bioRxiv preprint doi: https://doi.org/10.1101/2021.05.30.446358; this version posted May 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Cryo-EM Structure of the Photosynthetic LH1-RC Complex from

K. Tani1*, R. Kanno2, X.-C. Ji3, M. Hall2, L.-J. Yu4, Y. K im ura 5, M. T. Madigan6, A. Mizoguchi1, B. M. Humbel2 & Z.-Y. Wang-Otomo3*

1 Graduate School of Medicine, Mie University, Tsu 514-8507, Japan 2 Imaging Section, Research Support Division, Okinawa Institute of Science and Technology Graduate University (OIST), 1919-1, Tancha, Onna-son, Kunigami-gun, Okinawa 904-0495, Japan 3 Faculty of Science, Ibaraki University, Mito 310-8512, Japan 4 Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. 5 Department of Agrobioscience, Graduate School of Agriculture, Kobe University, Nada, Kobe 657-8501, Japan 6 School of Biological Sciences, Department of Microbiology, Southern Illinois University, Carbondale, IL 62901 USA These authors contributed equally to this work: K. Tani, R. Kanno *Corresponding authors, Email: [email protected]; [email protected]

Abstract: We present a cryo-EM structure of the light-harvesting-reaction center (LH1-RC) core complex from purple phototrophic bacterium Rhodospirillum (Rsp.) rubrum at 2.76 Å resolution. The LH1 complex forms a closed, slightly elliptical ring structure with 16 αβ-polypeptides surrounding the RC. Our biochemical analysis detected rhodoquinone (RQ) molecules in the

purified LH1-RC, and the cryo-EM density map specifically positions RQ at the QA site in the

RC. The geranylgeraniol sidechains of (BChl) aG coordinated by LH1 β- polypeptides exhibit a highly homologous tail-up conformation that allows for interactions with

the bacteriochlorin rings of nearby LH1 α-associated BChls aG. The structure also revealed key protein–protein interactions in both N- and C-terminal regions of the LH1 αβ-polypeptides, mainly within a face-to-face structural subunit. Our findings enable to evaluate past experimental and computational results obtained with this widely used organism and provide crucial information for more detailed exploration of light-energy conversion, quinone transport, and structure–function relationships in pigment-protein complexes.

Rsp. rubrum is an anoxygenic phototrophic purple bacterium with a long history as a model for the study of bacterial photosynthesis and related metabolic processes. It is unique among purple by producing both rhodoquinone (RQ) and ubiquinone (UQ)1 as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid sidechain by geranylgeraniol 2 (abbreviated as BChl aG) rather than phytol . Rsp. rubrum has a single pair of αβ-polypeptides in its core light-harvesting (LH1) complex and lacks both the peripheral light-harvesting (LH2) complex and reaction center (RC) cytochrome (Cyt) c subunit present in many ;

1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.30.446358; this version posted May 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

thus, Rsp. rubrum is one of the simplest phototrophic bacteria known, in terms of its photosynthetic light reactions. Despite many efforts, structures of both purified LH1 and the RC- associated core complex (LH1-RC) of Rsp. rubrum have not been obtained at high resolution, and no RC atomic structure is known.

Here we present a cryo-EM structure of the Rsp. rubrum LH1-RC complex at 2.76 Å resolution (Supplementary Table S1, Figs. S1–3). The LH1 complex forms a closed, slightly elliptical

double ring composed of 16 pairs of α(inner)β(outer)-polypeptides, 32 BChls aG and 16 all- trans-spirilloxanthins (Fig. 1). The arrangement of Rsp. rubrum LH1 α- and β-polypeptides overlaps with that of the LH1 from the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum (Fig. 1d)3. The N-terminal Met in the LH1 α-polypeptide is formylated (Supplementary Fig. S4)4, and the formyl groups were modeled in our structure. Both the LH1 α- and β-polypeptides of Rsp. rubrum exhibit helical conformations in the transmembrane regions, consistent with those observed by solution NMR (Supplementary Fig. S5)5. The Rsp. rubrum RC is surrounded by the LH1 complex with only a few close contacts on the periplasmic surface with residues near Ser34 in the LH1 α-polypeptides. There are no apparent strong interactions of the Rsp. rubrum RC with its LH1 polypeptides as occurs in Cyt c-bound LH1-RCs where the C- terminal domains of some LH1 α-polypeptides interact extensively with the Cyt c subunit6. This may explain why an LH1-only complex can be easily purified from Rsp. rubrum (Supplementary 7 Fig. S6a) . Cofactors in the Rsp. rubrum RC include four BChls aG, two bacteriopheophytins

(BPhe) aG, one 15-cis-spirilloxanthin, an RQ-10 at the QA site, and a UQ-10 at the QB site (Fig. 1c).

a b 112 Å

Periplasm

90˚ 105 Å

Cytoplasm

H-subunit

c d

Fe RQ UQ

Fig. 1 Structure overview of the Rsp. rubrum LH1-RC complex. (a) Side view of the LH1- RC parallel to the membrane plane. (b) Top view of the LH1-RC from the periplasmic side of the membrane. (c) Tilted view of the cofactor arrangement. (d) Superposition of Cα carbons of

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the LH1 αβ-polypeptides between Rsp. rubrum and Tch. tepidum (gray, PDB: 5Y5S). Color

scheme: LH1-α, green; LH1-β, slate-blue; L-subunit, magenta; M-subunit, blue; BChl aG in LH1

and special pair, red sticks; Accessory BChl aG, cyan sticks; BPhe aG, light-pink sticks; Spirilloxanthin, yellow sticks; UQ-10, blue sticks; RQ-10, green sticks; Fe, magenta ball. Phospholipids and detergents are omitted for clarity.

The BChl aG molecules in Rsp. rubrum LH1 form an elliptical, partially overlapping ring with average Mg–Mg distances of 9.3 Å within a dimer and 8.5 Å between dimers (Fig. 1c). These molecules are ligated by histidine residues (α-His29 and β-His39, Fig. 2) that have His–Mg distances of 2.3 Å and 2.0 Å, respectively (Supplementary Table S2). The geranylgeranyl

sidechains in the BChl aG associated with β-polypeptides form a tail-up conformation (Fig. 2a) with a much higher structural homogeneity compared with those of purple bacteria whose BChl a is esterified by a phytyl group (Supplementary Fig. S7). This may be due to the multiple double bonds present in the geranylgeranyl group, which results in a more rigid conformation.

This unique conformation allows the geranylgeranyl sidechains of the β-associated BChl aG to

interact with the bacteriochlorin ring of the α-associated BChl aG (Fig. 2b) where the methyl

group at geranylgeranyl g11 points to the macrocycle of a neighboring BChl aG at a distance of ~3.5 Å. This close proximity likely allows for π-π interactions between the double bonds in the

geranylgeranyl group and nearby bacteriochlorins. The C3-acetyl oxygen atoms in BChl aG form hydrogen bonds with the Trp residues (α-Trp40 and β-Trp48) of their associated polypeptides (Figs. 2c and 2d), in agreement with the results of resonance Raman spectroscopy8-10. The aromatic side chains of the Trp residues also interact with bacteriochlorins through π-π stacking, further stabilizing the LH1 complex.

a b LH1-α LH1-β

c d

LH1-α LH1-β

Trp48 Trp40 2.6Å 2.8Å His39

His29

Fig. 2 BChl aG in Rsp. rubrum LH1 complex. (a) Superposition of the bacteriochlorin rings of

3 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.30.446358; this version posted May 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

16 BChl aG molecules bound to the LH1 β-polypeptides. (b) Interactions between a

geranylgeranyl sidechain of the BChl aG (red sticks) bound to LH1 β-polypeptide and the

bacteriochlorin ring (tint sticks) of a BChl aG bound to the LH1 α-polypeptide. (c) Coordination

and hydrogen bonding of the BChl aG in LH1 α-polypeptide. (d) Coordination and hydrogen

bonding of the BChl aG in LH1 β-polypeptide.

The Rsp. rubrum RC is composed of L, M and H subunits with an overall protein structure and cofactor organization as reported in other purple bacteria (Figs. 3a and 3b). Our biochemical analyses of purified LH1-RC complexes using HPLC, absorption, and MALDI-TOF/MS spectroscopies identified approximately one UQ-9, ten UQ-10 and one RQ-10 to be present per

complex (Fig. 3c, Supplementary Fig. S8). The cryo-EM density map at the QA site is best modeled by the head group of an RQ-10 (Fig. 3d, Supplementary Fig. S3b). The aminobenzoquinone ring of RQ-10 is stabilized by formation of two hydrogen bonds between its carbonyl groups and a His residue (M-His218) and mainchain amide group (M-Ala259) in a loop region of the M-subunit (Supplementary Fig. S9). By contrast, the density map of UQ-10 at the

QB site was more diffuse (Supplementary Fig. S3b), indicating a more mobile nature of this

quinone. The special pair BChl aG molecules in the Rsp. rubrum RC are ligated by histidine residues (L-His174 and M-His201) with His–Mg distances (2.09 Å and 2.12 Å, respectively) and

BChl aG–BChl aG distance (7.76 Å) similar to those of Tch. tepidum (Supplementary Table S2, Fig. S10). While the C3-acetyl group in the Rsp. rubrum special pair L-BChl aG forms a hydrogen bond with a His residue (L-His169) as in all other purple bacterial RCs, no such

hydrogen bond is possible to the C3-acetyl group of M-BChl aG because the nearby residue is a Phe (M-Phe196) (Supplementary Fig. S10), as is also true of the RC from Rhodobacter (Rba.) sphaeroides. The corresponding residue is Tyr in RCs from the purple bacteria Blastochloris viridis, Tch. tepidum, Thiorhodovibrio strain 970 and Rhodopseudomonas palustris that also form a hydrogen bond with the M-BChl a C3-acetyl group.

a b

15-cis- spirilloxanthin M L

Fe UQ-10 RQ-10

H c d

UQ-10 L-His231 M-His218 RQ-10

UQ-9 RQ-10 LH1-RC 3.0Å

Fe 3.0Å

Absorbance atnm 270 (a. u.) Membrane L-His191 M-His265

M-Glu233 M-Ala259 20 30 40 50 Elution time (min)

4 Fig. 3 The RC complex of Rsp. rubrum. (a) Side view of superposition of Cα carbons for the RC protein subunits between Rsp. rubrum (colored) and Rba. sphaeroides (gray, PDB: 3I4D).

(b) Cofactor arrangement. Color code: special pair BChl aG, red; accessory BChl aG, cyan; BPhe aG, light-pink; 15-cis-spirilloxanthin, yellow; UQ-10, blue; RQ-10, green; Fe, magenta. (c) Reverse-phase HPLC chromatograms of the quinones extracted from purified LH1-RC and membranes. (d) The RQ-10 modeled at QA site and its interactions with the surrounding residues in the RC. Density map for the RQ-10 head group is shown at contour level of 4σ.

A total of 10 phospholipids (2 PG, 5 CL and 3 PE) were modeled in the cavities between the RC and LH1 based on the density map (Fig. 4a and 4b). Most CL molecules were located on the cytoplasmic side of the membrane with their head groups pointing toward the membrane surface, whereas most PG and PE molecules were distributed on the periplasmic side. This phospholipid distribution is similar to that observed in other LH1-RCs3, 6, 11, 12. Channels were found between every adjacent pair of LH1 αβ-polypeptides (Fig. 4c and 4d) and are composed of nine hydrophobic residues in each polypeptide (Supplementary Fig. S4). Similar channels have also been observed in other LH1 complexes with closed ring-like structures3, 6, 12, suggesting that these hydrophobic channels function as paths for quinone transport into and out of the complex. Such a function is supported by molecular dynamics simulation13 and biochemical analysis14.

a b Periplasm

Cytoplasm

c d

UQ-10

(QB site)

Fig. 4 Phospholipids, detergents and channels in LH1-RC complex. Top view (a) and side view (b) of the phospholipid and detergent distributions for CL (cyan), PG (magenta), PE (blue) and DDM (green). All proteins are shown in gray. (c) Surface representation of the channels in

LH1 ring with color codes: LH1-α, green; LH1-β, slate-blue; BChl aG, red; spirilloxanthin, yellow. (d) Cross section of a putative quinone channel illustrating the channel opening and the size of the benzoquinone head group of the UQ-10 (blue sticks) at QB site.

5

The Rsp. rubrum LH1 complex is particularly well known for its ability to form a highly stable structural subunit with an absorption maximum at 820 nm (Supplementary Fig. S6a)15. This so- called B820 subunit can be obtained from the native LH1 complex at high detergent concentrations or reconstituted from individually separated polypeptides including those from other species16-18 and various BChl analogues19, 20. The B820 subunit was determined by spectroscopic21, 22, biochemical23, 24 and small-angle neutron scattering25 methods to be a heterodimer composed of two interacting BChl a molecules linked to one αβ-polypeptide pair. Two different subunit forms can be distinguished from the LH1 complex as shown in Fig. 5a, one with a face-to-face configuration and another with a back-to-back configuration for the bacteriochlorins. Solution NMR26 and reconstitution27 experiments established that the B820 subunit has the face-to-face configuration with π-overlap at pyrrole rings III and V. a face-to-face b LH1-β back-to-back

3.5Å His39

His29 3.3Å

LH1-α Spirilloxanthin

c d α-Thr46 α-Trp5

α-Ala44 β-Arg46 β-Pro47 3.0Å β-His21

α-Arg37 β-Lys18

-Trp2 α 3.1Å

Fig. 5 Structural subunits in the LH1 complex. (a) Two structural subunits with different configurations for the BChl aG dimers. Color codes in the subunits: LH1-α, green; LH1-β, slate- blue; BChl aG, cyan; spirilloxanthin, yellow. Other polypeptides and pigments are shown in gray. (b) Structure of the face-to-face subunit showing coordination and cross hydrogen-bonding of the His residues to BChl aG molecules. (c) Major interactions in the N-terminal region within a face-to-face subunit. (d) Major interactions in the C-terminal region within a face-to-face subunit. Dashed lines indicate distances shorter than 3.5 Å.

Due to its structural simplicity and flexibility, the Rsp. rubrum B820 subunit has been thoroughly investigated using protease-modified polypeptides, mutants and chemically synthesized polypeptides27-30, the results from which predicted minimal requirements for stabilizing the subunit structure. These requirements included (i) a central α-helical transmembrane domain composed of 18 hydrophobic residues; (ii) a His residue for coordination and hydrogen bonding

6 to different BChl molecules; (iii) a Trp residue for hydrogen bonding to the BChl C31 carbonyl oxygen; and (iv) N-terminal regions of α- and β-polypeptides. The His interaction was estimated to account for over half of the stabilization energy of the B820 subunit, followed by hydrogen bonding by Trp residues30. Now, these predictions can be modeled by our high-resolution Rsp. rubrum LH1 structure. Both α- and β-polypeptides form transmembrane α-helices in the regions of 12–34 and 19–42, respectively (Fig. 1a, Supplementary Fig. S4). The BChl a-coordinating His residue also forms a hydrogen bond with the C131 carbonyl oxygen atom of the neighboring BChl a, and this cross-hydrogen bonding only occurs within the face-to-face subunit (Fig. 5b). Both Trp residues (α-Trp40 and β-Trp48) form hydrogen bonds with the C3-acetyl oxygen atoms of BChl a as was shown in Figs. 2c and 2d.

In addition to these pigment–protein interactions, our structure revealed significant protein– protein interactions in the N- and C-terminal domains of the Rsp. rubrum LH1 subunit. All amino acids at the N-terminal end of the α-polypeptide including their formyl groups can be traced in our cryo-EM density map, indicating a stable, ordered conformation (Supplementary Fig. S3b). The α-Trp5 forms a relatively strong hydrogen bond with β-His21, and α-Trp2 likely employs cation–π interactions31 with β-Lys18 in the N-terminal region (Fig. 5c). The α-Trp5 and β-His21 residues are conserved in all LH1 complexes reported, and the hydrogen bond between them has been observed in the LH1 structures of Tch. tepidum3, Trv. strain 9706 and Rps. palustris12. The α-Trp2 and β-Lys18 are either conserved or replaced with an aromatic (Tyr, His) and a cationic (Arg) residue, respectively, at corresponding positions in many LH1 complexes (Supplementary Fig. S4). Our Rsp. rubrum LH1 structure also explains previous observations that a stretch of 9–13 amino acids at the N-terminal end of the α- and β-polypeptides (especially the first Met of the α-polypeptide) is important for stabilizing the B820 subunit4, 27 and that the first three residues of the α-polypeptide likely maintain close interactions with the sidechain of His21 of the β-polypeptide27.

In summary, our high-resolution cryo-EM structure of the Rsp. rubrum LH1-RC definitively locates components of the complex with respect to each another, and reveals their intimate interactions and dependencies, thus providing a blueprint from which an even deeper understanding of light-energy conversion and quinone transport in photosynthetic organisms can be pursued. Our work also provides clues for how structure confers stability in pigment-protein complexes and broadens our understanding of the spectroscopy, photochemistry, and computational biochemistry of both the entire Rsp. rubrum LH1-RC complex and its simplified B820 subunit.

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ACKNOWLEDGMENT This research was partially supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Numbers JP21am0101118 (support number 1758) and JP21am0101116 (support number 1878), 17am0101116, 18am0101116, 19am0101116 and 20am0101116. R.K., M.H. and B.M.H. acknowledge the generous support of the Okinawa Institute of Science and Technology and the Japanese Cabinet Office. This work was supported in part by JSPS KAKENHI Grant Numbers JP16H04174, JP18H05153, JP20H05086 and JP20H02856, Takeda Science Foundation, and the Kurata Memorial Hitachi Science and Technology Foundation, Japan, and the National Key R&D Program of China (No. 2019YFA0904600).

Author Contributions Z.-Y.W.-O. and K.T. designed the work, K.T., R.K., X.-C. J. and M.H. performed the experiments, K.T., R.K., L.-J.Y., Y.K., M.T.M., A.M., B.M.H. and Z.-Y.W.-O. analyzed data, Z.-Y.W.-O., K. T. and M.T.M. wrote the manuscript. All authors edited and revised the manuscript.

Competing interests: The authors declare no competing interests.

Data availability: Map and model have been deposited in the EMDB and PDB with the accession codes: EMD-31258 and PDB-7EQD. All other data are available from the authors upon reasonable request.

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