Author response: The structure of plant photosystem I super-complex at 2.8 Å resolution

Full text Figures and data Side by side Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Most life forms on Earth are supported by solar energy harnessed by oxygenic photosynthesis. In eukaryotes, photosynthesis is achieved by large membrane-embedded super-complexes, containing reaction centers and connected antennae. Here, we report the structure of the higher plant PSI-LHCI super-complex determined at 2.8 Å resolution. The structure includes 16 subunits and more than 200 prosthetic groups, which are mostly light harvesting pigments. The complete structures of the four LhcA subunits of LHCI include 52 chlorophyll a and 9 chlorophyll b molecules, as well as 10 carotenoids and 4 lipids. The structure of PSI-LHCI includes detailed protein pigments and pigment–pigment interactions, essential for the mechanism of excitation energy transfer and its modulation in one of nature's most efficient photochemical machines. https://doi.org/10.7554/eLife.07433.001 eLife digest Most plants, green algae and some bacteria use a process called photosynthesis to convert energy from sunlight into the chemical energy they need to survive and grow. With this energy, these organisms use carbon dioxide and water to create organic matter and release oxygen into the atmosphere. Therefore, photosynthesis plays a major role in providing the basis for life on earth. During photosynthesis, molecules of pigments known as chlorophyll and carotenoid capture the light energy. These pigments are contained within large groups (or 'complexes') of proteins that sit in membrane structures within cells. Two of the protein complexes—called photosystem I and LHCI—interact with each other to form a 'supercomplex' that transfers energy to a small protein called ferredoxin. To achieve this, the light energy captured by pigment molecules is transferred to other pigment molecules so that the energy is funneled towards the center of photosystem I. Mazor et al. used a technique called X-ray crystallography to create a very detailed three-dimensional model of photosystem I and LHCI from pea plants. The model shows how the twelve proteins of photosystem I are arranged in relation to the four proteins of the LHCI complex. The super-complex contains more than 200 other molecules, which are mostly chlorophylls and carotenoids. Of these, 61 chlorophyll molecules and ten carotenoid molecules are found in LHCI. The model also provides detailed information about how the pigments interact with each other and with the proteins in the supercomplex. Mazor et al.'s detailed model may help us to understand how these interactions allow photosystem I to harvest light energy with almost 100% efficiency, and aid efforts to develop new technologies that harness light. https://doi.org/10.7554/eLife.07433.002 Introduction Oxygenic photosynthesis, in which the conversion of sunlight into chemical energy by plants, green algae, and cyanobacteria, occurs, underpins the survival of virtually all life forms. By producing oxygen and assimilating carbon dioxide into organic matter, this process determines, to a large extent, the composition of our atmosphere and provides essential food and fuel. Light photons are captured by pigments in very large membrane–bound complexes and the excitation energy is utilized to form NADPH and ATP. Two reaction centers, photosystem II (PSII) and photosystem I (PSI), drive this electron transport chain. PSII oxidizes water to produce oxygen and reduce membrane-embedded quinones. The reduced quinones are then utilized by the cytochrome b6f complex to produce a proton gradient across the membrane and to reduce the small copper protein plastocyanin (PC), the electron donor of PSI. After an additional photon is absorbed by any of the 200 antenna pigments of PSI, its energy migrates through this large network of connected pigments and eventually oxidizes P700, a special chlorophyll pair located at the center of PSI. The electron removed from P700 by this oxidation event migrates along an internal electron transport chain and finally reduces ferredoxin (Fd), the final electron acceptor of PSI. Reduced Fd is utilized by several cellular pathways, mainly the reduction of NADP to NADPH. This NADPH and ATP generated by the thylakoid ATP-synthase complex powers the Calvin cycle to produce carbohydrates. Oxygenic photosynthesis evolved over 3 billion years ago in cyanobacteria (Blankenship, 1992; Barber, 2004; Nelson, 2013). Later, approximately 1.5 billion years ago, the first photosynthetic eukaryotes appeared, eventually evolving into land plants roughly 0.5 billion years ago. The basic building blocks of photosynthesis are remarkably conserved. The architectures of both photosystems have been determined by numerous techniques, but X-ray crystallography has provided the most detailed structural information for the four large membrane complexes catalyzing oxygenic photosynthesis (Nelson and Ben-Shem, 2004; Nelson and Yocum, 2006; Croce and van Amerongen, 2013; Nelson and Junge, 2015). Structures at the highest resolution has been obtained for thermophilic cyanobacteria, but representative structures from eukaryotic chloroplasts, especially plants, are scarce (Jordan et al., 2001; Ben-Shem et al., 2003; Kurisu et al., 2003; Stroebel et al., 2003; Ferreira et al., 2004; Loll et al., 2005; Amunts et al., 2007, 2010; Umena et al., 2011). Here, we report the structure of plant PSI super-complex at high resolution. Results and discussion Structure determination The crystal structure of plant PSI was first reported at 4.4 Å resolution (Ben-Shem et al., 2003) and has been improved up to 3.3 Å resolution in the last decade (PDB 2WSC). This PSI preparation was limited to pea plants from the variety Alaska, and good crystals were hard to come by (Amunts et al., 2007, 2010). Therefore, we screened for new robust crystals that are abundant, stable, and much more uniform. The new crystals could be obtained from several pea plants varieties, a large proportion of them diffracted to 3 Å with several yielding higher resolutions. In contrast to the P21 symmetry of the previous crystal, the current crystal belonged to higher symmetry space group P212121. The organization of the PSI unit within the new crystal was also markedly different. In the P21 crystal the PSI-LHCI complex was organized as parallel layers in which the iron-sulfur clusters FX, FA and FB face the adjacent P700 (Figure 1A). The complexes inside the new crystal lattice were serially arranged in a crissed-crossed manner in which the polarity of each PSI unit contrasts another (Figure 1B). Consequently, the current crystals generated no net voltage (data not shown), whereas a voltage of up to 50 V was recorded (Toporik et al., 2012) upon illumination of dried P21 crystals placed on electron conductive material. Figure 1 Download asset Open asset Comparison of two PSI-LHCI crystal lattices. (A) The previous PSI-LHCI crystal in the P21 space group with a layered arrangement of the complex. This arrangement is capable of generating extremely high voltages upon illumination. (B) The new crystal lattice in the P212121 space group. Iron sulfur clusters are colored in red and the pigments of the internal electron transport chain in magenta (chlorophylls) and blue (quinones). PSI-LHCI complexes are arranged in a crissed-crossed manner from left to right. https://doi.org/10.7554/eLife.07433.003 The extreme size and complexity of the PSI assembly was a major obstacle for accurate and bias-free modeling. The best way to eliminate model bias in X-ray crystallography is to utilize experimentally measured phase information. Using the new, highly stable crystal form of PSI we were able to measure the weak native anomalous signal from the iron, sulfur, and phosphate atoms in the complex. Starting with a minimal model containing only the three natively bound iron-sulfur clusters, the entire structure was eventually re-built with more than 35,000 atoms (Figure 2 and Figure 2—figure supplement 1, see 'Material and methods' section for details). Figure 2 with 1 supplement see all Download asset Open asset Overall structure and organization of the plant PSI-LHCI supercomplex. (A) A view from the stromal side of the membrane of PSI-LHCI with the PsaL subunit pointing up. The PsaF and PsaJ subunits connecting in the middle of LHCI are colored in magenta and green, respectively. The three subunits of the stromal ridge, PsaC, PsaD, and PsaE, can be seen in the middle of the complex, colored cyan, pink, and blue, respectively. The two iron-sulfur clusters of PsaC can be distinguished as yellow and orange clusters in the middle of the complex. (B) Pigment organization in PSI-LHCI. The central pigments of the internal electron transport chain are colored red, chlorophylls of the core antenna green, chlorophyll a in LHCI in cyan, and chlorophyll b in magenta. Carotenoids, which are distributed throughout the complex, are colored in blue and lipids in key connecting points and conserved positions in the core, in orange. https://doi.org/10.7554/eLife.07433.004 Core subunits: PC binding site and implications for the state II PSI complex The structure of plant PSI includes 12 core subunits bound with four light-harvesting proteins comprising the LHCI antenna complex. The entire complex contains 214 prosthetic groups, including 156 chlorophylls (nine assigned as chlorophyll b), 32 carotenes, and 14 lipids, many of them located at key contact points of the complex. The core photosynthetic reaction centers have remained virtually unchanged over the entire 2 billion years of their evolution (Jordan et al., 2001; Ben-Shem et al., 2003; Amunts et al., 2010). Instead, the evolution of PSI is marked by the loss and gain of whole subunits from the complex (Scheller et al., 2001; Nelson, 2011; Nelson and Junge, 2015). Compared to our previous model (PDB 2WSC), the root-mean-square deviation (rmsd) between the plant and cyanobacterial core (PDB 1JB0) decreased from 1.1 Å to 0.55 Å. The majority of the changes made in the core subunits involved the configuration of extramembrane loops, which now closely resemble the cyanobacterial configuration. The exceptions to this role are found at the anchor points of LHCI to the core (discussed below) and at the interfaces between plant-specific subunits, such as the PsaH–PsaL interaction (Figure 3A). The dramatic change from trimer to monomeric organization that occurred in eukaryotes, was triggered by the addition of the PsaH subunit (Ben-Shem et al., 2003). A new configuration for PsaH shows that this subunit binds four other core subunits. Starting from the stromal side of the membrane, the N-terminus is tightly tucked between the N-terminus of PsaD and a eukaryotic-specific loop in the PsaL subunit. PsaH then enters the membrane surrounding PsaL to prevent PSI trimerization and associates with PsaI and PsaB via mostly hydrophobic interactions (Figure 3A). Figure 3 with 1 supplement see all Download asset Open asset PsaH connects the state II and the PC binding sites. (A) PsaL is presented in blue, and the path of the PsaH subunit (red) travels through its extended, eukaryotic specific, loop. The new PsaH chlorophyll (green, marked as H1) connects to the PsaL-coordinated chlorophyll and carotenoid (green and pink), as well as an additional chlorophyll trimer (middle left, marked as PsaA trimer), which is proposed to connect PSI to LHCII in state II. (B) A luminal view of the PSI surface at the PC binding site. The two hydrophobic helices are colored in grey (PsaA) and light blue (PsaB). The luminal PsaA loop is highlighted on the background of the entire PsaA subunit. All the ligands (with the exception of P700) are not shown. (C and D) Side by side view of the cyanobacterial (C) and plant (D) PC binding sites showing the extended PsaF helices that limit access from the membrane plane and the N terminus of PsaH (red), which parallels the configuration of the PsaA luminal loop. The approximate location of PC is indicated in blue. https://doi.org/10.7554/eLife.07433.006 Eukaryotes can modulate the distribution of excitation energy transfer between their two photosystems via a mechanism called state transitions (Lunde et al., 2000; Bellafiore et al., 2005; Rochaix, 2011; Rochaix et al., 2012). Under state II conditions, PSI associates with a mobile pool of the LHCII antennae, which increases its absorbance cross-section (Kargul et al., 2005; de Bianchi et al., 2010). Genetic studies suggest that PsaH, PsaL, and PsaK play important roles in this process (Scheller et al., 2001; Zhang and Scheller, 2004). Electron microscopy studies have identified the binding site of the additional antennae complexes along the PsaL/PsaH-PsaK side (Kargul et al., 2005; Kouril et al., 2005). A new chlorophyll bound by PsaH was identified at the current resolution. This new chlorophyll, together with pigments bound by PsaL, probably participates in energy transfer into the core (Figure 3A), suggesting that PsaH is not simply a 'landing pad' for LHCII, but is also important for energy transfer into the core during state II. Additional pigments bound by PsaA in close proximity to subunit PsaK (the structure of which is now almost completely defined) provided the first accurate description of this binding site and suggest a mechanism for energy transfer into the core antenna through the PsaK side (Figure 3—figure supplement 1). On the luminal side of the membrane, the new position of the N-terminus of PsaH suggests that this subunit also directly contributes to PC binding. One of the distinguishing characteristics of the eukaryotic PSI is the stable complex it forms with its electron donor PC, which results in a thousand-fold acceleration of the electron transfer rate (Bottin and Mathis, 1985). Three elements make up the PC binding site: the first is a positive patch located along the helix-turn-helix N-terminal domain of PsaF (Hippler et al., 1997, 1998; Ben-Shem et al., 2003). The second element is a hydrophobic patch composed of two parallel helices in PsaA and PsaB (Figure 2B) (Sommer et al., 2006; Kuhlgert et al., 2012). The third conserved feature of the PC binding site is a PsaA luminal loop protruding from the generally flat luminal surface of PSI. This loop is found in both plants and cyanobacteria PSI; the only exception being sequences from marine Prochlorococcus and their phages (Mazor et al., 2012). As seen in Figure 3C,D, the binding site of the cyanobacterial and plant complexes are similar. However, it is clear that the plant binding site is buried deeper in the complex, this is achieved by the extension of the PsaF N-terminal and by the new position of the N-terminus of PsaH, which forms a loop mirroring the conformation of the conserved luminal PsaA loop, suggesting a direct role for PsaH in PC binding. Core antenna, red pigments, and excitation energy transfer The PSI core is a highly efficient hub onto which diverse antennae systems connect such as phycobilisomes and IsiA-like assemblies in cyanobacteria and red algae and LHC type antennae in eukaryotes (Berera et al., 2009; Engelken et al., 2010; Nelson and Junge, 2015; Wahadoszamen et al., 2015). Remarkably, the core pigment organization is conserved across kingdoms despite this diversity in connected antenna (Amunts et al., 2007; Busch and Hippler, 2011; Croce and van Amerongen, 2013), which suggests that the connection points between the core and the antennae are conserved. The existence of red-absorbing pigments (or 'red traps') is a general property of PSI (Morosinotto et al., 2005; Wientjes et al., 2012). These pigments affect the rate of trapping in PSI and can affect the path of excitation migration in the complex. Most of the eukaryotic red pigments have been shown to reside at LHCI. However, red pigments may be lost from the core complex during the isolation of LHCI. The first high-resolution PSI structure from thermophilic cyanobacteria revealed the organization of the core antenna (Jordan et al., 2001). A stacked chlorophyll trimer supported by an extended loop in PsaB was the best candidate for one of the strong red absorbers in this complex (Jordan et al., 2001). PsaB sequences from eukaryotes and many cyanobacteria lack this extended loop, resulting in this chlorophyll trimer being lost, as has been shown in the plant and mesophilic PSI structures (Amunts et al., 2010; Mazor et al., 2014). At the current resolution, we observed new core chlorophyll bound between PsaG and Lhca1 and a newly discovered lipid (Figure 4A). This new chlorophyll restores the stacked chlorophyll trimer independent of the shortened PsaB loop and is responsible for one of the connection points between the core complex and the LHCI antenna, with a Mg–Mg distance of 12.5 Å between it and chlorophyll 1010 in Lhca1 (the nomenclature for LHCII is used to describe Lhcas [Standfuss et al., 2005]). On the stromal side of the membrane, an additional chlorophyll trimer first discovered in Synechocystis is also responsible for an antenna attachment point with a Mg–Mg distance of 13.7 Å between the core chlorophyll A40 and chlorophyll 1005 in Lhca1 (Figure 4—figure supplement 1). We suggest that chlorophyll trimers located at the periphery of the core antenna are extremely important for antenna attachment and are probably general attachment points to the core that are utilized not only by eukaryotes, but also by other antenna systems in cyanobacteria. Figure 4 with 2 supplements see all Download asset Open asset Antenna connections in PSI-LHCI. (A) The configuration of the PsaG-Lhca1 pigment connection shown from the luminal side of the membrane. The new stacked chlorophyll trimer (numbered B1231, B1232 and G1003) is shown. The N-terminus of PsaG (dark red) supports one of the chlorophylls making up this trimer. The entire trimer is connected with chlorophyll 10 (numbered 1010) in Lhca1 (blue). (B) The second LHCI-PSI connection between Lhca1 and PsaF (magenta) on the luminal side of the membrane is bound by a lipid (orange). (C) The Lhca3 (red) -PsaA connection. Two chlorophyll pairs mediate this interaction. At the lumen face, 13.7 Å separate chlorophyll 3010 from chlorophyll 1114. On the stromal side, chlorophyll 3005 and chlorophyll 1108 are 16.5 Å apart. (D) Lhca2 (blue)—PsaJ (green) connecting chlorophylls. https://doi.org/10.7554/eLife.07433.008 In contrast to the previous plant structures, which included a small pool of 'Gap chlorophylls', only six pigment pairs connect LHCI to the core antenna in the current structure. Lhca1 is the main connector for excitation transfer, harboring three chlorophylls that are within 14 Å of reaction center pigments (Figure 4A,B and Figure 4—figure supplement 1). This close proximity ensures efficient and fast energy transfer. Surprisingly, Lhca3 is also one of the main connection points with two such pairs (Figure 4C). The final excitonic connection between PSI to LHCI is located between chlorophyll J1302 bound by PsaJ and chlorophyll 2010 (A chlorophyll b molecule). The Mg–Mg distance of this pair is quite large (17.6 Å) however, since the gap between LHCI and PSI for some this distance can change to an efficient between the core and LHCI (Figure To the extremely fast and efficient energy transfer that PSI-LHCI through only six pairs of pigment molecules located at three sites. These sites connect to the core antennae at the PsaG and PsaK through Lhca1 and with Lhca2 a The structure of the LHCI complex structure includes the LHCI with complete structures of all four These structures the essential of the interactions each protein and the the red chlorophyll assembly in and and a pigment binding which is the site for the discovered at the luminal gap of LHCI Bianchi et al., 2010; et al., 2014). Overall view The LHCI is located on the PsaF side of the PSI On the stromal face of the membrane, the four conserved N-terminal connect each subunit to its through interaction with an loop (Figure that the second and supports a new chlorophyll site (numbered in and Lhca2 (Figure which with PsaG in the core complex, completely this loop and contains a in this (Figure One of the major changes in the structure of LHCI is the in the polarity of PsaG roughly an position to previous PSI-LHCI structures, the first Lhca1 the second one PsaB (Figure 4—figure supplement between each and the core are by small the first and are by between at the membrane point and a conserved located at a them (Figure Additional interactions are provided by a on the luminal side that binds 2 as it the membrane, mainly via hydrophobic interactions (Figure supplement 1). Figure with 1 supplement see all Download asset Open asset Lhcas connect to the PSI core through conserved structural (A) A view from the stromal side of the membrane of LHCI. connects to the one through its conserved N-terminal domain All connections to the core are by the the of the first into the membrane by a The extended loop is conserved in all Lhcas (B) interactions the of the binding site to The conserved interaction within the PsaF (magenta) which is and (C) of Lhca3 (red) and LHCII Lhca3 with the core via with PsaA and PsaK through extension (green in the highly conserved N The extension of loop is also seen at the left of the Figure with 1 supplement see all Download asset Open asset The within the provides a mechanism for (A and Structure of the (A) and (B) as from the membrane plane to the PSI-LHCI complex. The conserved connecting carotenoid in position a structural connecting the two subunits of the by chlorophyll to chlorophyll 9 left The position of the new chlorophyll site bound by loop of Lhca2 (numbered is also shown. (C and D) A new chlorophyll site connects the pigments of the (numbered is shown as from the luminal side of the membrane. The connections between the red pigment on the and position 9 in the adjacent subunit are marked with grey The new carotenoid is shown in This site the gap between LHCI and PSI and is The distance between and of chlorophylls systems is Å. Lhca3 is colored in A from the first is shown as 3005 is of the red-absorbing LHCII was on the structure and shown in is shown as a The side chain was from in LHCII to in important change is the to at position which chlorophyll 3005 to its by approximately The site in shown in a with colored in The basic is the is of the position Genetic in plants has revealed that each subunit has a binding site with the exception of the the Lhcas are not et al., 2006; Wientjes et al., The binding sites are identified in the current structure. At the PsaG of of Lhca1 with the first of Additional interactions occurred between a stromal loop of PsaB and the N-terminus of Lhca1 (Figure The contact point between LHCI and the core between the N-terminus of and the of the conformation of which is almost to the conformation found in cyanobacteria. This interaction of hydrophobic surrounding in the of both This binding site be between and and of PsaF and of form interactions in the middle of the hydrophobic binding site (Figure This is conserved in as but from the of the interaction. The interaction in the current structure not include any pigments. transfer from to the core probably through the pigment the Lhca2 is connected to the core almost through interactions with the N-terminus of PsaJ on the stromal side of the membrane (Figure supplement 1). The main contact point of Lhca3 is in the N-terminus of which a small patch 1 enters the membrane as in all other Lhcas (Figure The structure shows that this site is the main of binding to the In contrast to the previous PSI-LHCI Lhca3 the general of LHCII Å between the two from the LHCII are seen in key contact points small were extended from the conserved N-terminus domain to interactions with the core (Figure transfer and in All four Lhcas are remarkably to each other and to Most of the between them can be by their interaction such as the loss of loop in Lhca1 to its binding to Two key in pigment organization were found between and other The extended loop supports a new site (numbered to and Lhca2 (Figure and an additional chlorophyll by connects the two of each b pigments are bound by the proteins to and as antennae pigments. binding sites for chlorophyll b were in the high resolution structure of LHCII et al., 2004; et al., as well as in the structure of et al., 2011). We were able to chlorophyll b sites in LHCI on electron (Figure supplement 1). All proteins a to change to in positions to position of LHCII, this change the binding of b at three sites 12 and In with this change we that site 12 is by chlorophyll a in all Lhcas and sites 10 and are The distribution of chlorophyll b sites in LHCI is markedly with three sites located in Lhca2 and 2013), two sites found at Lhca1 and three sites at and and a site on Lhca3 These are with data which identified more chlorophyll b sites on Lhca2 then on Lhca3 et al., 2003). on 2 and 4 is probably a which can both chlorophyll a and chlorophyll The high of chlorophyll b pigments in Lhca2 and the that its connection to the core is by chlorophyll b shown in Figure is with our that Lhca1 and Lhca3 are the main for excitation energy transfer from LHCI to the PSI The of all but two chlorophylls were identified in the current structure. The most changes in LHCI to LHCII in two and are a is to the energy transfer within these LHCI subunits but the of this change be from the of the two contains a and red-absorbing subunit. The