Chapter Results and Discussion CHAPTER 4. RESULTS AND DISCUSSION 4.1 STRUCTURE OF MATURE ATFKBP13 REVEALS UNIQUE DISULFIDE BRIDGES 4.1.1 Overall Structure of AtFKBP13-S2 The AtFKBP13-S2 molecule reveals predominantly β-structures consisting of six β-strands and two α-helices (Fig. 4-1). The β-strands form an integral antiparallel β-sheet than constitutes the core of the protein. Figure 4-1. Structure of AtFKBP13-S2. The redox active disulfides are shown in ball-and-stick representation and the sulphur atoms are shown as yellow balls. 72 Chapter Results and Discussion The secondary structures are arranged in the order: β1 β4 β5a α2 β5b α1 β2 β6a β6b β3 (Fig. 4-2). The β5 strand of AtFKBP13 is split into two fragments, β5a and β5b, as in MtFKB17 [Suzuki et al., 2003] and HsFKB12 [Meadows et al., 1993]. However, the β5a strand of hFKB12 is formed only when a ligand (ascomycin) is bound [Meadows et al., 1993]. Otherwise it is disordered [Michnick et al., 1991; Moore et al., 1991]. Between β5a and β5b is the inserted α2 helix, similar to that of MtFKB17. The β6 strand pair, which is unique to AtFKBP13, is formed by strands β6a and β6b connected by a short loop. CEFSVSPSGL 15 AFCDKVVGYG 25 PEAVKGQLIK β1β1 55 GKPLTFRIGVG α2 35 AHYVGKLENG 45 KVFDSSYNR β4 65 EVIKGWDQGI β5b 75 LGSDGIPPML α1 β5a 85 TGGKRTLRIP 95 PELAYGDRG β2 105 115 125 AGCKGGSCLIP PASVLLFDIE YIGKA β6a 6a β6b β β3 Figure 4-2. Secondary structure elements of AtFKBP-13. The α-helix is represented by a cylinder and the β-strand is represented by an arrow. 73 Chapter 4.1.2 Results and Discussion Comparison with related structures A notable feature in the tertiary structure of AtFKBP13-S2 is the presence of two intra-chain disulfide bonds. These disulfide bonds, Cys5–Cys17 and Cys106–Cys111, form the two redox active motifs. These disulfides have no counterparts in other animal or yeast FKBPs. Eukaryotic FKB protein sequences indicate that this region is rather a unique feature of Arabidopsis FKBP13. Also, structural comparisons with other FKBPs indicate that AtFKBP13 has a conserved PPIase domain with additional strands (β6a and β6b) inserted at the C-terminus where Cys106 and Cys111 are located. C N Figure 4-3. Superimposition of the Cα backbones of AtFKBP13 (residues 5–129, blue), hFKBP12 (residues 1-107, magneta) and L. pneumophila FKBP25 (residues 496–612, green). The redox active-site disulfides of AtFKBP13 are shown in ball-and-stick representation. 74 Chapter Results and Discussion Alignment between AtFKBP13 and representatives of other FKBPs, namely hFKB12 [Wilson et al., 1995] and the Macrophage Infectivity Potentiator protein from Legionella pneumophila, LpMIP [Riboldi-Tunnicliffe et al., 2001] through the Dali server [Holm, and Sander, 1993] gives the r.m.s deviation values of 1.3 Å (with a Z-value of 17.5) and 1.4 Å (with a Z-value of 17.2), respectively. The core regions for these structures are similar (Fig. 4-3). The best matches are found in the regions that are directly involved in the prolyl isomerase activity. A few residues are conserved among these structures. These are the residues that form the substrate-binding pocket for the pipecolinyl ring of FK506 [Van Duyne et al., 1993], namely Tyr37, Phe47, Asp48, Phe59, Val66, Ile67, Trp72, Tyr99, Ile113, Leu119 and Phe121. These residues are essential for binding and maintaining the hydrophobic core of FK506 [Radzicka et al., 1992]. 4.1.3 Catalytic domain The catalytic domain of AtFKBP13 is composed of two sub-regions, the PPIase (residues 26-125) and Cys-Xn-Cys redox active motifs (residues 5-17 and 106-111). The PPIase active region follows the general extended PPIase fold, which consists of a fourstranded β-sheet and an α-helix inserted between them. In AtFKBP13, the corresponding β-sheet comprises strands β1-β5, helix α1 and a short 310 helix, α2 .The Cys-Xn-Cys motif regions are composed of two disulfides, found at the N and C-termini, respectively. The active site disulfide bond between Cys5 and Cys17 at the N-terminus is located at the β1 strand. Both Cys5 and Cys17 are partially solvent exposed. The second disulfide bond between Cys106 and Cys111 (between β6a and β6b) is fully solvent exposed. 75 Chapter Results and Discussion These disulfide bonds form additional secondary structures that are located on either side of the central β-sheet and reflect the intrinsic versatility and flexibility of this region. The two active sites that are involved in redox regulation exist in their oxidized state in the crystal structure as shown by their clearly defined electron density (Fig. 4-4). The theoretical dihedral energy for the N-terminal disulfide is 3.60 kcal mol-1 and for the C-terminal disulfide is 4.00 kcal mol-1, calculated with the program AMBER [Weiner et al., 1984], indicate the disulfide bonds are stable with less conformational strain. The corresponding value varies from 0.5-4.7 for most protein disulfide bonds [Darby and Creighton, 1995] and rarely reaches values over 5.0. Because the active site disulfide bonds are extremely stable, they may also act as strong reductants. Figure 4-4. Stereoview of the 2Fo-Fc maps contoured at the 1.2 σ level at the C-terminal disulfide region. The amino acid residues in the region are numbered. 76 Chapter Results and Discussion Despite this similarity, however, the two redox active disulfides show remarkable difference in their B-factors. The average B-factor for the side chain atoms (Cβ and Sγ) of the two cysteines is 39.32 for the N-terminal disulfide versus 26.22 for the C-terminal disulfide. Even though, all the residues in both redox active sites have very well defined electron density in the final 2Fo-Fc map, the side chain atoms of the two cysteines in the C- terminal disulfide have been better defined with more spherically shaped electron densities than those in the N-terminal disulfide. 4.1.4 Surface of AtFKBP13 In AtFKBP13, the residues around the two redox active sites form two grooves on the protein surface, the active site Grooves N and C (Fig. 4-5a). Groove C is built exclusively by residues from the C-terminus and is essentially hydrophobic. A B C Figure 4-5. (A) Surface charge distributions (blue for positive and red for negative charge) for the AtFKBP13 monomer. The dark red region indicates 77 Chapter Results and Discussion a potential of less than -12 kT/e, while dark blue indicates greater than 12 kT/e. The electrostatic potentials were calculated by GRASP. (B) Spacefilling representation in which the sulfur atoms of Cys-106 and Cys-111 are exposed on the surface of the molecule and (C) the S atom of Cys5 is fully exposed on the surface, and the sulfur atom of Cys17 is buried. The residues in the vicinity of Cys106 include Gly105, Pro114, Ser110, Leu112, and Ile113. The Sγ atoms of Cys106 and Cys111 are exposed on the protein surface (Fig. 4-5b). Unlike Groove C, the formation of Groove N involves residues both from the N and C-termini. While atom Sγ of Cys5 is fully exposed on the surface, the sulphur atom of Cys17 is buried (Fig. 4-5c). The redox active site loops C and N adopt an open confirmation in the crystal structure so that the Sγ atom and the substrate binding site are exposed to solvent. The two redox active grooves are adjacent to each other on the protein surface. Separately located in the two grooves with different accessibility, the two redox active sites probably function independently to some extent. The distance between the two active sites, measured between the Sγ atoms of Cys5 and Cys106 is 27.6 Å. 4.2 PAST WORK ON PRECURSOR ATFKBP13 Gupta et al. (2001) identified the first chloroplast FKBP from Arabidopsis and the Rieske protein, a component in the photosynthetic electron transport, as its putative target. The interaction between AtFKBP13 and the Rieske protein probably occurs before they are imported into the thylakoid because the mature proteins not interact with each 78 Chapter Results and Discussion other. Both yeast two-hybrid and in vitro protein interaction assays demonstrate that the full-length precursor proteins (or the cytoplasmic forms) interact. In addition, the intermediate (or stromal) forms of the two proteins also interact well. These results suggest that AtFKBP13 associates with the Rieske protein both before and after the import of the proteins into the chloroplast stroma. In addition, AtFKBP13 and Rieske intermediate forms also can interact after they are imported into the thylakoid lumen but before the thylakoid signal peptides are cleaved. After the cleavage of the peptide, the two matured proteins probably dissociate. 4.2.1 AtFKBP13 is targeted to the thylakoid lumen of chloroplasts by the ∆pHdependent pathway To determine whether AtFKBP13 is targeted to the chloroplast, and if so, what its suborganellar location is, protein import assays have been performed with isolated chloroplasts and the AtFKBP13 precursor as a substrate. The translated AtFKBP13 precursor is about 27 kDa (estimated by mobility in SDS/PAGE). When isolated intact, pea chloroplasts were incubated with the precursor protein in the presence of ATP, a 13 kDa protein was generated (Fig. 4-6A, lane 2). After incubation with the protease thermolysin (which under the used conditions does not penetrate the chloroplast envelope), intact chloroplasts were reisolated and fractionated. The resistance of the 13 kDa polypeptide to degradation by exogenously added thermolysin (Fig. 4-6A, lane 3) indicates that it is located within the chloroplast and is a product of the precursor protein import. Further analysis revealed that the AtFKBP13 protein was associated with the thylakoids (Fig. 4-6A, lane 5) and not in the stroma (Fig. 4-6A, lane 4). 79 Chapter Results and Discussion Figure 4-6. AtFKBP13 is targeted to the chloroplast thylakoid lumen by the ∆pH-dependent pathway. (A) Chloroplast import assay of AtFKBP13: translation products (lane 1), chloroplasts (lane 2), thermolysin-treated chloroplasts (lane 3), stromal fraction (lane 4), thylakoid fraction (lane 5), thermolysin-treated thylakoid fraction (lane 6), sonicated thylakoid membrane fraction (lane 7), soluble contents of thylakoid lumen (lane 8). (B) Western blot analysis of stromal (lane S) and thylakoid lumen (lane L) fractions of Arabidopsis chloroplasts by Coomassie staining (left), antiAtFKBP13 (middle), and anti-Plastocyanin (PC) antibodies (right). Approximate molecular masses (kDa) are shown on the side. Sonication of the thylakoid fraction liberated the 13 kDa polypeptide in a soluble form (Fig. 4-6A, lane 8), indicating that the protein is located in the thylakoid lumen. From this experiment, it was concluded that AtFKBP13 is a previously uncharacterized thylakoid lumen protein. 80 Chapter Results and Discussion The N-terminal extension of AtFKBP13 has the characteristic features of a thylakoid lumen protein presequence (Fig.4-7). It is bipartite, with the first region being hydrophilic in nature and enriched in basic and hydroxylated residues, which are features of a chloroplast envelope-transfer signal [Keegstra and Cline, 1999]. The twin arginine motif is present in the presequence of AtFKBP13 and is followed by a highly hydrophobic domain ending with AXA. These features suggest that AtFKBP13 may be translocated into the thylakoid lumen by a pH-dependent pathway. MSSLGFSVGT CSPPSEKRKC AEAINLRNKQ KVSSDPELSF AIIGFGFSIG RFLVNNSLNK ▼ LLDNVSALAE AQLSSCGRRE 50 GYGPEAVKGQ LIKAHYVGKL ENGKVFDSSY NRGKPLTFRI GVGEVIKGWD 150 QGILGSDGIP PMLTGGKRTL RIPPELAYGD RGAGCKGGSC LIPPASVLLF 200 TTSCEFSVSP SGLAFCDKVV 100 DIEYIGKA Figure. 4-7 Sequence of precursor AtFKBP13 protein. The amino acid sequence of the AtFKBP13 protein deduced from the cDNA. The double arginine motif is shown as bold and highlighted. The thylakoidal processing peptidase site (AxA) is shown in italics and the arrow indicates the putative cleavage site. The mature protein sequence is shown in black. 81 Chapter Results and Discussion There are no major rearrangements within the catalytic domains. Superimposition of only the catalytic domains of the reduced structure and the corresponding parts of the oxidized structure gives an r.m.s.d. of 1.478 Å for 16 Cα atoms (residue numbers 100116). In Fig. 4-12, the largest changes are in the position of the S atoms of the key cysteine residues: each S atom in the two thiol groups is shifted by about 0.5 Å compared with the disulfide form. The hydrogen-bond network in the active site is affected by the oxidation state of the thiol groups. The oxidized Cys5 S atom forms a strong hydrogen bond with the Cys17 amide, while the thiol S atom in the present reduced structure is located farther away. Cys17 makes other short contacts: as a proton donor with the carbonyl O atom of Thr90 (3.0 Å) and Cys17 Sγ with N of Lys19, Arg89 and Glu6. The reduction of the active site is also accompanied by a shift of the Cys17 S atom, which forms a short hydrogen bond with the O atom of the Thr90 side chain. The side chain conformations of the two cysteines are the same in AtFKBP13-(SH)2 and AtFKBP13-S2. Reduction of the disulfide bond requires an increase in the distance between the sulfur atoms of the two cysteines: the observed S–S distance in AtFKBP13-S2 Cys5–Cys17 and Cys106–Cys111 are 2.04±0.01 and 2.01±0.01Å, respectively as expected for a disulfide bond, whereas the corresponding distance is 5.18 Å and 2.81 Å for AtFKBP13(SH)2.The corresponding distance seen in reduced human thioredoxin is, surprisingly, 3.1 Å [Forman-Ka et al., 1991]. The backbone conformational change that occurs to accommodate the increase in the radius of sulfur upon reduction and the breakage of the disulfide bond also causes an increase in the Cα–Cα distances between the two cysteines, 95 Chapter Results and Discussion 6.208 (Cys5-Cys17) and 3.83 Å (Cys106-Cys111) for AtFKBP13-S2, compared with 6.988 (Cys5-Cys17) and 3.91 Å (Cys106-Cys111) for AtFKBP13-(SH)2, and the thiol of Cys5 is more solvent exposed in AtFKBP13-(SH)2. All surface features identified in the oxidized structure are also present in reduced AtFKBP13. Their general properties are nearly the same within allowable differences. There are some apparent changes in the local structure in the active site region upon the change of the reduction state. Small dihedral angle differences are also observed in the region of residues 107-109, which form a loop structure that closely contacts the active site. A significant difference in secondary structures between oxidized AtFKBP13-S2 and AtFKBP13-(SH)2 is found in α2, which in oxidized AtFKBP13 includes residues 50 to 53 and is absent in AtFKBP13-(SH)2. This region is converted to a loop. Another region where there is a difference in secondary structure is α1, which is one residue shorter in AtFKBP13-(SH)2. Interestingly, α2 in the oxidized structure contains the PPIase conserved residues namely Ser50 and Arg53. These shifts in secondary structures may account for the loss of PPIase activity. However, both variants occur in each of the conformers although with different proportions, notably, the structure of oxidized AtFKBP13 is better defined in this region. In addition to these changes in secondary structures, a few side-chains have large average displacement, in particular Asp48. In AtFKBP13-S2, Asp48 forms a salt bridge with Arg53. In reduced AtFKBP13, Asp48 is no longer at a suitable distance for such 96 Chapter Results and Discussion interactions. Due to the altered position of the active-site cysteines, the distance between Asp48 and Arg53 is longer in reduced AtFKBP13. 4.3.3 Changes in hydrogen bonding upon change of oxidation state No hydrogen bond constraints were included during refinement. There are no significant hydrogen bonding differences between the two forms of the protein outside the active site. Active site hydrogen bonding patterns for the two structures indicate that there is some degree of difference in the backbone conformation in the redox active site loop. Both structures have highly populated hydrogen bonds between Cys17 Sγ - Lys19 N, Cys17 O - Arg89 N and Cys17 NH - Thr90 0. Several of these groups form bifurcated hydrogen bonds. The crystal structures of both reduced and oxidized AtFKBP13 reveal a hydrogen bond between sulphur atom of Cys106 and the amide nitrogen of Cys 111. Reduced AtFKBP13 is also stabilized by a hydrogen bond between the Cys 111 thiolate and the main chain amide of Ser110. In addition, an electrostatic interaction is found in the crystal structure of reduced AtFKBP 13 between Arg102, Ile 113 and Lys45, which can further stabilize the reduced form. These interactions are also present in oxidized AtFKBP13, which is further stabilized by Pro115, Pro114 and Tyr51. Unlike other residues, proline cannot donate a main chain hydrogen bond and, unlike histidine, it cannot provide side chain stabilization of the thiolate by either electrostatic or hydrogen bond interactions. A number of backbone–side chain hydrogen bonds differ in the active site region. Unlike the disulfide bond in AtFKBP13-S2, the thiol groups in AtFKBP13-(SH)2 are 97 Chapter Results and Discussion capable of acting as hydrogen-bond donors. The cysteine sulfur atoms of both oxidation states can act as hydrogen-bond acceptors. The disposition of the groups in the active site loop makes the network of hydrogen bonds complex, especially in AtFKBP13-(SH)2. The thiol groups of Cys106 and Cys111 are most probably hydrogen bonded as donors to Ile113. It appears likely that the redox active site of AtFKBP13-(SH)2 includes two SH⋅⋅⋅O hydrogen bonds that are significantly populated. These are the most common hydrogen bonds observed for cysteine side chains in protein crystal structures. According to the structures, the Cys17 amide proton is not solvent-exposed in either form of the protein, but there appears to be additional hydrogen bonding in AtFKBP13-S2. The preferred hydrogen bond for AtFKBP13-(SH)2 is between the Cys17 NH and the Cys5 CO, with a N–H⋅⋅⋅O distance of 1.84±0.09 Å, an N–O distance of 2.80±0.04 Å and an N–H⋅⋅⋅O angle of 156±7°. The corresponding distances for AtFKBP13-S2 are quite similar, 2.12±0.08 and 2.86±0.06 Å, but the N–H⋅⋅⋅O angle is a little too acute (129°) for strong hydrogen bonding (cutoff value ≥ 135°). It thus appears likely that there is a significant amount of hydrogen-bond-like interaction between Cys17 NH and Cys5 CO in AtFKBP13-S2. Indeed, the presence of both the sulfur and oxygen atoms in close proximity to each other and to Cys17 NH could be the reason for the apparent distortion of the N–H .O hydrogen bond in AtFKBP13-S2. These observations can serve partially to explain the hydrogen exchange results for Cys17 NH. In AtFKBP13-S2 there are two hydrogen bond acceptors (Cys5 Sγ and Cys5 CO) in close 98 Chapter Results and Discussion proximity to this proton, giving greater protection than in AtFKBP13-(SH)2, where there is only one hydrogen bond acceptor (Cys5 CO), since the Cys5 thiol has swung out towards the solvent, out of hydrogen-bonding range. 4.3.4 Distance and dihedral angle restraints For both the oxidation and reduction reactions catalyzed by AtFKBP13, the primary site of reaction is probably the sulfur of Cys106 and Cys111. This is shown by the relative solvent accessibilities of the two cysteine side chains in the two oxidation states. Both AtFKBP13-(SH)2 and AtFKBP13-S2 have Cys17 completely buried. The thiol of Cys5 is relatively accessible, more so in AtFKBP13-(SH)2 (42.0315±1.8 Å2) than in AtFKBP13 –S2 (34.2512±3.7 Å2). This is illustrated in the space-filling representation of the active site region shown in Figure 4-5B and C. Using the insights provided by the high-resolution structures of the two forms of AtFKBP13, we can now attempt to rationalize stabilization by the presence of a positively charged group, the formation of a hydrogen bond between the Cys5 NH and Cys17 S. The structures show that there are no positively charged groups in the vicinity of the Cys5 thiol, which has moved out into the solvent from the pocket where it resides as part of the disulfide bond in AtFKBP13-S2. 4.3.5 N-terminal active site Both disulfides need to be reduced to inactivate AtFKBP13, it is obvious from our model how the N-terminal disulfide and extension is involved in the inactivation of the 99 Chapter Results and Discussion enzyme. In fact, the structure shows that the portions of the N terminus that can be seen are quite removed from the active-site. The overall similarity of the core of the oxidized, active AtFKBP13 and reduced, inactive AtFKBP13 structures suggest that there is not a conformational change transmitted through the framework of the enzyme upon reduction of the N-terminal disulfide, rather a direct interaction of the N-terminal extension with the active-site region is more likely. The structural data indicate that the N-terminal extension does not form a stable conformation. There is no density in our maps for the first residues. These first residues could easily reach to and obstruct the active site. It is noteworthy that mutants of the enzyme lacking the C-terminal disulfide show some catalytic activity suggesting that the oxidized N terminus may alter but not completely obstruct the substrate-binding site .The ability of either disulfide-constrained terminus to bind to the active site when the other temporarily dissociates by such a flip-flop or alternate intrasteric mechanism, would enable the activity of the reduced enzyme to be powerfully suppressed. 4.3.6 Reduction of the C-terminal disulfide causes localized changes The conversion of the protein from an inactive to an active conformation is facilitated by a number of factors. The juxtaposition of negative charges on the surface along with the close approach of submerged like positive charges will push the C terminus loop so that it will ill be able to return to its active conformation .Nevertheless, while reduction of the C-terminal disulfide and dissociation of the extension from the active site will enable the loop to change its conformation. 100 Chapter Results and Discussion 4.3.7 The role of disulfide bonds The thiol redox potential of the chloroplast stroma is comparable to that of the cytoplasm even in the dark [W. Bielawski & K.W. Joy,1986; Foyer et al.,1991]. The light-regulated chloroplast enzymes are unique in that they contain disulfide bonds in a reducing cellular environment. a common way of assaying chloroplast thioredoxins relies on the fact that DTT can reduce thioredoxin much faster than it can reduce the lightactivated enzymes, so that thioredoxins can catalyze the DTT-dependent reduction of these enzymes. Inspection of the AtFKBP13 shows that both regulatory disulfides are located on the outside of the molecule where they can be readily accessed from the solvent by both DTT and thioredoxin (Figure. 4-5C). The geometry of the AtFKBP13 disulfides are unstrained in terms of bond lengths, angles and dihedral conformations when compared to those commonly observed in proteins [Sowddhamini et al.,1989]. Reduction of a disulfide bond by thiol-disulfide exchange occurs in two steps: first the formation of a mixed disulfide between reductant and oxidant, followed by a further disulfide exchange that could produce products or revert to the initial reactants. The rate at which the extensions can change or dissociate from their oxidized conformation may determine the fate of the mixed disulfide intermediate. Thioredoxin's ability to reduce AtFKBP13 in vitro rapidly may reflect the possibility that thioredoxin can enhance dissociation of the extension, thereby increasing the proportion of thioldisulfide encounters that result in reduction of the regulatory disulfides. Mutants with only an N-terminal disulfide are less dependant on the presence of thioredoxin for reductive activation. This probably reflects the relatively disordered state 101 Chapter Results and Discussion of the N terminus and the ease with which the mixed-disulfide form of the N terminus can dissociate and drive the reaction to completion. By contrast, the C terminus is more firmly bound to the active site and the mixed-disulfide intermediate may not spontaneously dissociate from the active site without the assistance of thioredoxin. Although studies of the properties of the individual disulfides in mutants in which the other disulfide has been disrupted provide valuable information, it should be emphasized that the results should be interpreted with caution, as implicit in these studies is the assumption that disruption of one disulfide will not alter the properties of the other disulfide. As it is clear that both disulfides influence the active site, it is likely that conformation of the active site could in turn influence the properties of the disulfides. For example, if, as suggested above, the two termini bind mutually exclusively to the redox active site but the C terminus binds preferentially, then C-terminal disulfide mutants will allow the N terminus to bind exclusively in the redox active site. This could well slow the reactivity of the N-terminal disulfide of the mutant compared to the wild-type enzyme, but be of little physiological significance if the active site of the native enzyme is occupied by the C terminus and the then relatively disordered N-terminal disulfide is readily reduced. 4.4 DISCUSSION Redox reactions of AtFKBP13 and related enzymes are characterized by small conformational changes, which are in general localized to the active site. This is also true 102 Chapter Results and Discussion in AtFKBP13 when the structure of oxidized AtFKBP13 is compared to that of AtFKBP13-(SH)2. The high-resolution crystal structure of the AtFKBP13 protein [Gopalan et al., 2004] involved in redox regulation inside the chloroplast lumen, have been solved in the oxidized and reduced states (1U49 and 1Y00 PDB code, respectively). Reduced AtFKBP13 crystals were produced by adding 20 mM dithiothreitol (DTT) to the precipitant solution every 2–3 days to ensure AtFKBP13 was maintained in the reduced form. It can be noted from biochemical study that DTT alone at lower concentration (0.5mM) was a poor reducing agent (Table 4-1). But when used at higher concentration can reduce the protein which is evident from the electron density map of reduced structure. The overall structure of these models is conserved and the significant differences found are at the redox active regulatory cysteins and the secondary structure of residues 50-53. These structures demonstrate that reduction of the reactive cysteine residues does not disturb the geometry of the PPIase site in oxidized AtFKBP13 drastically. However, it alters the accessibility of the catalytic residues involved in PPIase activity to some extent. The significant change caused by the reduction is limited to the movement of the side chain Sγ of active site Cys5 ,Cys106 and Cys111 thus opening up the structure at the N-terminus. One of the most striking features of the reduced (inactive) form of the protein is the way in which the C-terminal extension undergoes conformational changes. When the disulfide bond is broken, the contacts in the active site are no longer strong enough to hold the extension. Other novel aspects of the structure include the N-terminal extension, its role and the changes that the enzyme may undergo upon reduction of the disulfide 103 Chapter Results and Discussion bond, which all collectively influence the regulation of the activation status of the enzyme in vivo. The present AtFKBP13 structure is the first example of a structure that shows how enzyme activity is regulated by disulfide-bond reduction and reoxidation in the chloroplast lumen. The crystal structure of another light-activated chloroplast enzyme, fructose-1,6-bisphosphatase, has been determined [Villeret et al., 1995]. However, that structure contains no disulfide bonds and is presumably of an active and reduced enzyme. So the structure reveals little about how oxidation inhibits activity. AtFKBP13 differs from NADP-malate dehydrogenase (NADP-MDH) [Carr et al., 1999], another light regulated chloroplast enzyme (which is active in its reduced state) in that oxidized AtFKBP13 can have substantial activity. 4.4.1 Regulatory active site and redox potential The two residues between the active-site cysteine residues have a large impact on the redox potential, not only in AtFKBP13 but also in other structurally related proteins [Grauschopf et al., 1995; Huber-Wunderlich and Glockshuber, 1998; Joelson, et al., 1990; Krause et al., 1991; Mossner et al.,1998]. This effect has been attributed to the influence of these residues on the pKa of the thiolate in the reduced form of the enzymes [Grauschopf et al., 1995]. Attempts to exploit such a relationship in the design of enzymes with a known redox potential by substituting the active site sequence of the wild-type protein to that of another member of the thioredoxin superfamily have been made [Huber-Wunderlich and Glockshuber,1998; Mossner et al., 1998]. These 104 Chapter Results and Discussion experiments show that while qualitatively in agreement with the design, such a simplified model does not predict the redox potential of the mutants with good precision. The crystal structure of oxidized AtFKBP13 reveals an intricate network of favorable interactions with the redox active site cysteins that are not present in reduced AtFKBP13. The greater stability of oxidized over reduced AtFKBP13 appears to be a result of greater stabilization of the disulfide (compared with the thiolate form) at the active site. This is affected by a network of hydrogen bond and electrostatic interactions. These favorable interactions are removed in reduced AtFKBP13. The interatomic distance between the two sulfur atoms in reduced AtFKBP13 is relatively short in the C-terminal active site (2.9–3.0 Å) compared to 3.8 Å for the hydrogen bonds observed in the neutron diffraction study of L-cysteine [Kerr et al.,1975] and the NMR [Qin et al., 1994] and crystal structures [Weichsel et al., 1996] of reduced thioredoxin. This shorter interaction could be an important component for both the stabilization of reduced AtFKBP13, as well as in the comparison of AtFKBP13 with other redox proteins such as thioredoxin. 4.5 BIOLOGICAL IMPLICATIONS Our results show how protein activity can be switched by Reactive oxygen species mediated disulfide bond formation. The possible regulatory role of a reversible disulfide bond formation in the redox-active proteins had been postulated [Weichsel et al., 1996]. Reversible disulfide bond formation in oxidoreductases such as thioredoxin 105 Chapter Results and Discussion [Weichsel et al., 1996] and AtFKBP13 introduces little structural transition since two redox-active cysteines are nearby in the reduced state of these proteins. The X-ray crystal structure of the reduced, inactive form of the light-regulated AtFKBP13 provides us with the first example of the crystal structure of a covalently regulated plant enzyme of the chloroplast lumen. The structure also illuminates a mechanism of covalent regulation by disulfide reduction and re-oxidation that may be unique to chloroplasts. Comparison of AtFKBP13 with other light regulated enzymes such as NADP-MDH and AND-dependent malate dehydrogenase (AND-MDH) structures shows how the light and redox regulation of an enzyme is achieved at the molecular level. However, these enzymes are present in the stroma and are active in their reduced state. The active form of the enzyme has the two cysteines joined in a disulfide bond as opposed to the NADP-MDH, which is active in the reduced state. The differences between AtFKBP13 and NADP-MDH are expected to reveal the factors responsible for the stabilization of the active sites of the two enzymes. AtFKBP13 has Nand C-terminal extensions when compared to the non-regulated AND-MDHs from all organisms. Each extension contains a disulfide bond. The disulfide bond at the C terminus causes the end of the polypeptide chain to turn and fold into the active site, providing the basis for an explanation of how the extent of light activation of the enzyme is adjusted according to the prevailing light intensity and photosynthetic rates (increasing at high light intensities and photosynthetic rates). The role of the N-terminal disulfide is less clear, but it may allow the N-terminal extension to interact with the redox active site in the absence of the C-terminal extension. 106 Chapter Results and Discussion The proteins present in the reducing compartments of all cells, which include the cytoplasm, mitochondria, and chloroplast stroma (in plant cells), contain very few disulfide bonds. All characterized proteins with disulfide bonds are from extracellular compartments or internal compartments in the secretory pathway, such as the endoplasmic reticulum and beyond or the periplasmic space of bacteria. The AtFKBP13 structure described here has two disulfide bonds that are able to form in a reducing cellular environment. 4.6 CONCLUDING REMARKS There are little structural changes between the oxidized and reduced structures of AtFKBP13.The conformational changes are restricted to some slight re-arrangements of the redox active site CXnC motif and residues 50-53. Cys5 is found exposed and Cys17 is buried in both the crystal structures, independent of their redox state. Therefore, oxidation and reduction are not related to a drastic re-orientation of Cys17 in AtFKBP13. DTT was found to play important role in redox regulation of protein and showed that activation was dependent on DTT concentration. Excess DTT (20 mM) directly deactivated the AtFKBP13 while at a lower concentration, less than mM DTT, the protein was in oxidized state .We, therefore, used this concentration of DTT in our studies to crystallized protein in reduced state. The results suggest that DTT reduced the S-S bond of AtFKBP13 and that the reduced dithiol causes localized changes resulting in the lost of enzyme activity. 107 Chapter Results and Discussion Figure 4-13. Proposed mechanism for the light-mediated regulation of AtFKBP13 in the thylakoid lumen. Trx, thioredoxin; PSI, photosystem I; PSII, photosystem II; Fd, ferredoxin. Rp is an undefined redox protein. The present results extend the capacity for redox regulation, long known for chloroplast stromal enzymes, to AtFKBP13, a resident of the thylakoid lumen. The results suggest that AtFKBP13, and possibly other redox-regulated proteins of the lumen, are photo-reduced by thioredoxin m via photosystem I on entering and traversing the stroma. In the case of AtFKBP13, reduction renders the enzyme inactive. It appears that, once imported into the lumen, AtFKBP13 is converted from the reduced, inactive state by 108 Chapter Results and Discussion photo-oxidation via a photosystem II reaction either directly by molecular oxygen or, indirectly, by way of a redox-active protein (Fig. 4-13). Such a mode of activation, in which an enzyme of the lumen is rendered active when oxidized in the light, contrasts with the mechanism functional in the stroma. The relation of electron transfer proteins of the lumen: HCF164 [Lennartz et al., 2001], thylakoid luminal protein [Lee et al., 2004], oxygen-evolving enhancer protein [Heide et al., 2004], and CcdA[Page et al., 2004] to the scheme depicted in Fig. 4-13 requires further work. Such mechanism is keeping well with the well-known role of light in enhancing the assembly of the photosynthetic electron transfer proteins that interact with AtFKBP13 [Malkin, 1992; Gupta et al., 2002]. The mechanism presented appears to be independent of the previously demonstrated interaction of the precursor form of FKPB13 with the Rieske protein [Gupta et al., 2002]. Whether the indicated light-induced activation would be reversed in the dark, as is the case for enzymes of the Calvin cycle or whether it represents a onetime only event occurring upon protein import remains to be determined. An additional and unanswered question centers on the identity of the redox protein proposed to function in Fig. 4-13. While thioredoxin has not been clearly identified in the lumen, one possible candidate that may act in this capacity is HCF164— a thioredoxin-like protein that is necessary for the formation of the cytochrome b6/f complex [Lennartz et al., 2001]. Finally, it will be of interest to learn how the recently described lumen analog of the prokaryotic thiol-disulfide transporter, CcdA, interfaces with redox regulation of resident proteins [Page et al., 2004]. 109 Chapter Results and Discussion As mentioned above, the mode of activation, in which a protein of the lumen is rendered active when oxidized in the light, contrasts with the mechanism functional in the stroma. Apropos this point, it is noted that one lumenal member of the cylophilin family (AtCYP20-2) has been found to be activated by oxidation in a manner akin to AtFKBP13 (unpublished data), whereas another, AtCYP20-3 of the stroma, is activated on reduction by thioredoxin m [Motohashi et al., 2003]. Current evidence thus suggests that the regulatory response of an enzyme to the redox change depends not only on its function, but also on its location within the cell. 110 [...]... no AJ 243 702) was sequenced on both strands to confirm its identity and was used in subsequent experiments to determine the interacting domains of the AtFKBP13 and the Rieske proteins A number of cDNA fragments encoding various domains of the AtFKBP13 or Rieske protein were fused in frame with either the DNAbinding or activation domain of the Gal4 protein in the vectors Different combinations of these... Gal4 DNA-binding domain to screen an Arabidopsis cDNA library in a Gal4 activation-domain vector Among the AtFKBP13 interacting clones that were sequenced, a number of them encoded the Rieske protein of various lengths fused in frame with the activation domain The longest one had four amino acid truncations in the N terminus The Rieske protein is an essential subunit of the cytochrome bf complex in the... significant difference in the Rieske mRNA levels among RNAi and control 86 Chapter 4 Results and Discussion plants, suggesting that AtFKBP13 affects the level of the Rieske protein by a posttranscriptional process 4. 2 .4 Mature FKBP13 is a target for reduction by thioredoxin The possibility of thioredoxin-linked reduction of AtFKBP13 arose from the finding of two solvent exposed disulfides in its structure... related protein present in the chloroplast stroma, has been found to be a thioredoxin target [Motohashi et al., 2001, 2003] We tested this possibility by applying the NADP/thioredoxin reducing system of E.coli combined with fluorescence gel analysis using mBBr as a thiol-specific probe In this assay, AtFKBP13 was incubated with thioredoxin and NTR in the presence of NADPH, a source of reducing equivalents... and Discussion in AtFKBP13 when the structure of oxidized AtFKBP13 is compared to that of AtFKBP13- (SH)2 The high-resolution crystal structure of the AtFKBP13 protein [Gopalan et al., 20 04] involved in redox regulation inside the chloroplast lumen, have been solved in the oxidized and reduced states (1U49 and 1Y00 PDB code, respectively) Reduced AtFKBP13 crystals were produced by adding 20 mM dithiothreitol... hydrogen bonds observed in the neutron diffraction study of L-cysteine [Kerr et al.,1975] and the NMR [Qin et al., 19 94] and crystal structures [Weichsel et al., 1996] of reduced thioredoxin This shorter interaction could be an important component for both the stabilization of reduced AtFKBP13, as well as in the comparison of AtFKBP13 with other redox proteins such as thioredoxin 4. 5 BIOLOGICAL IMPLICATIONS... mBBr, the proteins separated by SDS-PAGE and the fluorescence was recorded The reduction of disulfide(s) by the NADP/thioredoxin system was reflected by an increase in the fluorescence of the treated protein (Fig 4- 10) Owing to the absence of additional free cysteines in the sequence of AtFKBP13, the protein alone did not react with mBBr (Fig 4- 10, lane 1, control treatment) 87 Chapter 4 Results and... Discussion Figure 4- 10 Reduction of AtFKBP13 by the NADP_thioredoxin system from E coli Ctrl- control (FKBP13 alone); Cpl- complete thioredoxin system (NADPH+NTR+Trx) plus FKBP13; Trx- thioredoxin; -Trx, -NTR, and -NADPH lanes indicate the complete thioredoxin system in which each of these components was individually omitted Protein refers to the complete treatment in which the gel was stained with Coomassie... contrast, incubation of AtFKPB13 with the NADP/thioredoxin system for 20 minutes resulted in marked fluorescent labeling indicating disulfide reduction (Fig 4- 10, lane 2, complete treatment) Reduction was not observed when any one of the components of the system was omitted, i.e., thioredoxin, NTR or NADPH (Figure 4- 10, lanes 3-5) A similar experiment performed with mutant forms of AtFKBP13 in which... site of regulation by thioredoxin Activity was reduced almost 80% in the quadruple cysteine mutant (C5S/C17S and C106S/C111S) Following incubation and reduction of AtFKBP13 by Arabidopsis thioredoxin m and DTT, the PPIase activity was reduced by 55%, whereas DTT alone had a minimal effect These results suggest that AtFKBP13 PPIase activity can be modulated by the redox state in vitro 4. 2.6 Effect of . A number of cDNA fragments encoding various domains of the AtFKBP13 or Rieske protein were fused in frame with either the DNA- binding or activation domain of the Gal4 protein in the vectors lumen domain, of the Rieske protein is sufficient for interaction. Figure 4- 8. Interaction between AtFKBP13 and the Rieske protein determined by in vitro protein interaction. is a target for reduction by thioredoxin The possibility of thioredoxin-linked reduction of AtFKBP13 arose from the finding of two solvent exposed disulfides in its structure and from recent