Crystallographic and Computational Analysis of the Barrel Part of the

Jul 25, 2016 - (1) Approximately half of the 177 surface waters identified at 100 K are ... Grotthus-type proton transfer along the protein surface an...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/biochemistry

Crystallographic and Computational Analysis of the Barrel Part of the PsbO Protein of Photosystem II: Carboxylate−Water Clusters as Putative Proton Transfer Relays and Structural Switches Martin Bommer,*,† Ana-Nicoleta Bondar,*,‡ Athina Zouni,§ Holger Dobbek,† and Holger Dau*,∥ †

Institut für Biologie, Strukturbiologie/Biochemie, Humboldt-Universität zu Berlin, Unter den Linden 6, 10099 Berlin, Germany Fachbereich Physik, Theoretical Molecular Biophysics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany § Institut für Biologie, Biophysik der Photosynthese, Humboldt-Universität zu Berlin, Unter den Linden 6, 10099 Berlin, Germany ∥ Fachbereich Physik, Biophysics and Photosynthesis, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany

Downloaded via STEPHEN F AUSTIN STATE UNIV on July 28, 2018 at 14:27:19 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: In all organisms that employ oxygenic photosynthesis, the membrane-extrinsic PsbO protein is a functionally important component of photosystem II. To study the previously proposed proton antenna function of carboxylate clusters at the protein−water interface, we combined crystallography and simulations of a truncated cyanobacterial (Thermosynechococcus elongatus) PsbO without peripheral loops. We expressed the PsbO β-barrel heterologously and determined crystal structures at resolutions of 1.15−1.5 Å at 100 K at various pH values and at 297 K and pH 6. (1) Approximately half of the 177 surface waters identified at 100 K are resolved at 297 K, suggesting significant occupancy of specific water sites at room temperature, and loss of resolvable occupancy for other sites. (2) Within a loop region specific to cyanobacterial PsbO, three residues and four waters coordinating a calcium ion are well ordered even at 297 K; the ligation differs for manganese. (3) The crystal structures show water−carboxylate clusters that could facilitate fast Grotthus-type proton transfer along the protein surface and/or store protons. (4) Two carboxylate side chains, which are part of a structural motif interrupting two β-strands and connecting PsbO to photosystem II, are within hydrogen bonding distance at pH 6 (100 K). Simulations indicate coupling between protein structure and carboxylate protonation. The crystal structure determined at 100 K and pH 10 indicates broken hydrogen bonding between the carboxylates and local structural change. At pH 6 and 297 K, both conformations were present in the crystal, suggesting conformational dynamics in the functionally relevant pH regime. Taken together, crystallography and molecular dynamics underline a possible mechanism for pH-dependent structural switching.

I

pigments nor redox-active groups (Figure 1).5−9 Particularly important among these membrane-extrinsic proteins is the barrel-shaped PsbO gene product, previously denoted as 33 kDa protein. For recent reviews on the numerous, often seminal investigations of PsbO, see refs 10−17. The structure of PsbO protein is distinguished by its β-barrel domain, which extends pronouncedly into the lumenal space; PsbO is located relatively close to the Mn4Ca−oxo cluster, thereby breaking the

t is no exaggeration to state that oxygenic photosynthesis of cyanobacteria, eukaryotic algae, and plants powers life on earth.1 Light-driven water oxidation is a key step in oxygenic photosynthesis because it renders water available as a source of “energized electrons” (reducing equivalents) and protons. The photosystem II (PSII) cofactor−protein complex facilitates light-driven water oxidation at its electron donor side and plastoquinone reduction at its acceptor side (Figure 1).2−4 The water oxidation reaction itself is catalyzed at a Mn4Ca−oxo cluster bound to residues of membrane-spanning protein subunits, which is separated from the lumenal bulk phase by membrane-extrinsic proteins that carry neither light-absorbing © 2016 American Chemical Society

Received: May 3, 2016 Revised: July 6, 2016 Published: July 25, 2016 4626

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry

molecules.27 Importantly, the analysis of carboxylate−water clusters indicated that D224, an amino acid residue that is part of an extended hydrogen-bonding network leading to the manganese cluster, engages in persistent carboxylate−water bridging.27 The proton antenna functionality of PsbO may comprise (i) proton storage (to reduce the extent of acidification of the lumenal compartment), (ii) proton conduction along the protein surface of PsbO (to sites favorable for proton release), (iii) accelerated proton transfer to water molecules of the lumenal bulk, and/or (iv) neighboring protein subunits in the lumenal space of the thylakoids, which is densely packed with protein chains of PSII. Clusters of carboxylate side chains, together with histidine groups, are thought to be essential for proton antennas.28 Clusters of carboxylate residues, that is, aspartate and glutamate amino acid residues, are found at the surface of many proteins with bioenergetic functionality.29 This observation as well as previous work on the potential function of PsbO carboxylates as a proton antenna25−27 prompted us to investigate a truncated variant of the PsbO protein that consists of the prominent barrel structure with its hydrophobic interior and putative proton antenna residues at its exterior. Striving for insight into the structure and dynamics of the surface-exposed carboxylates and their water environment, we also discovered a putative pH-controlled structural switch involving deprotonation of a carboxylate dyad. In the work presented here, we focus on discussing clusters of carboxylate residues and surface water molecules. Detailed analysis of the role of other protonatable side chains may be undertaken on the basis of models and data we deposited in the PDB. In line with its important role in optimizing the function of PSII, the PsbO protein is the only membrane-extrinsic protein of PSII found in all organisms that employ oxygenic photosynthesis. (Yet PsbO is not absolutely essential for light-driven water oxidation by PSII.14,30) We decided to focus on the PsbO of thermophilic cyanobacteria, specifically Thermosynechococcus elongatus and Thermosynechococcus vulcanus, for which crystal structures of complete PSII have been determined. The PsbO protein consists of a β-barrel and several loops, some of which intricately link it to the PSII complex: the N-terminus (residues 1−15) connects PsbO to CP43, a large loop (residues 149−192) to D2, CP47, and PsbU, residues 220−231 to D2, while a loop comprising residues 55−63 reaches across the monomer−monomer interface of the dimeric PSII. In high-resolution crystal structures of T. elongatus/vulcanus PSII,5−9 both β-barrel and loop regions are well-resolved, except for the dimer-connecting loop. PsbO has been thought to be unfolded when detached from the PSII complex, and the crystal structure of PsbO alone has not been determined. However, recombinant T. elongatus PsbO has been shown by NMR to contain both well-folded and flexible domains.31 To study the PsbO surface carboxylates, we employ a robust PsbO model system, which is well-ordered, structurally defined, stable, and amenable to genetic manipulation. The truncated PsbO protein, consisting of the β-barrel region of PsbO, is denoted as PsbO-β.

Figure 1. Monomer view of the high-resolution dimeric PSII structure (PDB entry 3WU2).7 The vertical bar corresponds to 40 Å and indicates the extension of the thylakoid membrane. The subunits of the reaction center core (D1/D2) and of the PSII core antenna (CP47 and CP43) form the bulk of the transmembrane part of the PSII protein complex. CP47 protrudes into the lumen, along with the membrane-extrinsic subunits PsbU, PsbV, and PsbO. Chlorophyll cofactors are colored green. β-Carotene is colored orange, and atoms of the Mn4CaO5 cluster at the active site of water oxidation are shown as spheres (OEC, oxygen-evolving complex). The PsbO protein is composed of a β-barrel domain (blue) and loops, some of which connect PsbO to D1/D2, CP47/CP43, PsbU, and the other monomer (purple, right, along with the N-terminus on the left).

approximate C2 symmetry of the PSII protein complex (Figure 1) in cyanobacterial PSII,5−7 PSII from red algae,9 and PSII from higher plants.18 Variants of the PsbO protein are found in all organism groups capable of photosynthetic water oxidation (see review articles cited above); two or more PsbO isoforms can be present in angiosperms.19 First suggested to bind manganese, PsbO is now thought to be a manganese-stabilizing protein (MSP). This stabilizing role may involve protection against exogenous reductants as well as lowering of the calcium and chloride concentrations required for water oxidation. The enhanced binding of calcium to the catalytic Mn4Ca cluster in PSII when PsbO is present is well-documented and may relate directly to the protection against external reductants.14,20,21 However, the PsbO functionality goes beyond stabilization of the metal cluster and its protection against external reductants. Although the process is mechanistically ill understood, it has been shown by infrared difference spectroscopy that the PsbO protein is structurally coupled to transitions in the reaction cycle of the Mn4Ca−oxo cluster, especially in the so-called S1− S2 transition.13,22−24 In solutions of isolated plant PsbO, pH titrations revealed evidence of pH-dependent structural changes, as well as significant changes in the number of protons bound to PsbO. A possible involvement of PsbO in proton transfer is related to the presence of a significant number of aspartate and glutamate amino acid side chains on its surface, some of which are thought to function as a proton antenna.25−27 Recent molecular dynamics (MD) simulations of PsbO in water identified a number of carboxylate pairs that have rather stable interactions with hydrogen-bonding water

■

EXPERIMENTAL PROCEDURES Expression and Purification. Full-length PsbO has previously been produced in Escherichia coli, but its production was shown to be crucially dependent on the formation of the C19−C44 disulfide bond, which staples the N-terminus to the 4627

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry

Figure 2. Structure of PsbO-β at 100 K comprising the β-barrel domain (green) only. Loops colored gray were deleted in truncated PsbO-β. Lines represent their position in full-length PSII-bound PsbO (PDB entry 3WU2).7 Colored side chains indicate surface carboxylate clusters (SC6, SC7, and SC8, as identified in ref 46). (A) Close-up of the E97/D102 dyad.46 (B) Close-up of the Ca2+-binding site and cyano loop.61 Ordered solvent water molecules are shown as blue spheres. (C−F) Close-up views of water clusters around carboxylate side chains in SC7 (C and D) and SC6 (E and F). The model coordinates represent PsbO-β at 100 K, overlaid with the electron density for either 100 or 297 K. Both 0.8σ (light blue) and 1.2σ (dark blue) contour 2Fo − Fc electron density maps after occupancy refinement of placed water molecules are shown. Asterisks denote two water molecules within hydrogen bonding distance of a symmetry-related PsbO molecule within the crystal. Two conformers for D23 at 297 K are shown in panel F.

β-barrel.32 Previous strategies included periplasmic targeting,31,33,34 an N-terminal E. coli thioredoxin (trxA) tag,32,35 refolding,36 and the use of a disulfide-competent E. coli strain.37 The thioredoxin fusion has the advantage of robust expression of the fusion partner in E. coli.38 T. elongatus PsbO-β was placed at the C-terminus of E. coli thioredoxin, separated by a linker containing a tobacco etch virus (TEV) protease-cleavable hexahistidine tag in the pET-28a vector. See the Supporting Information and PDB entries for the protein sequence. The protein was expressed in E. coli and purified by nickel affinity and by size exclusion chromatography on a 24 mL GE Superdex 75 300/10 column. PsbO was isolated as a monomer with a minor peak corresponding to either dimer, uncleaved fusion protein or an impurity observed during the preparative size exclusion step. Approximately 5 mg of purified, cleaved PsbO-β was obtained per liter of E. coli culture. Crystallization. The protein was crystallized by vapor diffusion in a buffer containing 0.1 M MES-Na (pH 6) (varied), 0.2 M calcium acetate, and 30% polyethylene glycol 400. The structure was determined at 1.15 Å resolution at a cryogenic temperature (100 K) in space group P6155 with one monomer in the asymmetric unit. Data collection and refinement are summarized in Table S1. Cryogenic data collection is routinely

conducted to mitigate the detrimental effects of X-ray dose on the crystal. To investigate if the cryo-cooled structure differs from that at room temperature, a further data set was collected at room temperature (297 K) with an ∼100-fold reduced X-ray dose, resulting in a 1.5 Å structure from a similar crystal. To investigate the influence of pH, crystals were incubated in an otherwise identical crystallization solution containing a different buffer [such as bis-tris-propane (pH 10)] for 30−60 min prior to cryo-cooling. Molecular Dynamics Simulations. The protocol for MD simulations was similar to that used by Lorch et al.27 and is described in the Supporting Information. Briefly, MD simulations of PsbO-β at constant pressure and constant temperature were performed in water using the NAMD software39,40 with CHARMM force field parameters41−44 and TIP3P water.45

■

RESULTS PsbO-β Construct. While the PsbO loop regions are structurally well-defined within the PSII complex and possibly involved in PSII internal proton transfer pathways,25,27,46 they are likely unstructured for the free PsbO in solution.31 In initial experiments with full-length T. elongatus PsbO [purified in the 4628

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry

the interface of the β-barrel and D1/CP47 in PSII (approximately one-fourth of the total PsbO−PSII interface waters in PDB entry 3WU27) are resolved less well than those on the solvent-exposed surface. Furthermore, free and PSII-bound PsbO differed in the vicinity of crystal contacts in the new, more densely packed P6122 crystal form, which constrains the orientation of side chains and loop regions 20−28 and 72−76. These in turn affect, inter alia, the position of the side chains of E98, D99, and D141 located in the surface carboxylate clusters denoted as SC8 as well as D23 and D24 in SC6.46 While these regions are stabilized by crystal contacts, we have no reason to assume that their conformation is affected by the loop truncation and would differ between PsbO-β and full-length PsbO in solution. During crystallization and within the protein crystal, the PsbO concentration is extremely high (30 mg mL−1 PsbO in the crystallization drop and 600 mg mL−1 within the crystal). These high concentrations can favor formation of PsbO multimers that are unrelated to the oligomerization state of isolated PsbO-β in solution or of PSII-bound PsbO. In the PsbO-β crystals, two monomers are indeed packed in the form of a dimer, but neither stacking of aromatic side chains nor extensive hydrogen bond networks were identified (only three hydrogen bonds). When we overlaid the potential PsbO dimer on the structure of PSII (PDB entry 3WU2), we detected a clash with the opposite PSII monomer. In conclusion, the “weak” dimers present in PsbO-β crystals cannot support the presence of PsbO dimers in solution or PSII in its native membrane environment. An FTIR-detectable increase in β-sheet content upon binding of plant PsbO to PSII has been reported,52 which cannot be explained by major differences in the β-sheet content of the bound PsbO in the PSII holocomplex, as revealed by comparison of a recent structural model of plant PSII18 to the crystallographic model of thermophilic PSII (see Figure S1C). In ref 52, the authors discussed that the increase in β-sheet content is not fully reproducible but affected by prolonged storage of isolated PsbO, suggesting a metastable structure of spinach PsbO in solution. Investigation of the thermal stability of T. elongatus PsbO has revealed significant structural changes with a transition temperature of 76 °C,48 whereas spinach PsbO unfolds with a clearly lower transition temperature (∼50 °C). Thus, we consider it possible that partial loss (or loosening) of the β-barrel structure of isolated PsbO is observed in plant PsbO, but not in T. elongatus PsbO. Comparison of 100 K and Room-Temperature Structure. Cryo-cooling of protein crystals leads to a reduction in the size of both the protein and the crystal on the order of 1−2% and may trap both protein side chains and solvent molecules within subsets of naturally occurring conformations.53 The 100 and 297 K PsbO-β structures are the result of experiments that varied in only the buffer component of the crystallization solution, temperature, and X-ray dose. The unit cell (crystal size) was reduced by 2% in length upon cooling, but the packing and structure of PsbO remained the same with Cα atoms superimposable with a 0.3 Å rmsd (Figure S1). Of 18 minor changes initially identified between 100 and 297 K structures, 13 side chain rotamers and two loops were related to differences between PSII-bound and free PsbO at 100 K mentioned above (see D23 in Figure 2F for an example). For the remainder, side chain positions within PSII-bound PsbO systematically followed neither 100 nor 297 K structures of free PsbO. Thus, the constraints introduced by either PSII complex

same manner as described for the truncated construct (see Experimental Procedures)], mass spectrometry (MS) results indicated proteolytic cleavage after R162, R184, and R189 within the 149−192 loop, further supporting their unfolded nature in free PsbO. The same loop is susceptible to chymotrypsin and V8 protease digestion in recombinant T. elongatus PsbO,47 while enterokinase may cleave a loop corresponding to residues 220−231 in spinach PsbO.32 Nevertheless, expression of full-length PsbO from both cyanobacteria31,35,47,48 and plant,32,33,36 free from degradation as evidenced by silver-stain sodium dodecyl sulfate−polyacrylamide gel electrophoresis and MS,48 has been achieved in E. coli. Periplasmic expression,31,33,48 purification of inclusion bodies followed by refolding,36 heat shock,35 and the use of protease inhibitors32 may all reduce the level of exposure to E. coli proteases. Even though proteolytic cleavage can be avoided, we removed the susceptible regions because they are likely unstructured in solution. Aiming at a PsbO variant consisting of the solution-stable βbarrel only, we removed the N-terminus (residues 1−15) and loops (55−63, 149−192, and 220−231) from the sequence. Sequence numbers refer to those in full-length PsbO (such as in PDB entry 3WU27). For the sake of internal consistency, continuous numbering is used in PDB entries, and for comparison, residue numbers in truncated PsbO-β entries are shown in parentheses in Figure 2. The three loop regions protrude from alternating antiparallel β-strands within the barrel. Gaps created by their deletion were bridged by substitution with intrinsically stable Asn-Gly β-hairpins.49−51 The thus obtained minimal PsbO construct (PsbO-β) contained the complete β-barrel with 175 of the original 246 amino acid residues of full-length PsbO. As shown in the following, PsbO-β is especially well suited to serve as a structurally well-defined isolated model protein for investigation of surface-exposed carboxylate clusters. Although reconstitution of PSII with PsbO variants has been approached before,32 PsbO-β may not bind well to PSII because the eliminated loop regions are involved in numerous enthalpically favorable intersubunit contacts.10 Structural Comparison with PSII-Bound PsbO. Figure 2 shows the structure of PsbO-β at 100 K, while Figure S1 compares it to full-length PsbO bound to PSII.7 The overall fold of the β-barrel was the same in both structures, and Cα atoms could be superimposed with a rmsd of 0.7 Å. The βhairpin substituting for the loop present in full-length PsbO was resolved well in the case of the dimer loop (55−63); however, adjacent E54 and E64 side chain positions were perturbed. The β-hairpins substituting for loops 149−192 and 220−231 are partially resolved in the electron density, which from the observation of multiple crystals appeared to stem from at least two rotational conformers of internally stable hairpin structures. The first three residues were not resolved, so the first residue modeled is C19, which is stabilized by a disulfide bridge to C44. Differences from PSII-bound PsbO7 were seen in the backbone positions of loop 72−76 and β-strand 106−119, which are in contact with D1 and CP47 in the PSII complex. These residues show above-average anisotropic displacement of their atomic positions from their average within the crystal (Figure S2, in plane with the surface of the β-barrel). The changes perturb the positions of residues E54, E64, and E145 of the SC7 surface carboxylate cluster.46 These local changes likely result from the detachment of PsbO from its binding partners in PSII and are expected to also exist in solution. Consequently, waters lining 4629

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry

Figure 2 and Figures S3−S5 show a single model for protein residues and placed water molecules (obtained for the 100 K structure), but the electron density at these positions is shown for both 100 and 297 K data. Fading of electron density at 297 K is indicative of less populated sites (partial occupancy) or less order in the water structure (quasi-continuum of water positions in the time-averaged structure). On the basis of the counts of resolved water molecules and visual inspection of Figures S3−S5, we conclude that in the first hydration shell of the protein, surprisingly large numbers of water molecules can be detected in the room-temperature structure at approximately the same positions. This observation confirms that also in the PsbO protein (i) the low-temperature structure corresponds to a room-temperature configuration (of the protein and adjacent water molecules) with an especially low potential energy. (ii) At room temperature, the specific interactions between water molecules and the protein surface determine the time-averaged structure, the residue and water dynamics, and likely also protonation dynamics and proton binding energies (pKa values) in the protein−water interface region. A number of carboxylate clusters on the surface of PsbO, interspaced by water molecules, form potential proton transfer networks.27,46 To discuss the carboxylate clusters, we use the Surface Clusters (SC) identified in ref 46. One of these clusters (SC4) is harbored within the deleted loop region and is thus not part of the PsbO-β construct. Other surface clusters (SC6− SC8) are discussed below. Figure 2C−F shows hydration at the center of carboxylate clusters SC7 and SC6. The following linear chain of three glutamate side chains with a single interconnecting water molecule is well-resolved (Figure 2C):

formation or the crystal matrix outweighed the influence of temperature on a very flexible and often transient part of protein structure, the position of loops and side chains. The inside of the β-barrel is lined with hydrophobic residues, which may contribute to its exceptional stability. Only two water molecules could be placed inside the β-barrel at 100 K. Both are bound to Q170; only one water is resolved at room temperature and in PSII-bound PsbO.7 We are specifically interested in the interaction of the surface carboxylates with water molecules after cryo-trapping (at 100 K) and at room temperature. Care was taken to prevent an artificial structural influence due to formation of ordered water-ice within the crystal. The crystal solvent was a mixture of 70% water and 30% polyethylene glycol (PEG 400), such that a glass state was likely formed in the crystal for all extended volumes otherwise filled with water. Bulk solvent within the crystal thus could not have a periodicity that follows that of the protein component, and the average across all unit cells is represented by a flat solvent model.54 However, surface-bound waters adopted distinct, highly populated positions. We modeled 177 water molecules (including 15 assigned to symmetry-related PsbO proteins in the PDB file) on the surface of the 100 K structure (see Figure S3). Of these, 118 could be clearly assigned to the first hydration shell (between 2.5 and 3.2 Å of a protein oxygen or nitrogen atom) and 19 to the second hydration shell (in contact with a first-hydration shell water but not with amino acid residues of PsbO). A slightly smaller number of water molecules (121 and 140 in each monomer) were modeled on the surface of the β-barrel of PsbO attached to PSII at 1.9 Å resolution.7 In PsbO-β, the surface normally in contact with PSII lacks significant ordered hydration. Additionally, we expect 21 hydrogen bonds to symmetry-related PsbO molecules in the crystal to be occupied by water molecules in solution. In the 297 K structure, 79 water molecules were modeled, corresponding to approximately half of those in the first hydration shell (Figure S3), with waters bound by two PsbO proteins (crystal waters) and waters coordinated by a calcium the most highly ordered and conserved between 100 and 297 K. In contrast, only two of the second-hydration shell waters were modeled in the 297 K structure. The ability to observe water molecules in protein crystal structures is principally affected by (i) occupancy of a water site, which is related to the proximity of hydrogen bonding partners and the local water structure impacted by the temperature, (ii) uncertainty and/or mobility in the position of the water molecule, frequently related to the protein side chain to which it is bonded and described by its B factor, and (iii) overall resolution. We are interested in the resolvability of water molecules relating to local mobility and occupancy, but not in the influence of the global resolution. In Figure 2, we compare specifically the highest-resolution structures determined: a 100 K structure determined at 1.15 Å resolution and a 297 K structure determined at 1.5 Å resolution. The resolution difference might affect the outcome of this comparison. To (partially) disentangle the effects of resolution, sample, and temperature, we collected additional data sets at different resolutions at both 100 K (1.4−1.7 Å) and 297 K (1.5−1.7 Å) (Figures S4 and S5). Comparison of the thereby obtained 100 K PsbO-β structure with the 297 K structure at the same resolution shows distinct populations of water being affected by either overall resolution, a flexible, alternate occupancy side chain (D23), but most prominently room temperature versus cryogenic temperature.

H 2O−OCOE216 −H 2O−OCOE218−H 2O−OCOE232

This arrangement could facilitate especially fast, Grotthustype proton transfer along the protein surface. The Grotthus mechanism of proton transfer55−57 involves small shifts of protons within hydrogen bonds that occur without major movement of heavy (non-hydrogen) atoms, as exemplarily illustrated by the following reaction sequence: E216

−

OCOH−OH 2 − OCOE218

→ E216OCO− −H+OH 2 −− OCOE218

→ E216OCO−−H 2O−HOCOE218

In the example given above, small shifts of the individual protons result in ultrafast relocation of the protonated state (and of the corresponding positive charge) from the E216 side chain via an intervening water molecule to the E218 side chain. Figure 2E illustrates a more complex, triangular arrangement of three carboxylate residues and hydrogen-bonded water molecules. In recent MD simulations of full-length PsbO in water, we found complex dynamics of carboxylate−water interactions on the surface of PsbO.27 The carboxylate−water interactions are dynamic, and even for carboxylate side chains located within a short distance, there are fluctuations of the number of hydrogen-bonding water molecules that bridge the carboxylate groups.27 In general, short carboxylate−water bridges, whereby the carboxylate side chains connect via one or two hydrogenbonding water molecules, have high occupancy on a time scale of 200 ps.27 Examples of carboxylate pairs that have high 4630

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry

Figure 3. Structural snapshots illustrating the E97−D102 interaction. Crystallographic snapshots. (A) Structure of PsbO-β at 100 K together with 1.2σ 2Fo − Fc electron density maps at 1.15 Å resolution for E97, D102, and K123. The crystal was equilibrated in MES-Na (pH 6.0) and subsequently cryo-cooled. The 2.5 Å distance was consistent in multiple structures up to pH 8 at 100 K. (B) Structure at 100 K at 1.25 Å resolution for a crystal in bis-tris propane (pH 10). The same conformation was observed at pH 9. (C) Structure at 297 K together with 1.2σ and 0.8σ 2Fo − Fc electron density maps at 1.5 Å resolution in 50 mM MES-Na and 50 mM cacodylate-Na (pH 6). Mixed closed and open conformations were routinely observed at neutral pH (6−8). See Figure S6 for an overlay of panels A−C and a stereoview of panel C. (D and E) Coordinate snapshots from MD at 300 K. Illustration of protonation-coupled structural changes at the D102 site of truncated PsbO. The small yellow spheres indicate water oxygen atoms within 2.8 Å of the carboxylate oxygen atoms shown, and of K123 Nζ. (D) Coordinate snapshot from PsbOrtp with D102 protonated. (E) Coordinate snapshot from the simulation of PsbOrt, where D102 is negatively charged. The molecular graphics in panels D and E were prepared using VMD.69 (F and G) Interactions (distances) between E97 and D102 in PsbOrt (E) vs PsbOrtp (D). Time series of the distance between D102 Cγ/α and E97 Cδ/α are colored magenta for PsbOrt and cyan for PsbOrtp. Note that, in PsbOrtp, the distance between the carbon atoms of the carboxylate groups (F) is consistent with sampling of direct hydrogen bonding between the protonated D102 and the carboxylate groups of E97 (but E97 and D102 can also bridge via hydrogen-bonding water).

a result of its flexibility within the crystal. Atomic coordinates of the K123 side chain (Figure 3) thus show only one of several possible conformations. Amino acid residues E97 and D102 form a carboxylate dyad46 with a short E97 Oε2−Oδ2 D102 distance of only 2.5 Å (Figure 3). The dyad is conserved in PSII-bound PsbO structures with distances between 2.5 and 2.8 Å.7,36,44 This short distance suggests that one of the two carboxylates is protonated; for very short distances of 2.5 Å, the two carboxylate groups may share one proton in a low-barrier hydrogen bond. To probe the response of PsbO-β to the protonation state of the carboxylate pair, two independent MD simulations were pursued with (PsbOrtp) and without (PsbOrt) protonation of the carboxylate dyad. That is, D102 is protonated in PsbOrtp and negatively charged in PsbOrt; all other carboxylate residues were thought to be deprotonated in both simulation runs. The protocol for performing MD simulations of PsbO-β at constant pressure and constant temperature45 is similar to that used recently to study carboxylate/water dynamics of full-length PsbO27 (see the Supporting Information). Overall, the protein structure is well-preserved throughout the MD simulations, as indicated by the small values of the Cα rmsds computed relative to the starting crystal structure coordinates (Figure S2D). The overall stability of the PsbO-β structure in the current simulations likely arises from its predominantly β-sheet structure, without the long loops and

occupancy of water bridging include D205 and E210 (Figure 2E,F), E218 and E232 (Figure 2C,D), and D222 and D224.27 The observation from MD simulations of PsbO27 described above suggests that, in high-resolution structures of PsbO at room temperature, electron densities for water oxygen atoms may be found in the region between the carboxylate oxygen atoms. Indeed, comparison of the 100 and 297 K structures at the E218/E232 site (Figure 2C vs Figure 2D) confirms this prediction from room-temperature MD simulations of fulllength PsbO. For the E218E232 bridge, there is electron density for one or two water molecules, which is compatible with the simulations predicting a preference for bridging via one to two waters on the 200 ps time scale.27 Deprotonation of Carboxylate Dyad Results in Local Conformational Change. Carboxylate residues E97 and D102 are located in a short loop interrupting the β-sheet structure of the PsbO barrel (Figure 2A), which is conserved in PsbO from cyanobacteria to plants (E97-x-D/G99-G100-I101D102-F/Y103, where x stands for a linking, nonconserved residue10). The resulting kink folds two linked β-strands partially over the end of the barrel, creating a binding site for PSII. In the cyanobacterial PSII,7,58 this loop is connected to further PSII proteins: D99 is located within hydrogen-bonding distance of K381 of the CP47 subunit, while K123 hydrogen bonds with D102 and with S330 of CP47; F103 is within van der Waals distance of N332 in CP47. The electron density for K123 is incomplete in structures of free PsbO-β, presumably as 4631

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry

combination of positively and negatively charged side chains determines the putative proton antenna functionality. Metal-Binding Site in PsbO. A calcium ion is observed in the high-resolution structure of PSII-bound cyanobacterial PsbO60 at a site also implicated in higher-order assembly of PSII dimers.8 The ion is coordinated by residues N200, V201, and T138 and four water molecules, one of which is within hydrogen bonding distance of D141 (Figure S8). Residues T138 and D141 belong to the “cyano-loop”,61 which is conserved in cyanobacteria but not present in plants or algae. Residue T138 also forms part of the dimer−dimer contact in the rows of dimers model8 of the cyanobacterial thylakoid membrane,62 where PSII dimers are linked within the membrane via their CP47/CP43 proteins and, additionally, at the lumenal side via a patch of PsbO groups that includes T138. However, the PSII crystals used in the study were obtained only in the presence of sulfate, depleting the calcium site. Thus, the structure could not confirm a crucial role of bound Ca2+ in protein−protein interactions. Within the PsbO-β crystals, it was possible to deplete the calcium ion by prolonged incubation with 50 mM EDTA or to replace it by incubation with 10 mM lanthanum, gadolinium, or manganese chloride. In the crystals, calcium depletion or replacement did not alter the overall structure of PsbO. We note that the 7-fold coordination of the calcium ion is not maintained for Mn2+ binding, which is six-coordinate and binds directly to D134, without a bridging water molecule (Figure S8). An additional calcium ion mediates a crystal contact between three adjacent PsbO monomers in PsbO-β crystals at 100 K. This second calcium ion, which is included in the PDB entry, is not thought to play a role in solution or in PSII-bound PsbO.

termini of full-length PsbO. The observation here on the structural stability of PsbO-β is compatible with our previous simulations of a PsbO structure, including termini and long loops, in which PsbO contained ∼44% β-sheet structure.27 To assess the effect of the protonation state on the local dynamics of the protein and water interactions, we consider the distances that characterize the interaction between E97 and D102 (Figure 3). The distance between the Cα atoms of E97 and D102 is 9.2 Å in both crystal structures of PsbO-β. The distances between E97 Cδ and D102 Cγ are 4.2 and 4.4 Å in the structures of PsbO-β determined at 100 K and room temperature, respectively. As summarized below, comparison of simulations PsbOrt and PsbOrtp suggests that protonated D102 better preserves the distance between the E97 and D102 side chains, and that the protonation state has subtle but distinct effects on the local protein structure and on local protein−protein and protein interactions. When D102 is negatively charged, in PsbOrt, K123 and E97 sample salt bridge interactions (Figure 3E and Figure S7), and the distance between D102 Cγ and E97 Cδ increases to ∼8 Å (Figure 3F). In contrast, when D102 is protonated (PsbOrtp), the carboxylate groups of D102 and E97 remain closer to the starting crystal structure coordinates (Figure 3D,F), and K123 samples transient interactions with both E97 and D102 (Figure S7). The stronger interactions among E97, D102, and K123 in PsbOrtp compared to the strength of those in PsbOrt are associated with a somewhat shorter distance between the Cα atoms of D102 and E97 (Figure 3G). That is, these simulations suggest that the protonation state influences the local structure of PsbO. We note that water-mediated interactions between E97 and protonated D102 may also be possible; they are associated with a distance between the E97 and D102 carboxylates larger than that observed for direct hydrogen bonding. Because the MD simulations predict local structural change associated with deprotonation of the E97-D102 carboxylate dyad, we investigated this possibility on PsbO-β by highresolution protein crystallography. For PsbO crystals exposed to buffer solution at various pH values before freezing and crystallographic data collection at 100 K, we find that at pH >9, the loop containing D102 unwinds and the D102 carboxylate moves by 5 Å to an “open position” of the E97-D102 dyad. Despite the high resolution of the structure, attempts to deduce the protonation state of the carboxyl groups from bond lengths59 could not provide an unambiguous answer. At 297 K and pH 6−9, a mixture of both open and closed conformations was observed, presumably resulting from two PsbO-β populations with either protonation state of the carboxylate dyad. Positively Charged PsbO Residues. A large number of carboxylate side chains are observed at the surface of PsbO-β, namely, 9 Asp and 13 Glu side chains, most of which are closely spaced in clusters, as discussed above. In addition to these negatively charged groups, PsbO comprises 14 likely positively charged side chains (six Arg and eight Lys residues) and one possibly charged His side chain. In spite of the presence of numerous charged side chains, we detect only three wellresolved salt bridges (Figure S9), yet the properties of some carboxylate clusters are likely affected by hydrogen-bonded positively charged residues, as suggested by the cluster of three carboxylates and two lysine side chains shown in Figure 2C. It will be important in future investigations to address how the

■

DISCUSSION By truncation of the peripheral loops, which mediate binding of PsbO to PSII, a structurally well-defined and stable β-barrel protein is obtained. The high-resolution structures of the “free” PsbO β-barrel (PsbO-β) reveal a high degree of similarity to the PSII-bound PsbO with regard to protein backbone and side chain orientations; even some of the water molecules resolved at the protein surface are at the same positions in free and PSIIattached PsbO. Consequently, our PsbO construct and variants with modified surface-exposed residues could become useful as a model system for future high-resolution structural and indepth biophysical investigation of the clusters of protonatable groups at the protein−water interface of this important PSII protein. Several functions have been assigned to the PsbO of PSII (see the introductory section); some of these can be addressed by investigation of the isolated β-barrel part of the PsbO protein. We arrive at the following conclusions. (1) Approximately half of the 177 surface water molecules identified at 100 K were also resolved at 297 K, suggesting significant occupancy of specific water sites at room temperature, but loss of resolvable occupancy in others. This finding agrees well with MD simulations on hydrated PsbO at 300 K, which revealed that, on the time scale of tens of nanoseconds, there can be pronounced variations in the number and occupancy level of hydrogen-bonded water molecules connecting carboxylate side chains.27 For specific carboxylate pairs, preferred occupancies of one-water or two-water connections were detected, which is confirmed by the resolvability of a subset of surface water molecules in the 297 K structure. (2) Within a loop region present specifically in cyanobacterial PsbO (“cyano loop”), three amino acid residues and four 4632

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry

structural switching by reversible opening of the strong hydrogen bond between carboxylate side chains.

water ligands of a calcium ion of putative functional importance48,63,64 were particularly well ordered, even at 297 K; the ligation environment differs for manganese binding. In intact PSII, this metal-binding site could be involved in the delivery of manganese and calcium ions during assembly of the MnCa−oxo cluster at the water oxidation site, which is not directly accessible to the solvent phase. The plant PSII lacks this specific calcium-binding site, but other extrinsic PSII proteins absent in cyanobacteria may substitute for this functionality. A regulatory role for binding of calcium to the PsbO protein also has been proposed. We did not detect major changes upon comparison of PsbO structures with and without bound metal ions in the atomic-resolution structures or in MD simulations. However, this finding does not disprove a regulatory role for binding of calcium to the PsbO protein in the intact PSII. (3) Recent MD simulations identified carboxylate pairs that engage in high-occupancy bridges with water molecules.27 In the PsbO-β structure, we find water−carboxylate (Glu/Asp) clusters (that is, clusters of closely spaced Asp/Glu side chains bridged via hydrogen-bonding water), which may facilitate fast Grotthus-type proton transfer along the protein surface.29 When functioning as a proton emitting antenna, the cluster could promote the uphill transfer of a proton from a protonated protein side chain (pKa of ∼5) to a water molecule (formal pKa of −1.75).29 Such clusters could also represent proton storage sites that contribute to the buffer capacity of the lumenal space. The buffer capacity of the lumen is important, because overly severe acidification of the lumenal space (e.g., under fluctuating light conditions) reduces the quantum yield of the water oxidation reactions and favors the release of calcium from the Mn4Ca−oxo cluster and eventually its disintegration. Recent MD simulations identified carboxylate pairs that engage in highoccupancy bridges with water molecules.27 However, insight into the protonation state and titration behavior of the clusters of protonatable residues at the PsbO surface is required for definitive evaluation of their functionality. Future theoretical and experimental investigations along these lines are desirable. (4) In intact PSII, the first of the light-induced redox transitions of the MnCa−oxo cluster has been reported to associate with structural changes of the PsbO protein,13,22−24,65,66 but more specific details about the type, cause, and functional role of these structural changes are largely unknown. For isolated PsbO, evidence of pH-dependent and/ or calcium-dependent structural changes has been obtained.25,63,67,68 Our results suggest a possible pH-dependent structural switch mechanism in PsbO. Two carboxylate side chains, which are part of a structural motif interrupting two βstrands and connecting PsbO to PSII, are found to be directly linked via a strong hydrogen bond (2.5−2.8 Å) at pH 6 (100 K). This linkage was broken at pH 10 (100 K), resulting in a local structural change. At pH 6 and room temperature, both structures were present simultaneously in the protein crystal, pointing toward an apparent pK of the structural transition in the functionally relevant pH regime. MD simulations predict the same type of protonation-dependent local structure. In crystals of PsbO-β and within the ∼100−120 ns MD simulations presented here, we find that the structural changes are mostly local. This observation is compatible with the core of PsbO having a largely rigid β-barrel structure. Taken together, the results from crystallography and MD simulations presented here indicate an intriguing mechanism for pH-controlled

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00441. Detailed experimental procedures as well as Figures S1− S9 (PDF) Accession Codes

Crystallographic models and data for PsbO-β have been submitted to the PDB: 5G38 for 100 K and pH 6, 5G39 for 297 K and pH 6, and 5G3A for 100 K and pH 10.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Funding

This work was funded by the Deutsche Forschungsgemeinschaft (DFG) through the collaborative research center on ‘Protonation Dynamics in Protein Function’ (SFB1078), Projects A5-Zouni/Dobbek, C4-Bondar, and A4-Dau. A.-N.B. acknowledges funding from the Excellence Initiative of the German Federal and State Governments and an allocation of computing time from HLRN, the North-German Supercomputing Alliance (bec00063). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the members of the MX group at BESSY II (HZB, Berlin, Germany) and the P11 group at Petra III (DESY, Hamburg, Germany) for technical support, and we thank Chris Weise for provision of the MALDI-MS service at Freie Universität Berlin. We acknowledge access to beamlines of the BESSY II storage ring (Berlin, Germany) via the Joint Berlin MX-Laboratory sponsored by the Helmholtz Zentrum Berlin für Materialien und Energie, the Freie Universität Berlin, the Humboldt-Universität zu Berlin, the Max-DelbrückCentrum, and the Leibniz-Institut für Molekulare Pharmakologie; beam time at Petra III, P11, was provided by the Deutsche Elektronen-Synchrotron (Hamburg, Germany).

■

ABBREVIATIONS MD, molecular dynamics; PSII, photosystem II; PsbO, manganese-stabilizing protein or 33 kDa extrinsic protein, a subunit of PSII; PsbO-β, protein construct containing only the β-barrel domain of PsbO; rmsd, root-mean-square distance of (Cα) coordinates; SC4−SC7, carboxylate clusters at the surface of PsbO; PDB, Protein Data Bank.

■

REFERENCES

(1) Blankenship, R. E. (2002) Molecular mechanisms of photosynthesis, Blackwell Science, Oxford, England. (2) Renger, G., and Renger, T. (2008) Photosystem II: The machinery of photosynthetic water splitting. Photosynth. Res. 98, 53− 80. (3) Barber, J. (2009) Photosynthetic energy conversion: natural and artificial. Chem. Soc. Rev. 38, 185−196. 4633

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry (4) Williamson, A., Conlan, B., Hillier, W., and Wydrzynski, T. (2011) The evolution of Photosystem II: insights into the past and future. Photosynth. Res. 107, 71−86. (5) Zouni, A., Witt, H. T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature 409, 739−743. (6) Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J., and Iwata, S. (2004) Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831−1838. (7) Umena, Y., Kawakami, K., Shen, J.-R., and Kamiya, N. (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473, 55−60. (8) Hellmich, J., Bommer, M., Burkhardt, A., Ibrahim, M., Kern, J., Meents, A., Müh, F., Dobbek, H., and Zouni, A. (2014) Native-like Photosystem II Superstructure at 2.44 Å Resolution through Detergent Extraction from the Protein Crystal. Structure 22, 1607− 1615. (9) Ago, H., Adachi, H., Umena, Y., Tashiro, T., Kawakami, K., Kamiya, N., Tian, L., Han, G., Kuang, T., Liu, Z., Wang, F., Zou, H., Enami, I., Miyano, M., and Shen, J. R. (2016) Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga. J. Biol. Chem. 291, 5676−5687. (10) De Las Rivas, J., and Barber, J. (2004) Analysis of the Structure of the PsbO Protein and its Implications. Photosynth. Res. 81, 329−343. (11) Roose, J. L., Wegener, K. M., and Pakrasi, H. B. (2007) The extrinsic proteins of Photosystem II. Photosynth. Res. 92, 369−387. (12) Williamson, A. K. (2008) Structural and functional aspects of the MSP (PsbO) and study of its differences in thermophilic versus mesophilic organisms. Photosynth. Res. 98, 365−389. (13) Ifuku, K., and Noguchi, T. (2016) Structural Coupling of Extrinsic Proteins with the Oxygen-Evolving Center in Photosystem II. Front. Plant Sci. 7, 84. (14) Bricker, T. M. (1992) Oxygen evolution in the absence of the 33-kilodalton manganese-stabilizing protein. Biochemistry 31, 4623− 4628. (15) Popelkova, H., and Yocum, C. F. (2011) PsbO, the manganesestabilizing protein: analysis of the structure-function relations that provide insights into its role in photosystem II. J. Photochem. Photobiol., B 104, 179−190. (16) Ifuku, K. (2015) Localization and functional characterization of the extrinsic subunits of photosystem II: an update. Biosci., Biotechnol., Biochem. 79, 1223−1231. (17) Roose, J. L., Frankel, L. K., Mummadisetti, M. P., and Bricker, T. M. (2016) The extrinsic proteins of photosystem II: update. Planta 243, 889−908. (18) Wei, X., Su, X., Cao, P., Liu, X., Chang, W., Li, M., Zhang, X., and Liu, Z. (2016) Structure of spinach photosystem II−LHCII supercomplex at 3.2 Å resolution. Nature 534, 69−74. (19) Duchoslav, M., and Fischer, L. (2015) Parallel subfunctionalisation of PsbO protein isoforms in angiosperms revealed by phylogenetic analysis and mapping of sequence variability onto protein structure. BMC Plant Biol. 15, 133. (20) Miqyass, M., van Gorkom, H. J., and Yocum, C. F. (2007) The PSII calcium site revisited. Photosynth. Res. 92, 275−287. (21) Popelkova, H., Boswell, N., and Yocum, C. (2011) Probing the topography of the photosystem II oxygen evolving complex: PsbO is required for efficient calcium protection of the manganese cluster against dark-inhibition by an artificial reductant. Photosynth. Res. 110, 111−121. (22) Hutchison, R. S., Steenhuis, J. J., Yocum, C. F., Razeghifard, M. R., and Barry, B. A. (1999) Deprotonation of the 33-kDa, extrinsic, manganese-stabilizing subunit accompanies photooxidation of manganese in photosystem II. J. Biol. Chem. 274, 31987−31995. (23) Sachs, R. K., Halverson, K. M., and Barry, B. A. (2003) Specific isotopic labeling and photooxidation-linked structural changes in the manganese-stabilizing subunit of photosystem II. J. Biol. Chem. 278, 44222−44229. (24) Nagao, R., Tomo, T., and Noguchi, T. (2015) Effects of extrinsic proteins on the protein conformation of the oxygen-evolving

center in cyanobacterial photosystem II as revealed by Fourier transform infrared spectroscopy. Biochemistry 54, 2022−2031. (25) Shutova, T., Klimov, V. V., Andersson, B., and Samuelsson, G. (2007) A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II. Biochim. Biophys. Acta, Bioenerg. 1767, 434−440. (26) Bondar, A. N., and Dau, H. (2012) Extended protein/water Hbond networks in photosynthetic water oxidation. Biochim. Biophys. Acta, Bioenerg. 1817, 1177−1190. (27) Lorch, S., Capponi, S., Pieront, F., and Bondar, A.-N. (2015) Dynamic Carboxylate/Water Networks on the Surface of the PsbO Subunit of Photosystem II. J. Phys. Chem. B 119, 12172−12181. (28) Adelroth, P., and Brzezinski, P. (2004) Surface-mediated proton-transfer reactions in membrane-bound proteins. Biochim. Biophys. Acta, Bioenerg. 1655, 102−115. (29) Wraight, C. A. (2006) Chance and design–proton transfer in water, channels and bioenergetic proteins. Biochim. Biophys. Acta, Bioenerg. 1757, 886−912. (30) Burnap, R. L., and Sherman, L. A. (1991) Deletion mutagenesis in Synechocystis sp. PCC6803 indicates that the Mn-stabilizing protein of photosystem II is not essential for O2 evolution. Biochemistry 30, 440−446. (31) Nowaczyk, M., Berghaus, C., Stoll, R., and Roegner, M. (2004) Preliminary structural characterisation of the 33 kDa protein (PsbO) in solution studied by site-directed mutagenesis and NMR spectroscopy. Phys. Chem. Chem. Phys. 6, 4878−4881. (32) Nikitina, J., Shutova, T., Melnik, B., Chernyshov, S., Marchenkov, V., Semisotnov, G., Klimov, V., and Samuelsson, G. (2008) Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli. Photosynth. Res. 98, 391−403. (33) Seidler, A., and Michel, H. (1990) Expression in Escherichia coli of the psbO gene encoding the 33 kd protein of the oxygen-evolving complex from spinach. EMBO J. 9, 1743−1748. (34) Slowik, D., Rossmann, M., Konarev, P. V., Irrgang, K.-D., and Saenger, W. (2011) Structural investigation of PsbO from plant and cyanobacterial photosystem II. J. Mol. Biol. 407, 125−137. (35) Williamson, A. K., Liggins, J. R., Hillier, W., and Wydrzynski, T. (2007) The importance of protein-protein interactions for optimizing oxygen activity in photosystem II: reconstitution with a recombinant thioredoxin–manganese stabilising protein. Photosynth. Res. 92, 305− 314. (36) Wyman, A. J., Popelkova, H., and Yocum, C. F. (2008) Sitedirected mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties. Biochemistry 47, 6490− 6498. (37) Roberts, I. N., Lam, X. T., Miranda, H., Kieselbach, T., and Funk, C. (2012) Degradation of PsbO by the Deg protease HhoA Is thioredoxin dependent. PLoS One 7, e45713. (38) McCoy, J., and La Ville, E. (1997) Expression and purification of thioredoxin fusion proteins. Current Protocols in Protein Science, Chapter 6, Unit 6.7, p 6.7.1, Wiley, New York.10.1002/ 0471140864.ps0607s10 (39) Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kalé, L., and Schulten, K. (2005) Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781− 1802. (40) Kalé, L., Skeel, R., Bhandarkar, M., Brunner, R., Gursoy, A., Krawetz, N., Phillips, J., Shinozaki, A., Varadarajan, K., and Schulten, K. (1999) NAMD2: Greater Scalability for Parallel Molecular Dynamics. J. Comput. Phys. 151, 283−312. (41) Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., and Karplus, M. (1983) CHARMM: A program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187−217. (42) MacKerell, A. D., Bashford, D., Bellott, M., Dunbrack, R. L., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T., Mattos, C., 4634

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635

Article

Biochemistry

protonation states in proteins. Acta Crystallogr., Sect. D: Biol. Crystallogr. 63, 906−922. (60) Kawakami, K., Umena, Y., Kamiya, N., and Shen, J.-R. (2011) Structure of the catalytic, inorganic core of oxygen-evolving photosystem II at 1.9 Å resolution. J. Photochem. Photobiol., B 104, 9−18. (61) De Las Rivas, J., and Barber, J. (2004) Analysis of the Structure of the PsbO Protein and its Implications. Photosynth. Res. 81, 329−343. (62) Folea, I. M., Zhang, P., Aro, E.-M., and Boekema, E. J. (2008) Domain organization of photosystem II in membranes of the cyanobacterium Synechocystis PCC6803 investigated by electron microscopy. FEBS Lett. 582, 1749−1754. (63) Heredia, P., and De Las Rivas, J. (2003) Calcium-dependent conformational change and thermal stability of the isolated PsbO protein detected by FTIR spectroscopy. Biochemistry 42, 11831− 11838. (64) Kruk, J., Burda, K., Jemiola-Rzeminska, M., and Strzalka, K. (2003) The 33 kDa protein of photosystem II is a low-affinity calciumand lanthanide-binding protein. Biochemistry 42, 14862−14867. (65) Offenbacher, A. R., Polander, B. C., and Barry, B. A. (2013) An intrinsically disordered photosystem II subunit, PsbO, provides a structural template and a sensor of the hydrogen-bonding network in photosynthetic water oxidation. J. Biol. Chem. 288, 29056−29068. (66) Hong, S. K., Pawlikowski, S. A., Vander Meulen, K. A., and Yocum, C. F. (2001) The oxidation state of the photosystem II manganese cluster influences the structure of manganese stabilizing protein. Biochim. Biophys. Acta, Bioenerg. 1504, 262−274. (67) Shutova, T., Irrgang, K. D., Shubin, V., Klimov, V. V., and Renger, G. (1997) Analysis of pH-induced structural changes of the isolated extrinsic 33 kDa protein of photosystem II. Biochemistry 36, 6350−6358. (68) Shutova, T., Nikitina, J., Deikus, G., Andersson, B., Klimov, V., and Samuelsson, G. (2005) Structural dynamics of the manganesestabilizing protein-effect of pH, calcium, and manganese. Biochemistry 44, 15182−15192. (69) Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: Visual molecular dynamics. J. Mol. Graphics 14, 33−38.

Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, W. E., Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiórkiewicz-Kuczera, J., Yin, D., and Karplus, M. (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586−3616. (43) Mackerell, A. D., Feig, M., and Brooks, C. L. (2004) Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 25, 1400−1415. (44) Mackerell, A. D. (2004) Empirical force fields for biological macromolecules: overview and issues. J. Comput. Chem. 25, 1584− 1604. (45) Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., and Klein, M. L. (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926−935. (46) Bondar, A.-N., and Dau, H. (2012) Extended protein/water Hbond networks in photosynthetic water oxidation. Biochim. Biophys. Acta, Bioenerg. 1817, 1177−1190. (47) Tohri, A., Suzuki, T., Okuyama, S., Kamino, K., Motoki, A., Hirano, M., Ohta, H., Shen, J. R., Yamamoto, Y., and Enami, I. (2002) Comparison of the structure of the extrinsic 33 kDa protein from different organisms. Plant Cell Physiol. 43, 429−439. (48) Loll, B., Gerold, G., Slowik, D., Voelter, W., Jung, C., Saenger, W., and Irrgang, K. D. (2005) Thermostability and Ca2+ binding properties of wild type and heterologously expressed PsbO protein from cyanobacterial photosystem II. Biochemistry 44, 4691−4698. (49) Griffiths-Jones, S. R., Maynard, A. J., and Searle, M. S. (1999) Dissecting the stability of a beta-hairpin peptide that folds in water: NMR and molecular dynamics analysis of the beta-turn and betastrand contributions to folding. J. Mol. Biol. 292, 1051−1069. (50) Sibanda, B. L., Blundell, T. L., and Thornton, J. M. (1989) Conformation of beta-hairpins in protein structures. A systematic classification with applications to modelling by homology, electron density fitting and protein engineering. J. Mol. Biol. 206, 759−777. (51) Simpson, E. R., Meldrum, J. K., Bofill, R., Crespo, M. D., Holmes, E., and Searle, M. S. (2005) Engineering enhanced protein stability through beta-turn optimization: insights for the design of stable peptide beta-hairpin systems. Angew. Chem., Int. Ed. 44, 4939− 4944. (52) Svensson, B., Tiede, D. M., Nelson, D. R., and Barry, B. A. (2004) Structural Studies of the Manganese Stabilizing Subunit in Photosystem II. Biophys. J. 86, 1807−1812. (53) Fraser, J. S., van den Bedem, H., Samelson, A. J., Lang, P. T., Holton, J. M., Echols, N., and Alber, T. (2011) Accessing protein conformational ensembles using room-temperature X-ray crystallography. Proc. Natl. Acad. Sci. U. S. A. 108, 16247−52. (54) Afonine, P. V., Grosse-Kunstleve, R. W., Adams, P. D., and Urzhumtsev, A. (2013) Bulk-solvent and overall scaling revisited: faster calculations, improved results. Acta Crystallogr., Sect. D: Biol. Crystallogr. 69, 625−634. (55) de Grotthuss, C. J. T. (2006) Memoir on the decomposition of water and of the bodies that it holds in solution by means of galvanic electricity. Biochim. Biophys. Acta, Bioenerg. 1757, 871−875 [English translation of the original publication: de Grotthuss, C. J. T. (1806) Sur la decomposition de l’eau et des corps qu’elle tient en dissolution a l’aide de l’electricite galvanique. Ann. Chim. 58, 54−74]. (56) Agmon, N. (1995) The Grotthuss mechanism. Chem. Phys. Lett. 244, 456−462. (57) Wraight, C. A. (2006) Chance and designProton transfer in water, channels and bioenergetic proteins. Biochim. Biophys. Acta, Bioenerg. 1757, 886−912. (58) Suga, M., Akita, F., Hirata, K., Ueno, G., Murakami, H., Nakajima, Y., Shimizu, T., Yamashita, K., Yamamoto, M., Ago, H., and Shen, J.-R. (2014) Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses. Nature 517, 99−103. (59) Ahmed, H. U., Blakeley, M. P., Cianci, M., Cruickshank, D. W. J., Hubbard, J. A., and Helliwell, J. R. (2007) The determination of 4635

DOI: 10.1021/acs.biochem.6b00441 Biochemistry 2016, 55, 4626−4635