between human CyPA and the HIV-1 gag protein is required to promote the assembly of the viral core (Colgan et al., 1996). A number of structures of CyPA in complex with the various fragment lengths of the amino terminal domain of HIV-1 capsid (Gamble et al., 1996; Zhao et al., 1997) as well as with a series of hexapeptides (Vajdos et al., 1997) have been characterized. In contrast to all other peptide complexes, the proline residue of the HIV-1 gag peptide binds in the active site of the cyclophilin in the trans conformation. With longer peptides, one of the CyP loops moves in order to accommodate the peptide, whereas binding of smaller peptides leaves the CyP structures unaltered. 2.4.2.1 Structural features of cyclosporin A binding The crystal structures of CyPA-CsA complex reveal the formation of pentameric or decameric complexes, which however not seem to have any biological relevance, as monomers in complex with CsA are capable of causing immunosuppression (Pflugl et al., 1993; 1994). In a monomer of CyPA-CsA, six residues of CsA form close contacts with the CyPA active site, which is made up of the 13 residues: Arg55, Phe60, Met61, Gln63, Gly72, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122 and His126 (Ke and Huai, 2004). The binding is enhanced by conserved hydrogen bonds between CsA and CyPA. All peptide bonds of the bound CsA have the trans conformation in the complex structures. Most other cyclophilins have the active site residues well conserved and hence the binding of CsA basically remains the same. In cases where 65 two or more of the active site residues are different, the level of CsA binding appears to be significantly less. Trp121 Ile57 Phe60 Arg55 Met61 Leu122 Glu65 Phe113 Gly72 Gln111 Ala101 Ala103 His126 Asn102 Figure 17. The structure of human CyPA. α-helices are shown in red, loops in green and β-strands as yellow arrows. The residues of the active site that are involved in cyclosporine A (CsA) binding and PPIase activity are highlighted in blue as stick model. This figure was prepared with the PYMOL program. 66 2.4.2.2 Structural features of prolyl-peptide binding As discussed before, cyclophilins perform PPIase activity by catalyzing the cis-trans isomerization of a peptidyl-prolyl amide bond. The active site can bind to a peptide of varying lengths having a proline residue with at least one residue preceding the proline. The active site can bind to CsA as well. As CsA and the peptide essentially compete for the same active site, CsA binding leads to the complete loss of PPIase activity. Twelve active site residues of hCyPA that are located on the surface of its hydrophobic pocket and interact with a peptide substrate are more or less the same as that for CsA binding. The subtle difference is in the fact that Ile57 also takes part in binding the peptide whereas Gly72 and Ala103 not. Four hydrogen bonds between the peptide and the active site residues are known to stabilize the interaction. In addition, the side chain of Arg148 of CyPA also forms a hydrogen bond with the peptide. Most crystal structures reveal that the cis form of the proline peptide binds to CyP (Kallen et al., 1991; Zhao and Ke, 1996; Konno et al., 1996). But the N-terminal domain of HIV-1 capsid p24 is known to bind to CyPA in the trans peptidyl form (Zhao et al., 1997). The cyclophilin domain of CyP40 is quite similar to that of hCyPA and the active site residues are identical to CyPA, apart from His141 of CyP40 that corresponds to Trp121 in CyPA. Site directed mutagenesis of this residue in CyPA has shown that it plays a significant role in CsA binding, but not very important in the PPIase enzymatic activity (Liu et al., 1991). This observation is in line with the catalytic and CsA binding properties of bovine CyP40. 67 2.5 HOMOLOGS OF ATCYP38 Thylakoid lumenal proteins (TLP) are present in higher plants as well as lower forms of life such as the blue green algae. Thylakoid Lumen Protein 40 (TLP40) from spinach, rice and pea has a very similar sequence as that of AtCyP38 with 82% identity, Fig. 18. Such a high degree of identity suggests that they might perform very similar functions. Furthermore, AtCyP38 is known to have homologs in other plants, all of which are localized in the thylakoid lumen of chloroplasts. However, cyclophilins with the same domain organization of AtCyP38 have not been reported in any higher organism and hence these are thought to have evolved to perform functions that are specific to plants. AtCyP38 Spinach TLP40 Pea TLP40 Rice TLP40 VANPVIPDVSVLISGPPIKDPEALLRYALPIDNKAIREVQKPLEDITDSLKIAGVKALDS LTSPVLPDLAVLISGPPIKDPEALLRYALPIDNKAIREVQKPLEDITESLRVLGLKALDS AANSALSDLSVLISGPPIKDPGALLRYALPIDNKAIREVQKPLEDITDSLKISGVKALDS PLEPVIPDVSVLISGPPIKDPGALLRYALPIDNKAVREVQKPLEDITDSLKIAGVRALDS AtCyP38 Spinach TLP40 Pea TLP40 Rice TLP40 VERNVRQASRTLQQGKSIIVAGFAESKKDHGNEMIEKLEAGMQDMLKIVEDRKRDAVAPK VERNLKQASRALKNGKSLIIAGLAESKKDRGVELLDKLEAGMGELQQIVENRNREGVAPK VERNVRQASRTLKQGKTLIVSGLAESKKEHGIELIDKLEAGIDEFELILQDGIEALLDQN VERNVRQASRALSNGRNLILGGLAESKRANGEELLDKLAVGLDELQRIVEDRNRDAVAPK AtCyP38 Spinach TLP40 Pea TLP40 Rice TLP40 QKEILKYVGGIEEDMVDGFPYEVPEEYRNMPLLKGRASVDMKVKIKDNPN-IEDCVFRIV QRELLQYVGSVEEDMVDGFPYEVPEEYQTMPLLKGRAVVEMKVKVKDNPN-VDNCVFRIV RKNFLQYVGGIEEDMVDGFPYELPEEYRNMPLLKGRAAVDMKIKIKDNPKRVDECVFHIV QKELLQYVGTVEEDMVDGFPYEVPEEYSSMPLLKGRATVDMKVKIKDNPN-LEDCVFRIV AtCyP38 Spinach TLP40 Pea TLP40 Rice TLP40 LDGYNAPVTAGNFVDLVERHFYDGMEIQRSDGFVVQTGDPEGPAEGFIDPSTEKTRTVPL LDGYNAPVTAGNFLDLVERHFYDGMEIQRRDGFVVQTGDPEGPAEGFIDPSTEKPRTIPL LDGYNAPVTAGNFVDLVERHFYDGMEIQRADGFVVQTGDPEGPAEGFIDPSTEKIRTVPL LDGYNAPVTAGNFLDLVERKFYDGMEIQRADGFVVQTGDPEGPAEGFIDPSTGKVRTIPL AtCyP38 Spinach TLP40 Pea TLP40 Rice TLP40 EIMVTGEKTPFYGSTLEELGLYKAQVVIPFNAFGTMAMAREEFENDSGSSQVFWLLKESE EIMVEGEKVPVYGSTLEELGLYKAQTKLPFNAFGTMAMAREEFENNSGSSQIFWLLKESE EIMVEGEKAPVYGETLEELGLYKARQKLPFNAFGTMAMAREEFEDNSGSSQVFWLLKESE ELMVDGDKAPVYGETLEELGRYKAQTKLPFNAFGTMAMARDEFDDNSASSQIFWLLKESE AtCyP38 Spinach TLP40 Pea TLP40 Rice TLP40 LTPSNSNILDGRYAVFGYVTDNEDFLADLKVGDVIESIQVVSGLENLANPSYKIAG LTPSNANILDGRYAVFGYVTDNQDYLADLKVGDVIESVQAVSGVDNLVNPTYKIAG LTPSNANILHGRYAVFGYVTENEDFLADLKVGDVIESIQVVSGLHNLVNPSYKIAG LTPSNANILDGRYAVFGYVTENEDYLADLKVGDVIESIQVVSGLDNLANPSYKIVG Figure 18. Sequence alignment (by CLUSTALW) of AtCyP38 with TLP40 from spinach, pea and rice. The predicted helical domain is given in blue and the cyclophilin domain in red. The identical sequences are highlighted in yellow. 68 2.6 PURPOSE OF THIS STUDY A large body of genomic data for the Arabidopsis cyclophilin family has so far been collected and analyzed. In addition, proteomic studies have allowed accurate assignment of specific sub-cellular localization of some of these cyclophilins. So far, no plant cyclophilin structure has been determined. Structural characterization of plant cyclophilins is important to better understand their roles in plant biology. This approach is also highly valuable to study about their isoforms, which show unique primary sequences and their functions as multi-domain cyclophilin proteins. Even though functional characterization of cyclophilins is still at an early stage, it is clear that the chloroplast constitutes one of the chief intracellular sites of cyclophilin function. While two of the four lumenal cyclophilins show close association with photosynthetic complexes, accumulating evidence suggests that their function may comprise multiple roles in different redox states and 'maturation' states (i.e. precursor, intermediate and mature N-terminal cleaved forms) in both the stroma and lumen. The abundance of cyclophilin isoforms in and around the thylakoid membrane seems to be a testament to the highly dynamic nature of this multicomponent system, which must be maintained in a functionally competent state in order to meet the metabolic demands of the cell and whole plant. While they may be functionally redundant with respect to their PPIase activity, their diverged sequences suggest that they may play additional and highly specialized functions within this organelle. The possibility that chloroplast cyclophilins may have distinct functions that are dependent on their 'maturation' state raises the intriguing hypothesis that proteins destined for the lumen may play distinct roles throughout their import pathway, before reaching their final sub-cellular destination. 69 Though there are 29 CyP isoforms in Arabidopsis, not all are of multi-domain organization. CyP38 is the smallest among the various multi-domain cyclophilins in Arabidopsis. That makes it an ideal first candidate to be picked for a structural investigation. In addition, the CyP domain of this protein is highly divergent, with very low sequence identity to that of the well studied cyclophilins. Even though the mature form of AtCyP38 is finally localized into the thylakoid lumen, its intermediate form(s) during the import pathway exist in the thylakoid stroma. From the structural study of both forms of this protein, we will clearly identify the structural rearrangement, activation of the inactive and precursor protein to a mature and active protein and the mechanism of function of this multidomain plant cyclophilin. 70 CHAPTER MATERIALS AND METHODS 3.1 EXPRESSION AND PURIFICATION OF RECOMBINANT ATCYP38 3.1.1 Expression The genes encoding residues 1-437 (precursor protein), 37-437 (intermediate 1), 83-437 (intermediate 2) and 93-437 (mature protein) of AtCyP38 that were cloned into a pGEX vector were used for the expression of the respective GST fusion protein. The construct for precursor protein failed to express and the intermediate construct expressed only an insoluble protein. The constructs for intermediate and mature proteins could be expressed in soluble form and purified to homogeneity. We have crystallized the intermediate protein and attempts are underway to crystallize the mature protein. The gene encoding residues 83-437 of the AtCyP38 protein was cloned in the pGEX-KG vector, between the XhoI/XbaI sites. In this construct, the codons for leucine residues of positions 107, 111, 125, 140 and 154 of the protein were mutated to code for methionine residues by site-directed mutagenesis. Five individual replacement mutants as well as a mutant construct with all these five leucine residue codons mutated to those for methionine were generated. These were also used for expression of the GST fusion protein. We needed these mutant proteins for structure determination, vide infra. For all the constructs, the same expression protocol was used. A selected construct was first transformed into E. coli BL21 (DE3) cells and spread on to a Luria Bertani (LB)-agar plate containing 100 μg ml-1 ampicillin. The next day, one among the well-separated colonies was picked up from the plate, 71 inoculated into 50 ml of fresh LB broth having 100 μg ml-1 ampicillin (LB-Amp broth) and the inoculum was allowed to grow overnight at 37 oC. The next morning, this 50 ml inoculum was added to liter of fresh LB-Amp broth and used for standard expression works. Initial attempts to express the construct of this GST-fusion protein followed a standard protocol wherein the cells were grown to log phase at 37 oC and induced with IPTG at a final concentration of mM. This procedure gave only inclusion bodies of the protein and not even a trace of soluble protein. Further attempts to get the protein in soluble form were directed towards the usage of lower temperatures and lower doses of IPTG. A series of trials and standardizations resulted in the identification of a suitable condition for the AtCyP38 (83-437) construct’s expression in E. coli BL21 (DE3) cells. This final expression protocol, which is explained in the next paragraph, was used throughout the experiment. The inoculated liter LB-Amp broth in a liter flask was grown in a shaker incubator at a temperature of 30 oC until the OD600 of the culture medium reached a value between 0.5 and 0.6. The temperature was then brought down to 25 oC and the expression of the recombinant protein was induced by adding IPTG to a final concentration of 0.4 mM. After induction, cells were continued to be grown for hours at 25 oC. The cells were then harvested by centrifugation at 4,200 g for 10 min. The cell pellet was suspended in ice cold lysis buffer containing 50 mM TrisHCl (pH 7.5), 500 mM NaCl, mM EDTA, 1mM DTT, mM PMSF and subjected to sonication. The crude lysate was centrifuged at 42,400 g for 45 at oC and the supernatant was collected. SDS-PAGE analysis was carried out to confirm the presence of the soluble protein. 72 3.1.2 Affinity chromatographic purification The crude supernatant from the previous step was applied to a GST-affinity column (Glutathione Fast Flow; Amersham Biosciences) and the fusion protein was allowed to bind to the resin overnight at oC. The contaminant proteins that were loosely bound to the affinity column were removed by giving washes with high salt wash buffer [20 mM Tris-HCl (pH 7.5), 900 mM NaCl, mM DTT, mM EDTA] and low salt wash buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, mM DTT, mM EDTA]. SDS-PAGE of the various samples from the process was carried out to confirm the success of the purification step. 3.1.3 Thrombin cleavage The GST-tag was removed from the fusion protein, which was bound to the GST-column, by overnight on-column cleavage at oC using the thrombin protease (Amersham Biosciences) in the low salt buffer. Majority of the GST cleaved AtCyP38 protein was obtained in the flow through from the column and a little in the following wash with the low salt buffer. The GST-column purified and cleaved protein still had some amount of contaminant proteins, which were visualized in the SDS-PAGE analysis of the samples. 3.1.4 Size exclusion chromatography The GST cleaved protein was further purified by size exclusion chromatography on a pre-equilibrated HiLoad 16/60 Superdex-75 column (Amersham Biosciences) using the low salt wash buffer with mM PMSF. Fractions corresponding to the protein peak were analyzed by SDS-PAGE and pure fractions were pooled together and concentrated. The protein was stored at -80 oC in a buffer 73 containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, mM DTT, mM EDTA, mM PMSF at a concentration of about mg ml-1. 3.1.5 Analyses for purity and homogeneity The stored protein was analyzed by native-PAGE and Dynamic Light Scattering experiment to confirm its purity and homogeneity. MALDI-TOF mass spectrometry was performed to confirm the molecular weight of both the proteins. 3.2 EXPRESSION AND PURIFICATION OF SELENOMETHIONYLATED ATCYP38 3.2.1 Expression The selenomethionine derivatives of AtCyP38 and the mutants were produced in E. coli BL21 (DE3) cells (Doublie, 1997). Transformation and growth of inoculum followed the same protocol of the previous section. Cell pellet from 50 ml of the inoculum was suspended in ml of Minimal M9 medium and added to liter of the Minimal M9 medium having 100 μg ml-1 ampicillin in a liter flask. Cells were grown to an OD600 of 0.8 in a shaker incubator at a temperature of 30 oC and the following amino acids were added to the respective concentration in the parentheses: L-lysine (100 mg ml-1), L-phenylalanine (100 mg ml-1), L-threonine (100 mg ml-1), Lisoleucine (50 mg ml-1), L-leucine (50 mg ml-1), L-valine (50 mg ml-1) and Lselenomethionine (40 mg ml-1). The temperature was brought down to 25 oC and the expression of the construct induced by adding IPTG to a final concentration of 0.4 mM. Post induction cells were continued to be grown for hours at 25 oC. The other steps of the protocol remained almost the same as explained earlier. 74 3.2.2 Purification Extraction, purification, storage and analysis of the selenomethionine protein followed the same protocol as the one employed for the native protein, except for the composition of all the buffers wherein an additional mM DDT was incorporated, making the total DTT concentration as mM. The mass of the selenomethionine derivatized AtCyP38 protein and its mutants were obtained by MALDI-TOF MS experiments. 3.3 CRYSTALLIZATION AND TESTING OF CRYSTAL QUALITY 3.3.1 Hanging drop vapor diffusion method Crystallization trials were initially performed using the hanging drop vapor diffusion method at 20 oC with various commercially available crystallization kits. Crystals grew within two days in Hampton Crystal Screen-1 (Hampton Research), condition 41 [100 mM HEPES (pH 7.5), 20% w/v PEG 4000, 10% v/v isopropanol] and also in the flexible sparse matrix screen (Zeelen, 1999), condition 2D3 having 100 mM sodium citrate (pH 5.5), 20% w/v PEG 6000, 2.5% t-butanol. The mutant proteins were also crystallized in the same condition, even though the crystals were smaller in size. 3.3.2 Vapor batch method Vapor batch method of crystallization (Mortuza et al., 2004) was also attempted, based on the crystallization condition from the Hampton condition that gave crystals. For this, AtCyP38 protein, at a concentration of mg ml-1, was mixed with an equal volume of a crystallization solution containing 100 mM HEPES (pH 7.5), 20% w/v PEG 4000, and μl droplets were dispensed into 96-well vapor batch 75 plates (Douglas Instruments). Each droplet was covered with μl paraffin oil (Fluka). A total of about 14 ml of 5% (v/v) aqueous isopropanol was dispensed in the side wells of each tray and the tray was sealed with parafilm. The drops were then equilibrated overnight at 20 oC. Next day, the trays were opened and the 5% isopropanol was replaced by 10% isopropanol and the trays were sealed back and maintained at 20 oC. Similarly, another set of vapor batch plates were set up based on the flexible sparse matrix screening condition, which also yielded crystals. The AtCyP38 protein at a concentration of mg ml-1 was mixed with an equal volume of a crystallization solution containing 100 mM sodium citrate (pH 5.5), 20% w/v PEG 6000, and μl droplets were dispensed into 96-well vapor batch plates. Each droplet was covered with µl paraffin oil and a total of about 14 ml of 1.25% (v/v) aqueous t-butanol was dispensed in the side wells. The trays were sealed and the drops were equilibrated overnight at 20 oC. Next day, the trays were opened and the 1.25% t-butanol was replaced by 2.5% t-butanol and the trays were sealed back and maintained at 20 oC. Vapor batch crystallization of the selenomethionylated AtCyP38 followed the same strategy, except for the usage of protein sample having a slightly lower concentration, at only mg ml-1. In the case of mutant AtCyP38 proteins and their selenomethionine derivatives, slight modifications in the crystallization conditions were made for the vapor-batch method, in order to obtain better diffracting quality crystals. For this, the protein at a concentration of mg ml-1 (6 mg ml-1 for its selenomethionine derivative) was used. The remaining steps of crystallization using isopropanol or t-butanol nearly followed the corresponding steps that have been described above. 76 3.3.3 Cryo-protection of the crystals and testing of diffraction quality Initial tests for the diffraction quality and optimization of cryo-conditions for the crystals were carried out using a Rigaku R-axis IV+ area detector at the in-house facility, located in the Department of Biological Sciences, National University of Singapore. In the case of AtCyP38 protein and its selenomethionine derivative, single crystals from the vapor-batch condition were transferred to a fresh solution having 100 mM HEPES (pH 7.5), 25% w/v PEG 4000, 10% isopropanol, 20% glycerol and then flash-cooled in liquid nitrogen at -180 oC. The crystals from the other vaporbatch condition were transferred to a fresh solution having 100 mM sodium citrate (pH 5.5), 25% w/v PEG 6000, 2.5% t-butanol, 25% glycerol and flash-cooled in liquid nitrogen at -180 oC. Mutant AtCyP38 protein and its selenomethionine derivative crystals were cryo-protected and flash-cooled in a similar way with corresponding cryoprotectants supplemented with glycerol and flash-cooled in liquid nitrogen. 3.4 DATA COLLECTION Once crystal freezing conditions were standardized, cryo-cooled crystals were shipped to the National Synchrotron Light Source (Brookhaven National Laboratory, USA) facility in cryo-tanks. Native and MAD diffraction data sets were collected at the X25, X12B and X12C beam-lines with either a Brandeis B4 or ADSC Q315 charge-coupled device detector. Data collection of all the crystals was performed at 170 oC. For native AtCyP38 protein crystals, individual datasets of 180 images with an oscillation of one degree each were collected in one sweep at a wavelength of 0.95 Å and for the selenomethionine derivatives, datasets (180 images, o oscillation) were 77 collected at three different wavelengths, based on the respective selenium-absorption spectrum. Detailed data collection statistics are included in the next chapter. 3.5 DATA ANALYSIS AND STRUCTURE DETERMINATION The native and anomalous data were indexed, integrated and scaled using the HKL2000 program package (Otwinowski & Minor, 1997). The three-wavelength MAD method, with the selenomethionine data, was used for structure determination. Selenium positions were determined using the protein phasing program BnP (Weeks et al., 2002). The resulting phases were used for density modification and automated model building was attempted using the software ARP/wARP 6.0 (Perrakis et al., 1999). The program could trace only some short stretches of the structure and the sequence docking option did not work. All remaining residues were filled and sidechains were assigned manually using the O program (Jones et al., 1991). Refinement of the structure was carried out using CNS (Brunger et al., 1998) as well as Refmac5 of the CCP4 suite (Murshudov et al., 1997). Refinement started with the calculation of 2Fo-Fc and Fo-Fc maps. Positional and temperature refinement was performed iteratively until the structure was fully refined. Details of refinement and the results from various steps will be discussed in the next chapter. PROCHECK (Laskowski et al., 1993) which checks the stereochemical quality of protein structures was used for structure validation. Molecular structure figures were prepared using the program PYMOL (DeLano, 2002). The surface charge distribution figures were prepared with GRASP (Nicholls et al., 1991). 78 [...]... chemical structure of Cyclosporin A 36 Figure 10 The chemical structure of FK506 37 Figure 11 The chemical structure of Rapamycin 38 Figure 12 Peptidyl-prolyl isomerization reaction 41 Figure 13 Domain organization in Arabidopsis cyclophilins 51 Figure 14 The phylogenetic relationships of Arabidopsis cyclophilins 52 Figure 15 Arabidopsis cyclophilin isoforms & their sub-cellular localization 54 Figure 16 Structure. .. organization of AtCyP38 96 Figure 30 Overall structure of AtCyP38 (83-437) 97 Figure 31 Overall structure of AtCyP38 (83-437) in stereo view 98 Figure 32 Structure overlap for the helical bundle of AtCyP38 with PsbQ and cytochrome b562 10 0 Figure 33 Structure overlap of cyclophilin domains of AtCyP38 & hCyPA 10 1 Figure 34 Structure based sequence alignment of the cyclophilin domains of AtCyP38, bovine CyP40,... and E coli CyPB 10 3 Figure 35 The cyclophilin domain of AtCyP38 (83-437) 10 4 Figure 36 Surface charge features of AtCyP38 (83-437) 10 6 Figure 37 The amino acid sequence of precursor AtCyP38 10 8 xiv LIST OF TABLES Page Table 1 Crystal parameters and data-collection statistics for wt AtCyP38 88 Table 2 Crystal parameters and data-collection statistics for the multiple L→M mutant Table 3 Structure refinement... TM1367 Thermotoga maritima protein 13 67 TPR tetratricopeptide repeat xii LIST OF FIGURES Page Chapter 1 Figure 1 A unit-cell 5 Figure 2 Intersection of three (234) planes within a unit-cell 7 Figure 3 Interference of two waves 10 Figure 4 Derivation of Bragg’s Law 11 Figure 5 The reciprocal lattice 12 Figure 6 Ewald sphere 13 Figure 7 The anatomy of an X-ray diffractometer 16 Figure 8 The principle of. .. of size-exclusion chromatographic purification 82 Figure 23 SDS-PAGE of size-exclusion chromatography fraction 82 Figure 24 Native-PAGE of purified wt AtCyP38 (83-437) 83 Figure 25 Mass spectrometry for wt AtCyP38 (83-437) 84 Figure 26 Crystal of AtCyP38 86 Figure 27 Crystal of selenomethionylated AtCyP38 86 Figure 28 The sequence of AtCyP38 (83-437) 91 Figure 29 Secondary structural organization of. .. overlap of various cyclophilin domains 62 Figure 17 The structure of human CyPA 66 Figure 18 Sequence alignment (by CLUSTALW) of AtCyP38 with TLP40 Chapter 2 from spinach, pea and rice 68 xiii Chapter 4 Figure 19 SDS-PAGE showing the expression of soluble wt AtCyP38 79 Figure 20 SDS-PAGE showing the affinity chromatographic purification 80 Figure 21 SDS-PAGE showing the thrombin cleavage 81 Figure 22 Profile...SUMMARY Cyclophilin 38 (CyP38) is one of the highly divergent multi-domain cyclophilins from Arabidopsis thaliana A recombinant form of intermediate AtCyP38 (residues 83-437) was expressed in Escherichia coli and purified to homogeneity The protein was crystallized in the C22 21 space group using the vaporbatch technique with PEG 6000 and t-butanol as precipitants Crystals of recombinant AtCyP38 diffracted... process of ‘life’ 1. 2 PROTEIN CRYSTALLIZATION There are several bottlenecks in the determination of a crystal structure, of which obtaining a useful crystal is the most serious one If one cannot collect diffraction data of suitable quality, protein structure determination will not be possible Proteins have irregularly shaped surfaces, which result in the formation of large channels within a crystal. .. additional weakening of the scattering power of atoms by the temperature factor of the atoms Or, 14 2 fB = f e -B(sinθ/λ) (1. 6) where B is related to the mean displacement of a vibrating atom by the DebyeWaller equation B = 8π22 1. 3.9 (1. 7) Phase problem In order to know electron density distribution in a crystal, we evaluate Eq 1. 4 However, to evaluate Eq 1. 4, we need to calculate Eq 1. 5, which shows... macromolecular structure determination X-ray crystallographic techniques were first developed in 19 12 and initially applied to small molecules Based on its extraordinary success, the recording of the first diffraction pattern from a protein crystal, that of pepsin, was successfully attempted in the 19 30s (Bernal & Crowfoot, 19 34) However, the complete application of X-ray diffraction to protein structure . analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38) . ACTA Cryst. F 61: 10 87 -10 89. Vasudevan D, Radhakrishnan A, Luan S, Swaminathan K. 2007. Crystal structure of an intermediate form of Arabidopsis. 10 1. 3.6 Reciprocal space 11 1. 3.7 The Ewald sphere 13 1. 3.8 Fourier transform and structure factor 14 1. 3.9 Phase problem 15 1. 4 GEOMETRIC DATA COLLECTION 16 1. 4 .1 Data reduction 17 . reduction 17 iv 1. 5 STRUCTURE DETERMINATION 18 1. 5 .1 Phasing methods 18 1. 5 .1. 1 The Multi-wavelength anomalous dispersion (MAD) method 21 1. 5 .1. 2 Principle of anomalous scattering 23 1. 5.2 Phase