# Insights into energy quenching mechanisms and carotenoid uptake by orange carotenoid protein homologs: HCP4 and CTDH

Jenia Sklyar, Adjélé Wilson, Diana Kirilovsky, Noam Adir

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Jenia Sklyar, Adjélé Wilson, Diana Kirilovsky, Noam Adir. Insights into energy quenching mechanisms and carotenoid uptake by orange carotenoid protein homologs: HCP4 and CTDH. International Journal of Biological Macromolecules, 2024, 265, pp.131028. 10.1016/j.ijbiomac.2024.131028. hal-05142537

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Insights into energy quenching mechanisms and carotenoid
uptake by Orange carotenoid protein homologs: HCP4 and CTDH

Jenia Sklyar, Adjélé Wilson, Diana Kirilovsky, Noam Adir

<figure><img src="image_2.png" /></figure>

PII: S0141-8130(24)01833-6

DOI: https://doi.org/10.1016/j.ijbiomac.2024.131028

Reference: BIOMAC 131028

To appear in: *International Journal of Biological Macromolecules*

Received date: 13 January 2024

Revised date: 9 March 2024

Accepted date: 19 March 2024

Please cite this article as: J. Sklyar, A. Wilson, D. Kirilovsky, et al., Insights into energy quenching mechanisms and carotenoid uptake by Orange carotenoid protein homologs: HCP4 and CTDH, *International Journal of Biological Macromolecules* (2023), https://doi.org/10.1016/j.ijbiomac.2024.131028

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# Insights into Energy Quenching Mechanisms and Carotenoid uptake by Orange Carotenoid Protein Homologs: HCP4 and CTDH

Jenia Sklyar<sup>a,‡</sup>, Adjélé Wilson<sup>b,‡</sup>, Diana Kirilovsky<sup>b,*</sup>, and Noam Adir<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Photoprotection

Phycobilisome fluorescence quenching

Non photochemical quenching

Crystal structure

Cyanobacteria

## ABSTRACT

Photodamage to the photosynthetic apparatus by excessive light radiation has led to the evolution of a variety of energy dissipation mechanisms. A mechanism that exists in some cyanobacterial species, enables non-photochemical quenching of excitation energy within the phycobilisome (PBS) antenna complex by the Orange Carotenoid Protein (OCP). The OCP contains an active N-terminal domain (NTD) and a regulatory C-terminal domain (CTD). Some cyanobacteria also have genes encoding for homologs to both the CTD (CTDH) and the NTD (referred to as helical carotenoid proteins, HCP). The CTDH facilitates uptake of carotenoids from the thylakoid membranes to be transferred to the HCPs. Holo-HCPs exhibit diverse functionalities such as carotenoid carriers, singlet oxygen quenchers, and in the case of HCP4, constitutive OCP-like energy quenching. Here, we present the first crystal structure of the holo-HCP4 binding canthaxanthin molecule and an improved structure of the apo-CTDH from *Anabaena* sp. PCC 7120. We propose here models of the binding of the HCP4 to the PBS and the associated energy quenching mechanism. Our results show that the presence of the carotenoid is essential for fluorescence quenching. We also examined interactions within OCP-like species, including HCP4 and CTDH, providing the basis for mechanisms of carotenoid transfer from CTDH to HCPs.

<footnote><sup>a</sup> Schulich Faculty of Chemistry, Technion, Haifa 3200003, Israel</footnote>

<footnote><sup>b</sup> Université Paris-Saclay, CNRS, CEA, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif sur Yvette, France</footnote>

<footnote><sup>‡</sup>These authors contributed to the work equally and should be regarded as co-first author. * Corresponding authors.</footnote>
# 1. Introduction:

Cyanobacteria are significant in the evolution of life as they were the first organisms to perform oxygenic photosynthesis on Earth. Oxygenic photosynthetic begins with the absorption of light by a variety of pigments. In cyanobacteria (and red algae) the largest bed of light absorbing pigments are bound to the phycobilisome (PBS), which is a large antenna complex found on the stromal side of the thylakoid membrane [1][2]. Details on the PBS assembly and function have been described in detail in a number of recent reviews [3][4][5]. In most cyanobacterial species and under most growth conditions, the PBSs fill the stromal volume in between the thylakoid membranes [6],and thus can access all of the photochemical reaction centres (RCs) [7]. The ratio of PBS to Photosystem II (PSII), the main target of excitonic energy transfer (EET) from the PBS is quite high, providing a constant flux of energy for water oxidation and electron transfer. However, under different environmental conditions, excess EET may occur, leading to a disadvantage to the organism. If the cells are exposed to excess light radiation (beyond the biochemical capability of the photosynthetic apparatus to utilize the light-driven electron transfer processes), these same antennas can induce overexcitation of the RC of PSII, leading to significant damage, a condition called photoinhibition (PI) [8][9][10]. Conditions of overexcitation may occur over different timescales (fast or slow, transient or continuous), thereby requiring the organisms to maintain different protection mechanisms[11]. One such photoprotective mechanism that exists in cyanobacteria is non-photochemical quenching (NPQ), which induces photoprotective thermal dissipation, safely converting absorbed light energy into heat. In some species, the NPQ mechanism involves the Orange Carotenoid Protein (OCP) that physically attaches to the PBS, preventing energy transfer to the RCs [12][13][14][15][16]. NPQ via the OCP system will protect the RCs over relatively slow time scales (seconds to hours) and will operate continually for as long as the OCP is attached to the PBS.

The OCP is a 35kDa water-soluble carotenoid binding protein that has two domains: an α-helical N-terminal domain (NTD) and a C-terminal domain (CTD) with mixed α helical and β-sheet fold [17]. Upon high light exposure, the non-active state (called OCP<sup>O</sup> for its orange color) transforms onto an active state (called OCP<sup>R</sup> for its red color) [13]. In OCP<sup>R</sup>, the NTD disconnects from the CTD and now forms the active domain of the protein [18][19]. In OCP<sup>R</sup>, the CTD serves as the regulatory domain which can associate with the Fluorescence Recovery Protein (FRP) to facilitate the disassembly of OCP from the PBS [20][21]. The CTD was recently found to also participate in OCP<sup>R</sup> dimerization [22][23] when bound to the PBS. A stretch of amino acid residues serves to link the two domains together, and in OCP<sup>O</sup>, a non-covalently bound ketocarotenoid molecule is embedded within a pocket that spans the two domains. Carotenoids (Car) are hydrophobic pigment molecules that typically have 40 carbons and play an essential role in coloration and pigmentation, light harvesting in photosynthesis, photoprotection against oxidative damage, and serving as a structural component of cell membranes [24]. In the OCP, the Car molecule (typically echinenone, hydroxy-echinenone or canthaxanthin: ECN, hECN or CAN, respectively) plays a double role in photoprotection as singlet oxygen quenchers [25] and as excess energy dissipator by binding to the PBS. Due to their hydrophobic nature, carotenoid transfer in the aqueous environment is performed with the assistance of water-soluble carotenoid-binding proteins[26][27][28][29][30][31].

Light drives a conformational change to the Car that induces separation of CTD and the NTD domains, with the Car molecule sliding completely into the NTD [19]. The activated NTD binds to the PBS, preventing energy transfer to the RCs. This will continue until the OCP<sup>R</sup> spontaneously or actively dissociates from the PBS. The
kinetics of OCP<sup>R</sup> release can be enhanced by interaction between the CTD and FRP. Binding of the FRP also accelerates the reassociation of the NTD and CTD, thereby recovering the inactive OCP<sup>O</sup> [20][21].

Investigation of the activity of the OCP is facilitated by the ability to mimic the processes of NPQ *in vitro* [32].

Isolated PBS is highly fluorescent, while the PBS-OCP<sup>R</sup> complex is strongly quenched. *In vitro* experiments have led to several models of the OCP-PBS interaction [33][34][35].

More recently, the structure of the PBS-OCP<sup>R</sup> complex from *Synechocystis* sp. PCC 6803 was determined by cryo-electron microscopy (cryo-EM) [22]. The complex was formed *in vitro* as described above, and maps indicate that two OCP<sup>R</sup> dimers attach to the outside circumference of the core APC cylinders, without penetrating into the cylinders. Two CTD domains stabilize the OCP<sup>R</sup> dimerization interface. The structure revealed the specific residues involved in the NTD interaction with the PBS [22]. The closest distance between the Car molecule and the nearest PCB is ~10nm, a distance that is larger than typically found between Car molecules that quench chlorophyll pigments in other LHCs [36].

In recent years, new families of OCP (OCP2 and OCPX (or OCP3) and OCP homologs (HCPs and CTDHs) have been identified by genomic screening in various species of cyanobacteria [37][38]. Moreover, the structure and the function of OCPX(OCP3) and two OCP2s have been revealed, demonstrating that the structure of these proteins present unique structural characteristics that could explain their differences with OCP1 photoactivation and recovery [39][40].

Genomic screening discovered the existence of genes encoding for proteins that are homologous to either OCP-NTD or OCP-CTD, that were predicted to also potentially be carotenoid-binding proteins. NTD homologs have been termed Helical Carotenoid Proteins (HCPs), while the CTD homologs are collectively called CTDH proteins [41][42][43]. Biochemical and structural studies have proposed that the CTDH proteins function as Car carriers that initiate Car uptake and transfer from membranes [41][44]. The apo-CTDH structure from *Anabaena* sp. PCC 7120 (hereafter *Anabaena*) was determined to 2.75Å resolution, and analysis of the crystal lattice suggested that two types of functional dimeric organizations that may play a part in Car uptake. The two dimer types were denoted as head-to-head, where the interactions are formed via the Car binding cavity stabilized by a disulfide bond and back-to-back, a dimer formed by mostly by β-sheet interface interactions. At the end of the CTDH, an extended flexible C-terminal tail (CTT) was suggested to be responsible for putative different conformations of the apo-form and the holo-form of the CTDH, and may be the interacting element with the membrane [45].

Two CTDH clades and nine HCPs clades have been identified in cyanobacteria [46]. *Anabaena* possesses members of four clades of HCPs with putative diverse functions: carotenoid carriers (HCP1), reactive oxygen species quenchers (HCP2, HCP3, HCP4), and PBS energy quenchers (HCP4) [42][37]. Recent studies have shown that HCP1 is enriched with hydrophobic residues and can bind different Car types [37]. *Anabaena* HCP1 was proposed to have a role in Car transport or an unidentified function since it does not function as singlet oxygen (¹O₂) or as a PBS quencher [42][37]. However, this same protein from *Nostoc flagelliforme* (*N. flagelliforme*) exhibited robust ¹O₂ quenching activity [43], raising the possibility that various species may possess distinct HCP functions.

Several studies have reported the efficacy of HCP2 and HCP3 as effective quenchers of singlet oxygen in *Anabaena* [42] and for *N. flagelliforme* [43]. Notably, the crystal structure of HCP2 has been elucidated [47], while the structural details of HCP3 and HCP4 have remained unknown. Previous studies have shown HCP4's
distinct specificity for binding CAN [42]. Nonetheless, *AnaHCP4* displays low selectivity in its binding to the PBS, since it has been shown to be able to quench both the pentacylindrical *Anabaena* PBS and tricylindrical *Synechocystis* PBS [42][48]. This fact raises the question of whether HCP4 may be able to functionally associate with different binding sites to the PBS.

The main objective of this study is to experimentally explore the X-ray crystallographic structure of holo-HCP4 and elucidate its quenching mechanism. Additionally, this research aims to investigate the CTDH structure from different perspectives and suggest a mechanism of carotenoid transport between HCP4 and CTDH proteins. We describe here the structure of holo-HCP4, binding a CAN molecule, at a resolution of 1.9 Å. Furthermore, we have obtained an improved structure of apo-CTDH at 1.95 Å and studied the potential interaction between these two proteins. Building upon our analysis of the holo-HCP4 structure, we propose a mechanism for energy quenching by holo-HCP4 and an alternative mechanism for carotenoid transfer mediated by CTDH.

## 2. Materials and methods:

2.1. *Vector constructions for NTD, HCP and CTDH expression in E. coli cells*

pCDF-RCP-Syn-1-165Ctag, pCDF-HCP4-4941Ctag, pCDF-HCP1-1123-Ctag and pCDF-CTDHAna-4940Ctag plasmids construction were described previously in [19][42][41].

2.1.1. *pDuet-HCP4-CTDH C103F (pDuet-HCP4-4941Ctag-CTDHAna-4940-C103F)*

First, the *all4940* gene was amplified by PCR using genomic DNA of *Anabaena* sp. PCC 7120 as template and the primers Fall4940NdeI and Rall4940XhoI. The amplified fragment was cloned onto the NdeI and XhoI sites of *pDuet-HCP4-4941Ctag* plasmid to construct the *pDuet-HCP4-4941Ctag-CTDHAna-4940* vector. The point mutation C103F (see [41] for primers) were introduced in the *all4940* gene by site-directed mutagenesis to generate the plasmid *pDuet-HCP4-CTDH C103F*.

2.2. *OCP-like plasmids constructions*

2.2.1. *pDuet-OCP-like 1, 2 and 3*

The *Anabaena* sp. PCC 7120 genome region containing the *all4941* and *all4940* genes was amplified by PCR using the restriction sites-creating primers Fall4941NcoI and Rall4940NotI. This fragment was cloned into the pCDFDuet-1 plasmid. A His6-Tag was added in the 3' side of the *all4940* gene by site-directed mutagenesis. A fusion protein was constructed by deletion of the region between both genes including the stop codon of the *all4941* gene and the Met codon of the *all4940* gene. Then, a linker sequence coding for 29 amino acids (FDAAKLDGYTRVAEPVVPPKDISQRVQVT) was introduced by mutagenesis. First, 9 amino acids (RVAEPVVPP: 173-181) of *NIES 39* OCP linker were added between the two genes. Secondly, the last six amino acids (166-171) of the *all4941* gene were deleted and replaced by 10 amino acids (FDAAKLDGYT: 163-172) of the *Anabaena* sp PCC 7120 OCP linker. Thirdly, the resulting plasmid was used to construct the *OCP-like 1* gene by removing the first 13 amino acids (2-14) of the *all4940* gene and replacing it with 10 amino acids (KDISQRVQVT: 182-191) of the *Anabaena* sp. PCC 7120 OCP linker.

*OCP-like 2 and 3* genes were also constructed by replacing the amino acid C103 of the *all4940* gene respectively by 5 amino acids (FGAAV) or Phe using site-directed mutagenesis (see [41] for primers).
2.2.2. *pDuet-OCP-like 4*

NTDsyn (1-165), linker and *CTDHAna-4940* (15-140) were amplified by PCR using the *Synechocystis*
genome region coding OCP gene and *pDuet-OCP-like 2* as a template and two pairs of primers [F- NTDsyn and
R-NTDsyn-link; F-NTDsyn-link and R-Duet-CTDH]. The amplified fragments were cloned in pCDFDuet-1
vector linearized with Nde1 using the In-Fusion HD Cloning Kit (TaKaRa) to generate the *OCP-like 4* gene.

2.2.3. *pDuet-OCP-like 5*

HCP4 (1-165), linker and the *CTDsyn* (190-317) were amplified by PCR using the *Anabaena* sp PCC 7120 genome region coding *all4941* and *pCDF-OCPSyn-Ctag* as a template and two pairs of primers [F- HCP4 and R-HCP4-link; F-HCP4-link and R-Duet-CTDsyn]. The amplified fragments were cloned in pCDFDuet-1 vector linearized with Nde1 using the In-Fusion HD Cloning Kit (TaKaRa) to generate the *OCP-like 5* gene.
All plasmids were checked by sequencing. Primer sequences are listed in Table S1.

2.2.4. *pDuet-OCP-like 6*

HCP1 (1-165), linker and *CTDHAna-4940* (15-140) were amplified by PCR using the *Anabaena* sp PCC 7120 genome region coding *all1123* and *pDuet-OCP-like 2* as a template and two pairs of primers [F- HCP1 and R-HCP1-link; F-HCP1-link and R-Duet-CTDH]. The amplified fragments were cloned in pCDFDuet-1 vector linearized with Nde1 using the In-Fusion HD Cloning Kit (TaKaRa) to generate the *OCP-like 6* gene.

All plasmids were checked by sequencing. Primer sequences are listed in Table S1.

2.3. *Holoprotein production and purification in canthaxanthin-producing E. coli*

Expression and isolation were performed using protocols previously described [49].

## 2.4. Size Exclusion Chromatography

For the OCP-like species, the purified proteins were injected into a Superdex 75 HR 10/30 column (Pharmacia) previously equilibrated with 40mM Tris-HCl pH 8, 150mMNaCl. The AKTA Purifier FLPC system (GE Healthcare) was equipped with a UV detector set up at 280 nm, 510 nm and 560 nm and was running at a flow rate of 0.8 mL/min. To calibrate the column, the gel filtration calibration kit LMW (GE Healthcare) was used.
For holo-HCP4, the protein was loaded on a size exclusion column (Agilent SEC-5, 5µm 300Å, 7.8x300mm) and equilibrated with 40mM Tris, 25mM NaCl pH 8.0.

2.5. *Absorbance measurements in solution*

Absorbance spectra of PBS, OCPs and kinetics of photoactivity (illumination with 5000 µmol photons m⁻² s⁻¹ of white light at 15°C) of the OCP were performed in a spectrophotometer Specord S600 (Analytic Jena) in a 1-cm path-length cuvette. UV-vis Absorbance spectra was measured using NanoDrop™ Spectrophotometer (Thermo Scientific™).

2.6. *Isolation of PBS and NTD/HCP4-PBS complexes*

PBS from *Synechocystis* sp. PCC 6803 and *Anabaena* sp. PCC 7120 were purified as previously described by [32] and [48] respectively. Initially the NTDSyn/HCP4-PBS complexes were prepared in 1.4M phosphate by incubation of isolated PBSs for 30 min at 23°C in presence of NTDSyn/HCP4 (NTDSyn/HCP4:PBS ratio of 20). Then, the samples were loaded onto a sucrose gradient with 1.2 M phosphate to separate the resulting
NTDSyn/HCP4-PBS complexes from the unbound NTDSyn/HCP4. The gradient was centrifuged at 150,000g at 23°C for 3 h. The blue band was isolated from the gradient and its composition was analyzed by gel electrophoresis, absorbance, and fluorescence spectra.

## 2.7. Fluorescence measurements

Fluorescence yield quenching was monitored using a pulse amplitude modulated fluorometer (101/102/103-PAM, Walz). Measurements were made in a 1-cm path length stirred cuvette. The PBS (0.012 µM) quenching induced by OCPLs and HCPs (0.48 µM) was measured in 1.4 M potassium phosphate buffer (pH 7.5) at 23°C in darkness and in strong blue-green light (900 µmol photons m⁻² s⁻¹). The ratio of holo-OCPLs or holo-HCPs to PBS was = 40. The concentration of *Synechocystis* and *Anabaena* PBS were measured as described in [48]. Fluorescence emission spectra of PBS were recorded in a 1 cm-diameter cuvette at room temperature using the Cary Eclipse fluorescence spectrophotometer fluorometer (Varian). Excitation was made at 590 nm.

## 2.8. Protein and Immunoblot analysis

Proteins were separated by SDS-PAGE on 15% polyacrylamide in a TRIS-MES system [50]. Prior to loading (equal amounts of protein in each lane), PBS- samples were concentrated by precipitation with 10% (v/v) trichloroacetic acid. HCP4 protein samples were analyzed by SDS-PAGE on 14% polyacrylamide. The gels were stained with InstantBlueTM or Coomassie Brilliant Blue. The NTD/HCP4 were detected by a polyclonal antibody against All1123 (HCP1) or a polyclonal antibody against OCP. The binding of antibodies was monitored by an alkaline phosphatase colorimetric reaction (Bio-Rad AP Conjugate Substrate Kit).

## 2.9. Holo-HCP4 Crystallization and Data collection

Holo-HCP4 crystals diffracting to a resolution of 1.9Å were obtained by hanging drop vapor diffusion method at 20°C. 2µl of protein solution at concentration of 15 mg/ml in 40mM Tris, 25mM NaCl pH 8.0 buffer was mixed with 2µl of reservoir containing 0.095M sodium citrate tribasic dihydrate pH=5.6, 19% v/v 2-propanol, 19% v/v PEG 4000, 5% glycerol (Crystal Cryo HT screen, Hampton research). Purple rod crystals were obtained within a month. The obtained crystals were mounted on a cryo-loop (CrystalCap SPINE HT, Hampton Research) and flash-frozen in liquid nitrogen. X-ray diffraction data was collected at beamline I-04 at the Diamond Light Source (Oxfordshire, England). Diffraction data was integrated with XDS [51] and processed with CCP4 [52]. The X-ray crystallographic structure of the holo-HCP4 was determined by molecular replacement (MR) using a model produced by AlphaFold [53] as the search model. The AlphaFold model was obtained using the primary sequence of the HCP4 protein (RMSD of 0.418Å for 122 α carbons between the final holo-HCP4 structure and the initial model). The resulting solution was refined and rebuilt using phenix.refine [54] and COOT [55]. Statistics for diffraction data collection, structure determination, and refinement are summarized in Table S2.

## 2.10. Apo-CTDH Crystallization and Data collection

Apo-CTDH crystals diffracting to a resolution of 1.95Å were obtained by macroseeding technique [56] by hanging drop vapor diffusion method at 20°C. 1µl of protein solution at concentration of 0.85 mg/ml in 40mM Tris, 25mM NaCl pH 8.0 buffer was equilibrated with crystallization buffer containing 1µl of 44% Tacsimate
(being an optimized condition of INDEX HT screen, Hampton research). The solution was equilibrated for 4
hours, without any crystal growth during the incubation time. The drop was seeded with a single microcrystal.
The crystal was obtained in 1.5µl of protein solution at concentration of 2.1mg/ml in 40mM Tris, 25mM NaCl
pH 8.0 buffer with 1.5µl of reservoir containing 44% Tacsimate. Colorless rod crystals were obtained within few
days at 20°C. The crystals were mounted on a cryo-loop (CrystalCap SPINE HT, Hampton Research) and flash-
frozen in liquid nitrogen. X-ray diffraction data was collected at the at the European Synchrotron Radiation
Facility (ESRF) on beamline ID30-B. Diffraction data was integrated with XDS [51] and processed with CCP4
[52]. The X-ray crystallographic structure of apo-CTDH was determined by molecular replacement (MR) using
the apo-CTDH (PDB:6FEJ) as the homology model. The resulting solution was refined and rebuilt using
phenix.refine [54] and COOT [55]. Statistics for diffraction data collection, structure determination, and
refinement are summarized in Table S2.

## 2.11. *Docking analysis*

Docking analysis was performed using PyDock server [57], with the structure of the HCP4 and CTDH monomers as docking targets.

## 3. Results

Previous research has reported that cyanobacterial genomes that contain the HCP4 gene also always possess a gene encoding for a CTDH protein [46]. When the holo-HCP4 was heterologously expressed [49] in the absence of co-expressed CTDH, the resulting holo-HCP4 fraction was extremely low with a high concentration of the apo-HCP4 form (Fig. S1A). On the other hand, when the holo-HCP4 was co-expressed in the presence of the CTDH gene, much higher concentrations of holo-HCP4 were obtained (530/280 ratio of 1.1-2.1, Fig. S1B,C). CTDH has been proposed to be involved in the uptake and transfer of carotenoids from the membrane to the HCPs [41], and the fact that HCP4 has to be expressed in the presence of CTDH supports the idea that CTDH is essential for transferring carotenoids to HCPs. Whether the CTDH may assist other proteins in Car binding will be explored below.

Holo-HCP4 was purified by nickel affinity chromatography followed by size exclusion chromatography (SEC) purification. The fractions containing holo-HCP4 were characterized by SDS-PAGE and UV-Vis absorbance analysis (see Methods for details and Fig. S2). Spectroscopic analysis of the holo-HCP4 fractions showed an absorbance maximum of 530nm, which confirmed the binding of the carotenoid to the HCP4 protein (Fig. S2A). To determine the purity of the HCP4 fraction, the 530/280nm absorbance ratio of the holo-HCP4 to apo-HCP4 was calculated. In the process of isolating HCP4 for crystallization trials, different fractions were obtained. While it was shown that pure holo-HCP4 fractions should have a ratio of >2.5, [58][47] we performed crystallization trials on all fractions. The fraction that resulted in small but highly colored crystals actually had a lower ratio of 1.1, indicating that while a high fraction of the holo-HCP4 form was present, some apo-protein was also present. However, it is worth noting that analysis of the CAN occupancy in the crystal structure (described below) indicated that no apo-HCP4 was present in the crystals. Even slight differences between holo- and apo forms of the HCP4 may have resulted in the crystallization driven purification of holo-HCP4. However,
the presence of some apo-HCP4 in the solution may have prevented the growth of larger crystals. Fortunately,
the diffraction quality of the small crystals was sufficient to determine the structure.

3.1. *Structural analysis of holo-HCP4 crystal structure*

The holo-HCP4 structure was determined to a resolution of 1.9Å using an initial structural model produced by AlphaFold [53][59] as the search model for molecular replacement (MR). The structure contains two monomers in the asymmetric unit (RMSD of 0.144 Å for all α carbons). Each monomer binds a single CAN molecule (Fig. 1A) as evidenced by the strong Fo-Fc difference electron density observed for the CAN molecules in the binding pocket (Fig. 1B). There is currently no evidence to support a physiological function for an HCP4 dimer, and it was previously suggested that the protein is active in its monomeric form [41][42]. Data collection and refinement statistics are shown in Supplementary Table 2. The electron density maps lack clear density for residues 1-13 (Chain A) and 1-16 (Chain B), and thus these are not present in the structure. The remaining residues of the N-terminal tail (up to Pro24) lack secondary structure but have clearly interpretable density. Disordered termini are quite typical for many crystal structures, and the reason for the disorder (true functional flexibility or crystal lattice issues) cannot always be ascertained experimentally. We however note that the flexibility of the termini of HCP4 may play important functional roles (as previously shown for the CTDH). A major difference between the monomers was found between residues 57-64, which is modeled as a somewhat distorted α-helix in monomer B. This same stretch of residues in monomer A could be modeled into the electron density map, however is without clear secondary structure. The overall position of this stretch of residues is nearly identical, and it forms one side of the opening to the CAN binding channel, and its flexibility may play a role in CAN uptake. This is the first instance of more precise visualization of this stretch of residues as they are lacking in secondary structure in OCP-NTD and other HCP structures, in some cases completely absent in the maps (and thus not present in the coordinates deposited in the PDB) [19]. From our analysis of the crystal packing, the order observed in this stretch of residues in monomer B is only weakly affected by adjacent monomers in the lattice. We thus use the more ordered B monomer for further analysis of the structure, unless otherwise noted.

When comparing the HCP4 structure to other HCPs/OCP NTD structures, the differences are on the same order
(Cα RMSD of ~0.95Å±0.1), indicating that the observed differences in function are not due to major changes to
the three-dimensional structure (Table S3). HCP1 for instance does not have the ability to quench PBS
fluorescence [42], while HCP4 does (Fig. S3). The homology on the sequence level is less conserved than the
structural conservation, and these differences in the sequence lead to significant changes in the surface
electrostatic potential. In the HCP1 structure, the surface charge is mostly negative, with a strong negative
surface potential in different sections of the surface when compared to the same areas of the surfaces of HCP4
or other HCPs (Fig. S4). These extremely negative patches may prevent association with negative surfaces on
the PBS, and consequently may prevent the binding of HCP1 to the PBS in a fashion that would enable
quenching. On the other hand, in the HCP4 structure, the surface charge distribution is mild, which may enable
the protein to associate and bind to the PBS (Fig. S4).

The CAN molecule is symmetric, both sides terminating with identical β rings, that in the bound state are
adjacent to two significantly different surface electrostatic potential distributions. This creates two distinct faces
of the protein, which we denote as face 1 surrounding the aperture containing the β1 ring (containing the oxygen atom denoted O1 in the molecular structure) and face 2 around β2 ring (containing the oxygen atom denoted O2 in the molecular structure) (Fig. 2). The charge on face 1 is highly positive, while that of face 2 is weakly negative (i.e. mostly hydrophobic). The different charge distributions on the two aromatic rings, and the mode of binding of the OCP to the PBS [22][23] suggests that association to the PBS is most likely specific to face 1.

The positive patch on HCP4 face 1 is formed by residues Ser106, Asn107, Asn108, Arg159, and Lys160 (Fig. S5). In the NTD of SynOCP, Arg155 (AnaHCP4 Arg159) is considered critical for OCP photoactivation and for OCP<sup>R</sup> binding to the PBS [19][60][61]. Lys156 (AnaHCP4 Lys160) was suggested to play a role in binding to the negative residues in the APC core along with Arg155 [42]. Additionally, it has been proposed that HCP4 is the only HCP capable of binding to the PBS, as only in this isoform these residues are exposed, enabling interaction with the PBS [42]. We examined the importance of these residues by comparing the HCP4 structure with HCP1 and HCP2. In HCP1, the comparable Arg residue (Arg147) appears to be on the surface of the protein, but its positive potential is somewhat negated by the presence of the adjacent Asp148. In the crystal structure of HCP1, the stretch containing residues 40-48 was not modeled, however, taking into account the positions of the last visible residues (Gly39 and Ser49), this stretch may actually cover this Arg residue preventing possible interactions. In the HCP2 structure, Arg163 is followed by Ser164, located on the N-term tail, and it is not on the surface of the protein. Those findings are in line with [42], in which was demonstrated that the Arg155 in HCP2/3 forms intramolecular salt bridges, hindering interactions with other residues. In RCP (PDB:4XB4), Arg155 with Asn156 is on the protein's outside surface, creating the required positive patch. These results can support the proposal that a positive surface is necessary for positioning the protein at a site on the PBS that enables quenching.

Recently, the structure of the PBS-OCP complex was determined by Cryo-EM [22][23]. It is certainly feasible that the residues conserved between the OCP NTD and the HCP4 are those that interact with the PBS and are thus critical for HCP4 function. This Cryo-EM structure shows that Thr52-Ala60 (OCP PDB:7SCC) are the major interacting residues between the OCP-NTD and the ApcA/ApcB monomers in the PBS core [22][23]. In AnaHCPs, this loop contains several conserved residues such as Ile51, Thr52, Ala55, and Ala58 (AnaOCP NTD numbering) [42]. Interestingly, HCP4 is the only HCP in which those residues contain a structured short α-helical segment (residues 57-59), making the CAN cavity more open and less buried inside the protein. This loop creates a mostly hydrophobic surface in face 1 around the β1 ring, on the opposite side of the positive patch. In AnaHCPs, which are unable to quench the PBS energy, this is a loop with non-conserved residues. In HCP2, the conserved amino acids are completely missing in this sequence, creating a mostly negative surface. In the HCP1 structure, these residues are absent from the crystal structure, which may indicate a highly flexible loop. The more open pore that exposes the CAN β2 ring to interaction may be one of the key components of HCP4's function. In the AnaAPC core a series of negatively charged amino acids next to a hydrophobic surface can possibly be the binding site to the PBS (Fig. S6), with the positive and the hydrophobic surface on face 1 of the HCP4, as was proposed in [60].

Analysis of the distribution of surface electrostatic potential within the CAN cavity shows significant difference between the different HCPs and thus may have a role in the mechanism of quenching by the HCP4 protein. In the HCP4 structure, the electrostatic charge distribution around the β1 ring is positive, while it is negative
around the β2 ring (Fig. 3A). This charge distribution is similar to the distribution of charges found in the *AnaOCP* and RCP ligand cavities (Fig. 3D). On the other hand, in the HCP1 and HCP2 cavities, the charge distribution is mostly negative along the entire carotenoid chain (Fig. 3B,C). This is consistent with the surface charge distribution of the proteins. The positive charge around β1 ring in the HCP4 structure may be responsible for the binding of HCP4 to PBS, which is absent in HCP1 and HCP2, preventing them from binding to PBS and quenching its energy (Fig. 3).

It has been previously shown that OCP undergoes a conformational change upon photoactivation. CAN changes from a bent (in OCP<sup>O</sup>) conformation to a more planar (in OCP<sup>R</sup>) conformation. The β1 and β2 aromatic rings in OCP<sup>O</sup> are oriented perpendicular to one another (Fig. 3G) [15][16][19]. In the HCP4 structure (which is functionally equivalent to the NTD in OCP<sup>R</sup>), CAN is in a cis-cis configuration, with β1 and β2 CAN rings oriented in the same plane, with a minor twist of the rings relative to each other (Fig. 3E). The planarity of the HCP4 CAN and the relative orientations of the β1 and β2 CAN rings are similar to those found in the active OCP<sup>R</sup> CAN configuration (Fig. 3F), which quenches the PBS. The HCP4 is constitutively active and does not require photoactivation [42].

Even though the HCP1/2 proteins are also homologous to the OCP-NTD, the β1 and β2 rings in these proteins exhibit a perpendicular orientation, with the HCP1 CAN molecule also displaying bending, resembling the OCP<sup>O</sup> configuration (Fig. 3H,I), and both proteins do not quench the PBS EET transfer. During OCP photoactivation, the β rings switch to a more planar conformation, allowing access to the aromatic ring for binding to enable closer proximity to the closest PBS chromophore.

## 3.2. *The HCP4 N-terminal tail*

HCP4 has an extended N-terminal tail (NTT) (Fig. 2). The structured tail is also present in *AnaOCP*, OCPX1, and OCPX2. HCP1 and HCP2 lack a structured NTT, and their overall structure is more globular. We propose that the HCP4 tail may play a role in HCP4 quenching activity. In the PBS-OCP structure [22], the N-terminal tail is oriented towards the ApcA subunit and to the rod-core linker CpcG. Although the tail is not modeled in the structure, it appears that it is directed towards the inside of the APC hexamer, possibly between the two trimers. Assuming that HCP4 binds to the PBS in a similar way, there are two possible pathways for PBS energy quenching. The first option is that the HCP4 positive residues on face 1 bind to the negative residues of the APC core through electrostatic interactions. The NTT then binds to complementary charges on the APC core trimer and the CAN β1 ring approaches close to the chromophores in the core, quenching excess energy. In a second possibility, the finger-like NTT inserts in between the APC monomers, shifting the APC core monomers and altering the structure of the core, as proposed for the OCP NTD quenching mechanism [44]. This might also enable a closer proximity between the CAN and PBS chromophore, enabling HCP4 to dissipate the excess energy and preventing it from reaching the reaction centers. Further examination through mutagenesis analysis is necessary to explore the significance of the NTT in quenching activity.

## 3.3. *Interaction of holo and apo HCP4 with the PBS*

OCP<sup>O</sup> is unable to bind to PBS since the face of interaction containing Arg 155 (essential for binding) interacts with the CTD and is not free [60]. Only the red open photoactivated form of OCP containing the carotenoid in the NTD subunit can bind to the PBS and induce PBS fluorescence quenching. Apo-OCP is unable to bind PBS
[61][49]. High concentrations of apo-OCP are unable to hinder holo-OCP binding and fluorescence quenching
[49]. To further understand the role of the carotenoid in protein binding and PBS fluorescence quenching,
SynPBSs were incubated with apo and holo-HCP4 and NTD-OCP (see Materials and Methods for details) and
then the complexes were reisolated in a sucrose gradient. The re-isolation is needed to remove the unbound
proteins. Fig. 4A and 4B show the room temperature fluorescence spectra after re-isolation of SynPBS
complexes. No significant fluorescence decrease was observed when the PBSs were incubated in the presence of
apo-proteins (Fig. 4A, 4B). By contrast, a significant quenching (~75%) was detected in the complexes formed
with the holo-proteins.

Fig. 4D,4E and 4F show the protein analysis of the isolated complexes by Coomassie blue-stained SDS-PAGE and detection of HCP4 and NTD by immune-blot using different antibodies: anti-*Arthrospira* OCP (which recognize both HCP4 and Syn-NTD), anti-*Synechocystis* OCP (which recognizes only Syn-NTD) and anti-*Anabaena* HCP1 (which recognize only HCP4). Since the molecular weight of the NTD paralogs is similar to those of PC and APC α- and β-subunits, they cannot be seen in the Coomassie blue-stained gel. Immuno-blot analysis showed that both the holo and apo proteins bound to the PBS. While the apo and holo SynNTD are similarly bound to SynPBS, apo-HCP4 bound less than the holo-HCP4. When the experiment was repeated with Ana PBS, no differences in binding were observed when apo and holo HCP4 were used (Fig. 4C). Nevertheless, fluorescence quenching was observed only in the complexes containing holo-HCP4 (Fig. 4C). These results clearly showed that NTD or HCP4 binding to the PBS is not sufficient to induce PBS fluorescence quenching. The presence of the carotenoid is essential to induce the decrease in PBS fluorescence (or energy transfer in vivo).

3.4. Carotenoid transfer and uptake from membranes

3.4.1. Improved apo-CTDH structure

It has been found that HCP4 cannot directly uptake carotenoids from the membrane without the aid of a
carotenoid carrier such as the CTDH [41][42]. In a previous study, the apo-CTDH structure was determined to
2.75Å [44]. We have now obtained crystals with superior diffraction and can thus present an improved Ana wild
type (WT) apo-CTDH crystal structure at 1.95Å (PDB: 8QX7). The asymmetric unit contains two monomers,
which interact with each other through the β-sheets interface in a back-to-back conformation (Fig. S7). Each
monomer binds malonic acid from the crystallization liquor (MLA) within the ligand binding cavity.

3.4.2. CTDH and CTD

To understand the difference between OCP NTD/CTD interaction and CTDH binding to HCP4, we compared
*Ana*CTD (PDB:5HGR) and the CTDH structure. The primary structural difference is observed in the interface
loop between the CTD and the NTD. In the CTD, this loop presents a short α-helical structure, while in the
CTDH structure, the homologous loop (comprising residues 100-103) is shorter with more closed conformation
(Fig. S8). In the CTD-NTD interface residues from the CTD flexible loop would be central to stabilization:
Thr52-*Asn281*, Ile58-*Phe278*, and Pro56-*Trp277* (NTD residues in italics). The shorter CTDH loop, however, is
incapable of forming these stabilizing interactions, that would result in a more open and less stable HCP4-
CTDH conformation. This, however, may be functionally beneficial, as the complex should only form transiently, allowing the CAN transfer process, followed by disassociation.

### 3.4.3. HCP4-CTDH interaction and carotenoid transport

In our investigation of carotenoid transport from CTDH to HCPs, we utilized the PyDock server [57] to conduct a docking analysis of the HCP4 structure onto the apo-CTDH structure followed by structural analysis of the putative interface (Fig. 5). The docking analysis is based on free energy calculations of protein-protein interactions, and the interface with the lowest energy shows a possible similarity between the interface binding of CTDH and HCP4 and the canonical NTD/CTD interaction of OCP (Fig. 5B). Notably, due to clashes in interface loops, the docked structures exhibit a more open conformation compared to the NTD/CTD interaction in OCP, characterized by an increased distance between conserved amino acids at the NTD/CTD interface (PDB: 5HGR residues N104-T277, R155-E245/246, and P22-F229). Interestingly, the NTT of HCP4 adopts a configuration that envelops the surface of CTDH, featuring complementary opposite charges, as well as the charges on the protein surfaces around the Car cavity (Fig. 5C), thereby possibly fostering the formation of a stable CTDH-HCP4 complex, likely sustained until carotenoid transfer is complete. It is conceivable that the CTDH C-terminal tail may also interact with HCP4 or the tail of HCP4, thereby contributing to the transient stability of the complex (Fig. 5B). However, we suggest that this complex should be transitory, as the HCP4 does not require photoactivation for its activity.

In the past, we have shown that *in vitro*, HCP4 does not form stable complexes with CTDH or CTD-OCP when incubated together, although holo-CTDH is able to transfer its carotenoid to apo-HCP4. A stable complex is not formed in the *E. coli* cells that express the HCP4 and CTDH genes. As mentioned above, under these conditions, higher concentrations of holo-HCP4 were isolated since the CTDH uptakes the carotenoid from the membrane and transfers it to the HCP4. Thus, to better understand the interactions between HCP4 and OCP-NTD with CTDH or CTD-OCP, we constructed a novel category of hybrids called OCP-like proteins. These OCP-like entities were isolated from *E. coli* cells in which fusion genes were expressed. These genes were built with HCP4 or *Syn NTD-OCP* genes combined with either mutated or native *AnaCTDH* or *SynCTD-OCP* genes. The different combinations were fused using a sequence coding for a linker domain (linker sequence of *Syn*OCP, comprised of 36 amino acids, see Materials and Methods for details). In *AnaCTDH* (clade 2) residue 103 is Cys while in the CTD-OCP and the CTDH (clade 1) this residue is Phe. In addition, all CTDH proteins lack 4 amino acids after amino acid 103 compared to CTD-OCP. To study whether these differences between CTDH and CTD could affect the interaction between HCP4 and the NTD with the CTDH, two mutations were also introduced in the CTDH gene: C103F CTDH and C103F+ 4 amino acids CTDH.

Fig. S9 shows the absorbance spectra and the color of the different OCP-like proteins: OCPL1 (HCP4-CTDH), OCPL2 (HCP4-CTDH C103F+4aa), OCPL3 (HCP4-CTDH C103F), OCPL4 (NTD-CTDH) and OCPL5 (HCP4-CTD). All the spectra were different indicating that the position of the carotenoid and its interactions with the protein were different. None of the OCP-like proteins are orange like the OCP<sup>O</sup>. Their colors are red-orange (OCPL1, OCPL3), violet (OCPL4) or red-rose (OCPL2, OCPL5) resembling the color of holo-HCP4 (Fig. S9C, S2B). The absorption spectrum of OCPL1 exhibits similarities to the spectrum of OCP<sup>O</sup>, characterized by three distinct vibrational bands; however, in OCPL1 these bands are red-shifted, with two
maxima at 487 and 516 nm as opposed to 476 and 496 nm in the OCP. OCPL1 also contains a shoulder at 550 nm (which is absent in OCPº). The spectrum of OCPL3 also presents the vibrational bands similar to OCPL1, but the absorption at 487 nm is lower and at 550 nm is higher when compared to OCPL1. The spectra of all the other OCP-like species lost the resolution of the vibrational bands, presenting large maxima at 523-530 nm (OCPL2 and OCPL5), like the HCP4 (530nm), or 545 nm (OCPL4). The red-shifted band of OCPL4 suggests that this sample contains a large quantity of OCP dimers in which the carotenoid is shared by two CTDHs. None of the OCP-like proteins changed their absorbance spectra upon illumination with high intensities of blue or white light, pointing out that they are not photoactive proteins. These results indicate that the position of the carotenoid and its relationship with the OCP amino acids in the OCPº is essential to allow effective photoactivation.

We then tested the ability of the OCP-like proteins to bind to PBSs and to induce PBS fluorescence quenching. The analysis of the quenching activity of the OCP-like species revealed distinctive characteristics. HCP4 exhibited a remarkable quenching activity while HCP1 did not have any quenching activity (Fig. 6, Fig. S3), consistent with prior proposals [42]. OCPL4 (NTD-CTDH) was unable to induce any PBS fluorescence quenching, suggesting (as indicated by the absorbance spectrum) that the carotenoid is mostly bound and shared by the CTDH of OCPL4 dimers and not by NTDs. The other OCP-like proteins were able to induce some PBS fluorescence quenching but less than the free HCP4 indicating that the presence of CTDH hindered the attachment to the PBS. OCPL1 (HCP4-CTDH) induced 20% reduction in fluorescence, indicative of minimal quenching activity suggesting a close conformation of the protein with rather strong interactions between HCP4 and CTDH. Nevertheless, we cannot discount the possibility that the high concentration of apo-protein present in this sample could affect the binding of the holo-protein and decrease in quenching. Conversely, OCPL5 (HCP4-CTD) showed the most substantial quenching activity, with fluorescence rapidly diminishing by over 60%. This observation suggests that the interaction between CTD and HCP4 is weak and that the interaction with the PBS induced opening of the protein, or the protein was already in an open conformation with the carotenoid in the HCP4. OCPL3 (HCP4+CTDH C103F) and OCPL2 (HCP4+CTDH C103F+4aa) also induced a final quenching of around 60%, surpassing the 20% fluorescence reduction seen in OCPL1 (HCP4-CTDH) (Fig. 6). Previous studies proposed that the C103F mutation enables enhanced carotenoid dynamics and a preference for carotenoid transport [41]. In addition, these modifications could generate a weaker interaction between HCP4 and CTDH. OCPL6 (HCP1-CTDH) induced 18% fluorescence quenching suggesting that the addition of the CTDH helps a weak binding of HCP1 to the PBS (Fig 6). The amplitude of fluorescence quenching induced in darkness and under illumination was identical confirming that light has no influence on the OCP-like conformation and that the proteins are not photoactive (Fig. S10A). Interestingly, the presence of FRP largely prevented PBS fluorescence quenching induced by OCPL5 (HCP4-CTD). When OCPL and FRP were premixed before addition to the PBS sample, the inhibition of fluorescence quenching was ever larger (Fig S10B) indicating that FRP could bind to the CTD of OCPL5 and inhibit its binding to the PBS. In addition, when FRP was added to an already quenched OCPL5-PBS complex, it induced an increase of fluorescence indicating that the FRP helped the detachment of OCPL5 from the PBS (Fig. S10C). The fluorescence of OCPL2 (HCP4-CTDH C103F+4aa) remains unaffected by the presence of FRP, consistent with expectations, as OCPL5 is the only one containing the CTD (Fig. S10C).
Thus, the effect of the FRP is either by binding to the CTD of the free protein which decreases the quantity of protein able to interact with the PBS (Fig. S10B), or by helping the protein detachment from the PBS (Fig. S10C). However, the presence of the FRP does not change the absorption properties of OCPL5, indicating that FRP is unable to change the conformation of the protein inducing a change in its color and that this OCP homolog does not reassemble into an OCP<sup>O</sup>-like structure (Fig. S10D).

Size exclusion analysis was used to further characterize the OCP-like proteins. The size was compared to the closed OCP<sup>O</sup> (apparent MW: 40kD, detected at 510 nm) and the open OCP<sup>R</sup> (apparent MW: 60kD, detected at 560 nm), as also shown in [62]. The results revealed that OCPL1, OCPL2 and OCPL3 have a size that is slightly larger than native OCP (Fig. 7) suggesting a weaker interaction between HCP4 and CTDH compared to the NTD-CTD interaction in OCP. Moreover, depending on the position of the carotenoid shared by HCP4 and CTDH (probably in OCPL1 and OCPL3) or more in HCP4 (probably in OCPL2) the size of the protein changes (Fig. 7). In OCPL4, a large peak corresponding to a dimer of the open form was observed. This must be related to the dimers in which two CTDH shared a carotenoid and had a maximum absorbance at 550 nm. In OCPL5 preparation, dimeric open forms were also observed but they absorbed more at 510 nm than at 560 nm suggesting that in these dimers the carotenoid is bound to the HCP4 and not to the CTDs (Fig. 7).

Taking together the absorbance spectra of different OCP-like proteins with PBS fluorescence quenching efficiency and SEC experiments, we can propose that the position of the carotenoid molecule within the OCP-like heterodimer appears to play a pivotal role in its function as fluorescence quencher. When the carotenoid molecule is shared between HCP4 and CTDH and the interaction between them is rather strong, the carotenoid cannot attain the appropriate position inside HCP4 and the protein remains closed, unable to induce fluorescence quenching (OCPL1). When the interaction between HCP4 and CTDH (or CTD) is weaker (OCPL2, OCPL3 and OCPL5) interaction with the PBS helps in the opening of the protein and the carotenoid transfer into HCP4.

Then, when the carotenoid resides inside HCP4 in the correct position, quenching activity can be effectively performed (Fig. 6).

Our proposition suggests that HCP4's interaction with the PBS is facilitated by the positive patch on face 1 (Fig. S5) when the β1 ring is exposed from the ligand cavity (Fig. 2). The results of the docking analysis align with this hypothesis: face 1 remains free for interaction with the PBS, and the overall structure remains sufficiently open for energy quenching (Fig. 7B,C,E). These findings underscore the significance of the entire Car molecule's precise positioning within HCP4 for its quenching activity.

As already mentioned above, SEC analysis showed that the conformation of the HCP4-CTDH heterodimer (OCPL1) occupies an intermediate state between the open OCP<sup>R</sup> and the closed OCP<sup>O</sup> conformations (Fig. 7, Fig. S11). The proposed model depicting the interaction between HCP4 and CTDH involves the interaction of the positively charged patch on face 1 with a negatively charged patch on the CTDH surface (Fig. 5A). Notably, the β1 pore on face 1 of HCP4 is larger than the β2 pore, suggesting that the carotenoid would likely be initially threaded into the cavity through the β1 interface. Additionally, if the HCP4-CTDH indeed behaves as an OCP-like protein, the presumed CAN cavity within CTDH is seamlessly connected with the CAN cavity in HCP4 (Fig. 5B), enabling carotenoid transfer between the two proteins. We hypothesize that CTDH must exist as a monomer during the carotenoid transfer process, as this scenario allows the formation of an elongated tunnel between HCP4 and CTDH (Fig. 5B).
There are however clear differences between OCP and OCP-like assemblies. Phe151 is a conserved residue found in all HCPs 1-9 [37]. Notably, in the *AnaOCP* NTD, this position is occupied by Gln147, and in *SynOCP*, it is Ser147. The presence of Phe151 in HCP4 assumes significance for the HCP4-CTDH interaction. Within HCP4, Phe151 is outward-facing and has the potential to interact with Trp102 situated on the CTDH loop, thereby contributing to the stabilization of the HCP4-CTDH interaction during carotenoid transfer (Fig. 5E).

## 4. Discussion

### 4.1. HCP4 interaction with the PBS

Prior studies have reported at least nine clades of OCP NTD homologs and two OCP CTD homologs [46]. Remarkably, among the four NTD paralogs identified in *Anabaena* (HCP1-HCP4), only HCP4 exhibits the capability for PBS binding and energy quenching. Our study presents the crystal structure of holo-HCP4 and a potential energy quenching mechanism of the PBS, which is initiated by the interaction of the holo-HCP4 face 1 with the PBS core.

The holo-HCP4 crystal lattice presents two potential associations between monomers (each monomer binding a CAN molecule) that could indicate the possibility of the existence of a stable HCP4 dimer (including the dimer found in the asymmetric unit). However, the functional unit of the holo-HCP4 is most likely a monomer, as no physiological function was found for the dimeric form. All HCPs share a mostly α-helical structure and possess a similar globular configuration. However, despite their structural similarities, the diverse functions of each HCP imply that the overall structure alone does not account for their distinct functionalities. Holo-HCP4 exhibits two faces on its protein surface with significantly different electrostatic potentials. We suggest that face 1, featuring a mostly positive potential, binds to the PBS to allow energy quenching. This positive potential patch is not prominently observed in other HCPs, suggesting their potential inability to bind (correctly) to the PBS. Our study's findings are consistent with [22][23] who elucidated the structure of the PBS-OCP<sup>R</sup> complex, identifying OCP residues involved in the interaction with quenched PBS. The analogous residues in the holo-HCP4 situated on face 1, can engage in interactions with the PBS core. Sequence comparison of HCP1 and HCP2 with the HCP4 revealed various alterations in potential PBS interacting residues compared to the HCP4. Some of the changes result in amino acids with different chemical characteristics (Table S4). Moreover, several of these residues are positioned on the surface of face 1 of the HCP1/2 proteins. This observation supports the proposed binding site being on face 1.

In the PBS-OCP<sup>R</sup> structure, the OCP NTD is positioned towards the ApcA core. Holo-HCP4 possesses a structured NTT, like that found in other OCPs, which is absent in other NTD homologs. We suggest that this tail may contribute to the energy quenching mechanism, either by bringing HCP4 and APC chromophores into proximity or by inducing physical alterations in the core structure.

Another unique property of the holo-HCP4 structure is the electrostatic charge distribution within the CAN binding channel. Surrounding the β1 ring, there is a positive charge distribution, while the area around the β2 ring exhibits a negative charge, establishing two distinct environments adjacent to the β rings. This stands in contrast to the CAN cavity charge distribution in HCP1/HCP2, which is predominantly negative. The distinct
charge distribution in the holo-HCP4 channel is posited to be a key characteristic for PBS binding and energy quenching.

The Car type and its configuration are also an essential aspect of the functionality of the HCPs. The planarity of the holo-HCP4 CAN β rings (which aligns with the OCP<sup>R</sup> conformation), differs significantly from the perpendicular β rings configuration in HCP1/HCP2 (similar to the OCP<sup>O</sup> configuration). This difference in carotenoid configuration may indicate another structural element in holo-HCP4 that is not found in other HCPs. Carotenoid and keto-carotenoid synthesis, such as CAN, is elevated under stress conditions like high light and UV-B radiation [63], as well as HCPs levels [37][64]. On the other hand, under low light or non-stress conditions, it is essential to maintain a low concentration of HCPs to prevent the quenching of the PBS.

Activation of photoprotective mechanisms is crucial to prevent an excess of light from reaching the reaction centers. NPQ represents one such photoprotection mechanism induced by holo-HCP4. The significant increase in CAN concentration under UV-B radiation [63], may explain the exclusive binding of CAN by holo-HCP4, which is more abundant under stress conditions, and its binding is more stable compared to other carotenoids (similar to the superior stability of CAN compared to ECN within the CTDH [65]). The photoprotection mechanism should be fast to prevent irreversible photodamage. This raises the question of whether HCP4 acquires the carotenoid immediately after protein synthesis, transforming into a holo-protein, or whether, during high light radiation when photoprotection is required, apo-HCP4 receives the carotenoid from the CTDH, converting into holo-HCP4. HCP4 activation mechanism and the HCP4 disassembly from the PBS requires further investigation.

The fact that HCP4 (and NTD) can bind to the PBS even in the absence of carotenoid (however relative affinities were not determined) suggests that the carotenoid could play a role in binding or in the correct structure and/or electrostatic charge distribution of HCP4 (and NTD). More importantly, our results clearly demonstrate that the presence of the carotenoid is essential for PBS fluorescence quenching. The apo-proteins are able to bind to the PBS but the bound proteins did not induce fluorescence quenching. Although a role of the carotenoid in the fluorescence quenching process was proposed in the past [10], different mechanisms have also been suggested [11][66][67], this is the first time that it is clearly demonstrated that binding of HCP4 and NTD to the PBS is not sufficient to induce a quenched state.

## 4.2. Carotenoid transfer from membrane to HCP4 by CTDH

The fashion by which organisms protect themselves from different types of stress is typically multi-stage and tightly controlled, as the protection mechanisms interfere with normal physiological processes. NPQ by the OCP or HCP4 must be activated only when necessary, so as not to interfere with the normal processes of energy transfer from the PBS to the RCs. OCP activity is controlled by the association of the CTD and NTD domains, that prevent association of the protein with the PBS. Only upon prolonged illumination with strong light is OCP<sup>O</sup> converted to OCP<sup>R</sup>, allowing association and quenching. Quenching by OCP<sup>R</sup> can then be stopped by the activity of the FRP, that releases the OCP from the PBS, allowing reconversion to OCP<sup>O</sup>. The HCP4 protein however is lacking such control. It is constitutively active and cannot be released by FRP. Thus, one would predict that other control mechanisms are required – on the levels of protein expression and assembly into the holo-protein and perhaps by controlled degradation, as well. In this study, we wished to try and identify critical structural determinants in the HCP4 and CTDH that lead to controlled activity, especially in the assembly steps.
Static crystal structures can provide insight into possible dynamic pathways, while biochemical analysis can provide dynamics, with lower molecular details. A more complete picture thus requires the combination of both strategies.

It has been previously shown that photosynthetic membranes contain a variety of carotenoid molecules, that serve different purposes and bind to different molecules. The thickness of the hydrophobic membrane permits the presence of free carotenoids within the lipid bilayer, exhibiting different orientations based on the carotenoid type. The aliphatic chain is typically buried within the lipid bilayer, while the polar groups face outward to the hydrophilic environment [68]. The orientation of CAN in the membrane can vary, either being perpendicular or parallel to the membrane surface [69]. The perpendicular orientation can allow the carotenoid carrier (such as CTDH) to approach the carotenoid and extract it directly from the membrane, by interacting with the protruding polar groups of the CAN. Indeed, the carotenoid uptake by CTDH from the membranes was shown in several previous works [44][45][65][70]. In addition, CTDH is able to transfer the carotenoid to HCPs and OCP[41][65]. Here we confirmed the role of CTDH in the assembly of holo-HCP4. We have shown that the presence of CTDH strongly increased the concentration of holo-protein in *E. coli* cells.

Previously, different hypotheses about how the CTDH binds and transport carotenoids have been suggested [41][71]. Our higher resolution apo-CTDH structure is consistent with previous studies that have reported [44] the presence of two types of potential CTDH dimers. One dimer type can be stabilized by a disulfide bond, termed head-to-head. In this assembly, two CTDH proteins mimic the OCP in shape, and would contain a continuous cavity for carotenoid binding. The presence of the disulfide bond would also allude that this crystallographically identified dimer is physiologically relevant. The second dimer type seen in the crystal structure is between each monomer's β-sheets, termed back-to-back. It was previously suggested that the back-to-back apo-CTDH dimer uptakes the carotenoid molecule, and reorganizes to a head-to-head conformation which encapsulates the carotenoid until the delivery to the HCP/OCP [44]. In our apo-CTDH structure, a molecule of malonic acid (MLA) from the crystallization liquor tightly binds to the internal cavity at the site where the carotenoid binds in the OCP-CTD. If the MLA molecules indeed represent the site of the binding of the β-rings, the holo-CTDH cannot be formed in the exact orientation of the head-to-head dimer in the crystal, since the CAN chain is much longer than the distance between the two MLA molecules (Fig. S12). The CTDH head-to-head conformation may have a different biological function, such as stabilizing the protein until required for transport. We have attempted to obtain holo-CTDH crystals, and while in some cases purple crystals were obtained, they did not exhibit clear presence of bound carotenoid in the electron density maps. This observation suggests that the carotenoid is very loosely bound or is highly flexible, aligning with the protein's role as a carrier for carotenoids.

Small-angle X-ray scattering (SAXS) experiments have elucidated the structure of holo-C-terminal domain-like Car proteins (CCPs), indicating that the two CTD monomers adopt a head-to-head position with a space and twist between them. This differs from the head-to-head apo-CTDH crystal structure, and enables the accommodation of the Car inside the created cavity [72]. While the structure of the holo-CCP can perhaps give insight into the holo-CTDH structure, further study is necessary to actually obtain the structure of the holo-CTDH. We propose that the CTDH is either active in the back-to-back conformation, or perhaps even as a monomer during carotenoid transfer. It is also possible that the head-to-head conformation (with a twist and a
space between the monomers, as observed in the structure of the holo-CCP) exists during Car transport, stabilizing the holo-CTDH monomer until the Car is transported to the HCP4.

Unlike in the CTDH head-to-head dimer, docking analysis of the CTDH and HCP4 monomers shows that the cavity that can be formed between the two proteins, can be modeled as a continuous void from the CTDH monomer to the HCP4, allowing the CAN molecule to be transferred. A recent study carefully followed carotenoid uptake from artificial liposomes by the apo-CTDH [73]. It was shown that AnaCTDH dimerization does not affect the carotenoid transfer and that the carotenoid transfer is faster for CAN than for ECH. These results support our suggestion that holo-CTDH has to pass through a stage where the head-to-head dimer opens up to present the cavity, either by monomerization or by moving a monomer to a second position as a back-to-back dimer) during carotenoid transfer. A model for the process of Car uptake based on the results presented here is shown in Fig. 8.

### 4.3. OCP-like species

Since the interaction between the HCP4 and CTDH is not stable, studying this process poses challenges. To better understand this interaction, OCP-like proteins were constructed in which the interaction was stabilized by a linker between those proteins. The fusion protein containing HCP4 and native CTDH (OCPL1) is apparently in a weakly closed conformation (i.e. not as compact as in native OCP) in which the carotenoid is shared between the two proteins with a rigid conformation (suggested by the presence of vibrational bands in its absorbance spectrum). However, OCPL1 protein is reddish (compared to OCP<sup>O</sup>) and the vibrational bands are red shifted. As predicted by the models, the interaction between the HCP4 and CTDH was weaker than in OCP giving a more open structure (Fig. 7, S11). While the native CTDH seemed to impose a rigid structure on the Car, the modifications of CTDH (C103F and C103F+4aa) induced a larger mobility of the carotenoid (supported by the disappearance of distinct vibrational bands and red shifted absorbance band in OCPL2 and OCPL3) and a partial transfer of the Car into the HCP4. None of these OCP-like proteins was photoactive indicating that the interaction of the carotenoid with specific amino acids in the NTD and the CTD are essential for the photoactivation. Nevertheless, the interaction of these proteins (presenting a weaker interaction between the subunits) with the PBS induced a partial opening of the protein allowing the holo-HCP4 binding to the PBS and fluorescence quenching (Fig. 4). In the fusion proteins, the interaction between HCP4 and CTD (OCPL5) or between NTD and CTDH (OCPL4) was so weak that most of the proteins were in an open conformation with the Car in the HCP4 (in OCPL5) or in the CTDH (OCPL4). A recent study with chemically modified OCP that limited OCP<sup>O</sup> opening to form a complete OCP<sup>R</sup> also showed some photoactive quenching ability [74].

It was proposed that fusion of primitive genes HCP4 and CTDH gave rise to a primitive OCP [37]. Our results suggest that the ancestor HCP4 and CTDH were very different from the proteins that presently exist or that the fusion of genes and addition of a linker were not sufficient to give a photoactive OCP. Mutations were needed to allow photoinduction of protein opening and transfer of the carotenoid into the NTD.

## Funding
This work was supported by the Israel Science Foundation (ISF 1596/21 to N.A.) and was supported by grants to D.K. obtained from the Agence Nationale de la Recherche (RECYFUEL [ANR-16-CE05-0026] and DynOCP [ANR-18-CE11-0005-03]), and by the Centre National de la Recherche Scientifique (CNRS) and the Commissariat à l'Energie Atomique et énergies alternatives (CEA).

**CRediT authorship contribution statement**

**Jenia Sklyar:** Methodology, Investigation, Validation, Writing – original draft, Writing – review & editing.

**Adjélé Wilson:** Methodology, Investigation, Writing – review & editing. **Diana Kirilovsky:** Project administration, Conceptualization, Writing – review & editing. **Noam Adir:** Project administration, Supervision, Conceptualization, Writing – review & editing, Resources, Funding acquisition.

**Declaration of competing interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Data availability**

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the following accession codes: 8QX5 (Holo-HCP4) and 8QX7 (Apo-CTDH). All other data associated with the study are available upon request.

**Acknowledgments**

We would like to thank Dr. Anat Shahar from the National Institute for Biotechnology in the Negev (NIBN), as well as the beamline staff of I04 at the Diamond Light Source (Oxfordshire, UK) for data collection and their support. We would like to thank the staff of the European Synchrotron Radiation Facility (ESRF) and EMBL Grenoble for assistance and support in using beamlines ID30-B. We thank the staff of the Technion Center for Structural Biology (TCSB), Lokey Center for Life Sciences and Engineering; We would like to thank Dr. Yael Pazy-Benhar for helping with data collection, and Dikla Hiya for the help in protein crystallization.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at

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[73] K. Likkei, M. Moldenhauer, N. N. Tavraz, E. G. Maksimov, N. N. Sluchanko, and T. Friedrich, "Lipid composition and properties affect protein-mediated carotenoid uptake efficiency from membranes," *Biochim. Biophys. Acta - Biomembr.*, vol. 1866, no. 1, p. 184241, Oct. 2024, doi: 10.1016/j.bbamem.2023.184241.

[74] Y. B. Slonimskiy, E. G. Maksimov, E. P. Lukashev, M. Moldenhauer, T. Friedrich, and N. N. Sluchanko, "Engineering the photoactive orange carotenoid protein with redox-controllable structural dynamics and photoprotective function," *Biochim. Biophys. Acta - Bioenerg.*, vol. 1861, no. 5-6, 2020, doi: 10.1016/j.bbabio.2020.148174.
## Figure Legends

**Fig. 1: Holo-HCP4 crystal structure.** (A) Ribbon diagram of the two holo-HCP4 monomers in the asymmetric unit. Each monomer binds a single CAN molecule (depicted in beige stick representation). (B) section of the Fo-Fc difference electron density map (contoured at 3σ) obtained following MR with the initial model lacking the CAN molecule, overlayed over the coordinates of the CAN molecule.

**Fig. 2: Holo-HCP4 surface charge distribution around β1 and β2 CAN rings,** colored by electrostatic potential and calculated by APBS as implemented in Pymol (scale from -3.3 kT/e (red) to +3.3 kT/e (blue)). The protein shown from above face 1 and (after rotating by 180°) from face 2 (surrounding the β1 and β2 rings, respectively).

**Fig. 3: Different CAN configurations and electrostatic charge distributions exist within CAN binding cavity in HCP4 and other HCPs.** (A) HCP4 (PDB:8QX5) (B) HCP1 (PDB:5FCX) (C) HCP2 (PDB:6MCJ) (D) RCP (PDB:4XB4). The electrostatic potential was calculated by APBS as implemented in Pymol (scale from -3.3 kT/e (red) to +3.3 kT/e (blue)). All the Car molecules are in stick representation. Hydrogen atoms positions were not experimentally determined and are shown for visualization only. (E) HCP4 CAN configuration. (F) OCP<sup>R</sup> (PDB:7SCC) CAN configuration. (G) OCP<sup>O</sup> (PDB: 5HGR) CAN configuration. (H) HCP1 CAN configuration. (I) HCP2 CAN configuration. HCP4 CAN has a planar conformation, similar to the active OCP<sup>R</sup> form.

**Fig. 4: Interaction of apo/holo HCP4 and NTD with the PBS.** Syn PBS (A and D) and Ana PBS (C and F) were incubated (30 min) in darkness in the presence of Holo-HCP4 and Apo-HCP4 in 1.4 M phosphate. The Syn PBS were also incubated (30 min) in darkness in the presence of Holo-NTD and Apo-NTD in 1.4 M phosphate (B and E). The complexes were isolated using sucrose gradient centrifugation and the blue band was collected and analyzed. Panels A, B and C show fluorescence spectra of isolated SynPBS/AnaPBS (black) and re-isolated PBS after incubation in the presence of Holo-HCP4 and Syn Holo-NTD (red) or Apo-HCP4 and Syn Apo-NTD (blue). Panels D, E, and F show the polypeptide composition of the blue band isolated from the different sucrose gradients corresponding to the fluorescence spectra shown in panels A, B and C. Coomassie blue stained SDS-PAGE (upper panels) and antibody detection of HCP4 and NTD (lower panels) are shown: D) Syn PBS interaction with HCP4: 1-isolated HCP4, 2-Syn PBS/holo-HCP4, 3-Syn PBS/apoHCP4, 4-Syn PBS alone; E) Syn PBS interaction with Syn NTD: 1-isolated NTD, 2-Syn PBS/holo-NTD, 3-Syn PBS/apoNTD, 4-Syn PBS alone; F) Ana PBS interaction with HCP4: 1-isolated HCP4, 2-Ana PBS/holo-HCP4, 3-Ana PBS/apoHCP4, 4-Ana PBS alone. The HCP4 and NTD-Syn proteins were detected using different antibodies: a: anti-Athrospira OCP, b: anti-Ana OCP, c: anti Syn OCP and d: the anti-AnaHCP1 (anti-All1123). The isolated HCP4 and Syn NTD were also shown in the gel and western blot. Each lane contains 10 µL of a solution containing 0.5 µM PBS.

**Fig. 5: Docking result of HCP4 onto Apo-CTDH structure.** (A) HCP4-CTDH top 10 docking results, showing the possible interaction of HCP4 and CTDH. The purple surface represents HCP4, while the ribbon representation in different colors depicts the CTDH's possible interactions with HCP4 based on the predictions from PyDock [57]. (B) Cartoon representation of the potential HCP4-CTDH interaction. (C) Surface electrostatic potential of HCP4-CTDH showing the ligand cavity formed between HCP4 and CTDH during interaction. The HCP4 and CTDH monomers are rotated in 90° relative to each other. HCP4 and CTDH exhibit contrasting charges on their protein surfaces, allowing their interaction upon carotenoid transfer. (D) Side view of the CTDH-HCP4 complex showing the suggested carotenoid cavity during Car transfer. HCP4 Phe151 and CTDH Trp102 residues are highlighted. (E) Enlarged view of the interaction between HCP4 Phe151 and CTDH Trp102.
**Figure 6: Synechocystis PBS fluorescence quenching induced by different OCP-like proteins and HCPs.**
OCPL1 (HCP4-CTDH) in pink, OCPL2 (HCP4-CTDH C103F+4aa) in green, OCPL3 (HCP4-CTDH C103F) in blue, OCPL4 (NTD-CTDH) in violet, OCPL5 (HCP4-CTD) in black, OCPL6 (HCP1-CTDH) in red, holo-HCP1 (in orange) and holo-HCP4 inazure. OCP-like proteins were added (indicated by an arrow) to a solution of PBSs in 1.4 M phosphate in darkness. The decrease in the fluorescence was monitored in a PAM fluorometer.

**Fig. 7: Size exclusion (SEC) analysis of OCP-like proteins.** SEC chromatograms measured at 560 nm (OCP<sup>R</sup> form) and 510 nm (OCP<sup>O</sup> form). Under the experimental conditions used (150mM NaCl, 8°C, proteins at a concentration of 7µM), *SynOCP* appears as a monomer with an apparent MW of 37-40 while the red photoactivated OCP<sup>R</sup> has an apparent MW of 66. The red OCP was obtained by pre-illumination with high intensities of white light. Illumination was maintained during throughout the analysis. (A) OCPL1 (HCP4-CTDH). (B) OCPL2 (HCP4-CTDH C103F+4aa). (C) OCPL3 (HCP4-CTDH C103F). (D) OCPL4 (NTD-CTDH). (E) OCPL5 (HCP4-CTD).

**Fig. 8: Schematic representation of the suggested models for carotenoid transfer between CTDH and HCP4.** CAN is shown in brown, holo-CTDH in dark orange, apo-CTDH in light brown, apo-HCP4 in light purple and holo-HCP4 in purple. (A) Apo-CTDH monomer uptakes CAN from the membrane into the ligand cavity (1). It is possible that, during this process, certain monomers adopt a twisted head-to-head conformation to stabilize the CAN molecule in the aqueous environment. Then CAN is transferred to the apo-HCP4 (2), leading to the formation of a HCP4-CTDH heterodimer (3). The HCP4-CTDH heterodimer disassembles, and apo-HCP4 transforms into of the holo-HCP4 form and the holo-CTDH reverts to its apo- form (4). (B) A monomer from the apo-CTDH dimer, arranged in a back-to-back conformation, uptakes CAN from the membrane (1). One of the CTDH monomers contains the CAN molecule (2), which is transferred to the apo-HCP4, forming HCP4-CTDH heterodimer (3). The HCP4-CTDH heterodimer separates, forming holo-HCP4 and apo-CTDH (4).
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 8</figcaption></figure>
Supplementary data

# Insights into Energy Quenching Mechanisms and Carotenoid uptake by Orange Carotenoid Protein Homologs: HCP4 and CTDH

Jenia Sklyar<sup>a,‡</sup>, Adjélé Wilson<sup>b,‡</sup>, Diana Kirilovsky<sup>b,*</sup>, and Noam Adir<sup>a,*</sup>

<sup>a</sup> Schulich Faculty of Chemistry, Technion, Haifa 3200003, Israel

<sup>b</sup> Université Paris-Saclay, CNRS, CEA, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif sur Yvette, France

<sup>‡</sup>These authors contributed to the work equally and should be regarded as co-first author.

<sup>*</sup> Corresponding authors:

**Noam Adir:**

Schulich Faculty of Chemistry, Technion, Haifa 3200003, Israel

Email: noam@ch.technion.ac.il

Tel: +97248292141

**Diana Kirilovsky:**

Université Paris-Saclay, CNRS, CEA, Institute for Integrative Biology of the Cell (I2BC),

91198 Gif sur Yvette, France

Email: diana.kirilovsky@cea.fr

Tel: 33 1 69 08 95 71
Supplementary Tables

<table><caption>**Table S1: Sequences of primers used in this study.** Listed primers for construction of plasmids for expression in *E. coli* as described in the Methods section.</caption><thead><tr><th>Primer names</th><th>Primer sequences</th></tr></thead><tbody><tr><td colspan="2"><strong>1- Construction of pDuet-HCP4-CTDH C103F plasmid</strong></td></tr><tr><td>Fall4940NdeI</td><td>5'-GCA TTC CAT ATG AAA GCT GCT GAG TCT CTC CCT AAT ATC-3'</td></tr><tr><td>Rall4940XhoI</td><td>5'-GCCGGC CTCGAG CTT ATT GAT GAC AGC GCA-3'</td></tr><tr><td colspan="2"><strong>2- Construction of OCP-like 1, 2 and 3 plasmids</strong></td></tr><tr><td>Fall4941NcoI</td><td>5'-GA TAT ACC ATG GGC ACA TTC ACT TCT GAT TCC GC-3'</td></tr><tr><td>Rall4940NotI</td><td>5'-CAT TAT GCG GCC GC CTT ATT GAT GAC AGC GCA CCC-3'</td></tr><tr><td>Fall4940HIS</td><td>5'-CAC CAC CAC CAC CAC CAC TAG TCT TGA TTA ATT ACT TTA ACT TTG-3'</td></tr><tr><td>Rall4940HIS</td><td>5'-GTG GTG GTG GTG GTG GTG CTG TTC ACG ACG CAA AGC CAG TAA CTC-3'</td></tr><tr><td>Fall4941-40 fus</td><td>5'-GTTGACCCCTTAGCTGATAAAGCTGCTGAGTCTCTCCCTAATATC-3'</td></tr><tr><td>Rall4941-40 fus</td><td>5'-ATCAGCTAAGGGGTCAACACCCATATCAGCTAC-3'</td></tr><tr><td>FOCP-like</td><td>5'-CGCGTTGCTGAGCCCGTAGTTCCTCCCAAAGCTGCTGAGTCTCTCCC-3'</td></tr><tr><td>ROCP-like</td><td>5'-GGGAGGAACTACGGGCTCAGCGATGCGATCAGCTAAGGGGTCAACAC-3'</td></tr><tr><td>F-Link A</td><td>5'-TTTGATGCAGCTAAACTAGACGGCTACACACGCGTTGCTGAGCCCGTAG-3'</td></tr><tr><td>R-Link A</td><td>5'-TGTGTAGCCGTCTAGTTTAGCTGCATCAAAACCCATATCAGCTACTAC-3'</td></tr><tr><td>F-Link B</td><td>5'-AAAGATATTTCCCAGCGTGTTCAGTCACCATTGCAGGAATCACAGAGCC-3'</td></tr><tr><td>R-Link B</td><td>5'-GGTGACTTGAACACGCTGGGAAATATCTTTGGGAGGAACTACGGGCTCAG-3'</td></tr><tr><td colspan="2"><strong>3- Construction of OCP-like 4 plasmid</strong></td></tr><tr><td>F-Duet-NTDsyn</td><td>5'-GAAGGAGATATACATATGCCATTCACCATTGACTCTGCCCGC-3'</td></tr><tr><td>R-NTDsyn-link</td><td>5'-CTAGTTTAGCTGCATCAAAGCCCATGTCCACCACGGCATTACGCAATAC-3'</td></tr><tr><td>F-NTDsyn-link</td><td>5'-GCCGTGGTGGACATGGGCTTTGATGCAGCTAAACTAGACGGCTAC-3'</td></tr><tr><td>R-Duet-CTDH</td><td>5'-GAGATCTGCCATATGCTTATTGATGACAGCGCACCCCGATTGGCA-3'</td></tr><tr><td colspan="2"><strong>4- Construction of OCP-like 5 plasmid</strong></td></tr><tr><td>F-Duet-HCP4</td><td>5'-GAAGGAGATATACATATGACATTCACTTCTGATTCCGCTTCAACC-3'</td></tr><tr><td>R-HCP4-link</td><td>5'-GTCCTTACCAGCGGTGAAACCCATATCAGCTACTACTTTGCGGAGAAC-3'</td></tr><tr><td>F-HCP4-link</td><td>5'-GTAGTAGCTGATATGGGTTTCACCGCTGGTAAGGACGGCAAACGCATCGCTGAG-3'</td></tr><tr><td>R-Duet-CTDSyn</td><td>5'-GAG ATC TGC CAT ATG TGT AAC ATT AAC TTA TGA TAT TGA TTT G-3'</td></tr><tr><td colspan="2"><strong>5- Construction of OCP-like 6 plasmid</strong></td></tr><tr><td>F-Duet-HCP1</td><td>5'-GAA GGA GAT ATA CAT ATG ACT TTT ACA CAA ACA AAC GAT CCC-3'</td></tr></tbody></table>
<table><caption>**Table S2.** Data collection and refinement statistics.</caption><tr><td>R-HCP1-link</td><td>5'-C TAG TTT AGC TGC ATC AAA ACC CAT TGG GGA AAC GTA ATC ACG GAA AAG-3'</td></tr><tr><td>F-HCP1-link</td><td>5'-C GTT TCC CCA ATG GGT TTT GAT GCA GCT AAA CTA GAC GGC TAC-3'</td></tr></table>

<table><thead><tr><th></th><th>Holo-HCP4<br>(PDB:8QX5)</th><th>Apo-CTDH<br>(PDB:8QX7)</th></tr></thead><tbody><tr><td>Wavelength (Å)</td><td>0.9537</td><td>0.9763</td></tr><tr><td>Resolution range (Å)</td><td>29.73-1.90 (1.90-1.94)</td><td>47.78-1.95 (1.95-2.00)</td></tr><tr><td>Space group</td><td>P 41 2 2</td><td>C 1 2 1</td></tr><tr><td>Unit cell</td><td>59.47 59.47<br>183.78<br>90.00 90.00<br>90.00</td><td>111.93 52.92 48.60 90.00 96.88<br>90.00</td></tr><tr><td>Total reflections</td><td>710429 (43746)</td><td>135114 (8977)</td></tr><tr><td>Unique reflections</td><td>27148 (1679)</td><td>20695 (1410)</td></tr><tr><td>Multiplicity</td><td>26.2 (26.1)</td><td>6.5 (6.4)</td></tr><tr><td>Completeness (%)</td><td>99.9 (99.1)</td><td>99.7 (97.4)</td></tr><tr><td>Mean I/sigma(I)</td><td>25.0 (4.4)</td><td>12.8 (2.4)</td></tr><tr><td>Wilson B-factor (Å<sup>2</sup>)</td><td></td><td></td></tr><tr><td>R-merge</td><td>0.089 (0.797)</td><td>0.076 (0.677)</td></tr><tr><td>R-meas</td><td>0.092 (0.827)</td><td>0.091 (0.806)</td></tr><tr><td>R-pim</td><td>0.024 (0.221)</td><td>0.049 (0.433)</td></tr><tr><td>CC1/2</td><td>1.000 (0.955)</td><td>0.998 (0.843)</td></tr><tr><td>Reflections used in<br>refinement</td><td>27068 (2620)</td><td>20679 (2586)</td></tr><tr><td>Reflections used for R-free</td><td>1357 (115)</td><td>1994 (156)</td></tr><tr><td>R-work</td><td>0.1897 (0.2424)</td><td>0.1950 (0.2907)</td></tr><tr><td>R-free</td><td>0.2128 (0.3364)</td><td>0.2421 (0.3702)</td></tr><tr><td>Number of non-hydrogen<br>atoms</td><td>2786</td><td>2022</td></tr><tr><td>Macromolecules</td><td>2586</td><td>1911</td></tr><tr><td>Ligands</td><td>2</td><td>2</td></tr><tr><td>Solvent</td><td>200</td><td>111</td></tr><tr><td>Protein residues</td><td>313</td><td>250</td></tr><tr><td>RMS (bonds, Å)</td><td>0.008</td><td>0.008</td></tr><tr><td>RMS (angles, °)</td><td>1.03</td><td>0.95</td></tr><tr><td>Ramachandran favored (%)</td><td>99.03</td><td>96.75</td></tr><tr><td>Ramachandran allowed (%)</td><td>0.97</td><td>3.26</td></tr><tr><td>Ramachandran outliers (%)</td><td>0.00</td><td>0.00</td></tr><tr><td>Rotamer outliers (%)</td><td>0.78</td><td>1.01</td></tr><tr><td>Clashscore</td><td>7.73</td><td>4.71</td></tr><tr><td>Average B-factor (Å<sup>2</sup>)</td><td>30.38</td><td>37.47</td></tr></tbody></table>

Statistics for the highest-resolution shell are shown in parentheses.
<table><caption>**Table S3. Structural comparison between AnaHCP4 and HCPs/OCP homologs.** The table shows Cα structural alignment between Ana holo-HCP4 and: Ana HCP1, *Tolypothrix* HCP2 (Toly HCP2), *Synechocystis* PCC6803 RCP (*Syn* RCP), Ana OCP NTD structures, with the number of the aligned residues in parenthesis.</caption><thead><tr><th rowspan="2">PDB code</th><th rowspan="2">Protein</th><th>Cα RMSD (Å)</th><th>Cα RMSD (Å)</th></tr><tr><th>Chain A</th><th>Chain B</th></tr></thead><tbody><tr><td>5FCX</td><td>Ana HCP1</td><td>1.11 (122 res)</td><td>1.056 (116 res)</td></tr><tr><td>6MCJ</td><td>Toly HCP2</td><td>0.725 (106 res)</td><td>0.832 (110 res)</td></tr><tr><td>4XB4</td><td>Syn RCP</td><td>0.859 (123 res)</td><td>0.882 (122 res)</td></tr><tr><td>5HGR</td><td>Ana OCP NTD</td><td>0.975 (130 res)</td><td>0.914 (125 res)</td></tr></tbody></table>
<table><caption>Table S4: Comparative analysis of PBS interacting residues with SynOCP and AnaOCP based on the model by Domínguez-Martín et al. (2022) [1], compared with equivalent residues from HCP4 face 1, HCP1, and HCP2. Amino acids exhibiting distinct characteristics from the interacting residues of both OCP and HCP4 are marked in yellow.</caption><thead><tr><th>Interacting residue on the PBS</th><th>SynOCP (3MG1) interacting residue</th><th>AnaOCP NTD (5HGR) interacting residue</th><th>HCP4 interacting residue</th><th>Is the interaction can be done by HCP4, in comparison to SynOCP and AnaOCP?</th><th>HCP2 interacting residue</th><th>HCP1 interacting residue</th></tr></thead><tbody><tr><td>Pro68 (APCA)</td><td>Ile53</td><td>Val53</td><td>Pro57</td><td>yes</td><td>Pro41</td><td>Pro44</td></tr><tr><td>Gln60<br>Ly61 (APCA)</td><td>Pro56</td><td>Pro56</td><td>Thr60</td><td>yes</td><td>Pro44</td><td>Pro47</td></tr><tr><td>Ly61 (APCA)<br>Arg62</td><td>Gly57</td><td>Gly</td><td>Gly61</td><td>No change</td><td>Asn45</td><td>Gly57</td></tr><tr><td>Gly69<br>Ly61 (APCA)<br>Arg62</td><td>Ala58</td><td>Ala</td><td>Ala62</td><td>No change</td><td>Ser46</td><td>Ala58</td></tr><tr><td>Thr50<br>Glu54 (APCB)</td><td>Ser60</td><td>Ser60</td><td>Arg64</td><td>yes</td><td>Asp48</td><td>Thr51</td></tr><tr><td>Asn47<br>Thr50<br>Leu140<br>Arg131</td><td>Gln62</td><td>Gln</td><td>Gln66</td><td>No change</td><td>No residue</td><td>Ala64</td></tr><tr><td>Thr50<br>Glu54<br>Val136<br>Leu140 (APCB)</td><td>Leu63</td><td>Phe63</td><td>Leu57</td><td>yes</td><td>Ile65</td><td>Pro62</td></tr><tr><td>Ser139 (APCB)</td><td>Thr97</td><td>Thr97</td><td>Ser100</td><td>yes</td><td>Gly84</td><td>Glu90</td></tr></tbody></table>
<table><tr><td>Lys58<br>Ser139<br>(APCB)</td><td>Ser100</td><td>Thr100</td><td>Ile104</td><td>no</td><td>Ala87</td><td>Ser93</td></tr><tr><td>Ser139<br>(APCB)</td><td>Trp101</td><td>Trp101</td><td>Leu105</td><td>yes</td><td>Leu88</td><td>Leu94</td></tr><tr><td>Glu54</td><td>Ser102</td><td>Ser</td><td>Ser106</td><td></td><td>Ser89</td><td>Gly95</td></tr><tr><td>Arg249<br>(CPCG)</td><td>Pro103</td><td>Pro103</td><td>Asn107</td><td>yes</td><td>Pro90</td><td>Asp96</td></tr><tr><td>Glu54<br>(APCB)</td><td>Ile105</td><td>Ile105</td><td>Thr109</td><td>yes</td><td>Ala92</td><td>Thr98</td></tr><tr><td>Lys234<br>(CPCG)</td><td>Glu146</td><td>Asp146</td><td>Asp80</td><td></td><td>Glu133</td><td>Gly139</td></tr><tr><td>Tyr73<br>(APCA)</td><td>Ser147</td><td>Gln147</td><td>Phe151</td><td>yes</td><td>Phe134</td><td>Phe140</td></tr><tr><td>Gly74<br>Glu75</td><td>Gly148</td><td>Gly</td><td>Gly152</td><td>No change</td><td>Asp135</td><td>Glu141</td></tr></table>

Supplementary Figures
A

Plasmid: 4941HIS

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

B

Plasmid: 4941HIS-4940

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

C

Plasmid: 4941HIS-4940C103F Stag

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

**Fig. S1: Expression of *Anabaena* HCP4 gene (All4941) in *E. coli* cells in the absence or presence of the CTDH gene (All4940).** The presence of the CTDH gene and its expression allowed the synthesis and isolation of larger holo-HCP4 quantities (based on 530/280 nm ratio). (A) Absorbance spectra of holo-HCP4 expression in the absence of CTDH gene. (B) Absorbance spectra of holo-HCP4 expression in the presence of CTDH gene. (C) Absorbance spectra of holo-HCP4 expression in the presence of CTDH C103F gene. All the spectra presented herein are after the first stage of purification (Ni-affinity column). Each expression construct is shown above the appropriate spectra.

A

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

C

<figure><img src="image_19.png" /></figure>
D

<figure><img src="image_20.png" /><figcaption>**Fig. S2: Holo-HCP4 purification and characterization.**</figcaption></figure>

(A) SPS-PAGE of holo-HCP4 purified by Size exclusion (SEC). Lane 1- holo-HCP4 fraction.

(B) Holo-HCP4 fraction which was crystallized. (C) Absorbance spectra of holo-HCP4 fraction used for crystallization. SEC results indicated the presence of monomeric form of the holo-HCP4. The SDS-PAGE analysis showed a primary band at around 19kDa (lane 1), which is in line with the HCP4 calculated molecular weight (MW) of 19.2 kDa. (D) Holo-HCP4 crystals (scale bar = 300µm).

<figure><img src="image_21.png" /><figcaption>**Fig. S3: Anabaena PBS fluorescence quenching induced by different HCPs.** HCP1 (black open circle), HCP2 (blue open square), HCP3 (green close circle) and HCP4 (red open triangle).</figcaption></figure>
A

<figure><img src="image_22.png" /></figure>

B

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

**Fig. S4: Holo-HCP4 and holo-HCP1 surface electrostatic potential.** Calculated by APBS as implemented in Pymol (scale from -3.3 kT/e (red) to +3.3 kT/e (blue)). (A) Holo-HCP4 coordinates from this study. (B) Holo-HCP1 (5FCX). Both proteins are shown from above face 1 and 2 (surrounding the β1 and β2 ring).
<figure><img src="image_26.png" /><figcaption>**Fig. S5.** Holo-HCP4 residues on face 1 create a positive patch adjacent to the CAN cavity aperture on the HCP4 surface. Residues suggested to interact with the PBS are highlighted.</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>**Fig. S6:** Superposition of *AnaAPC* hexamer (PDB:7EYD) with *SynOCP*<sup>R</sup> (PDB:7SCC). (A) Superposition of *AnaAPC* hexamer with the *SynAPC* hexamer from *SynPBS-OCP*<sup>R</sup> complex (PDB:7SCC). The *SynOCP*<sup>R</sup> is superimposed onto the *SynAPC* hexamer (cartoon representation colored in gray). (B) Close-up view of the potential holo-HCP4 binding site to the *AnaAPC*.</figcaption></figure>
<figure><img src="image_28.png" /></figure>

**Fig. S7: Apo-CTDH structure binding MLA.** (A) Ribbon diagram of apo-CTDH in a back-to-back conformation, binding MLA (represented by sticks) in the Car binding cavity. (B) Two neighboring asymmetric units showing the head-to-head conformation (Top dimer). Cys residues are represented in spheres form a disulfide bond that is shared by two adjacent asymmetric units.

<figure><img src="image_29.png" /></figure>

**Fig. S8: CTDH and CTD structure comparison.** Superimposition of apo-CTDH (yellow) and AnaCTD (pink, PDB:5HGR) monomers showing the interacting loop with the NTD (indicated by an arrow).
<figure><img src="image_30.png" /><figcaption>**Fig. S9: Absorbance spectra and visible color of different OCP-like proteins.** (A) Absorbance spectra of OCP-like species: OCPL1 (HCP4-CTDH) in red and OCPL4 (NTD-CTDH) in black. (B) Absorbance spectra of OCPL2 (HCP4-CTDH C103F+4aa) in red, OCPL3 (HCP4-CTDH C103F) in blue and OCPL5 (HCP4-CTD) in black. (C) The visible color of OCP-like proteins as described in the panel.</figcaption></figure>
A

<figure><img src="image_31.png" /></figure>

B

<figure><img src="image_32.png" /></figure>

C

<figure><img src="image_33.png" /></figure>

D

<figure><img src="image_34.png" /></figure>

**Fig. S10: FRP effect on isolated OCPL and on PBS induced fluorescence quenching (A) PBS**
fluorescence quenching induced by OCPL3 (blue), OCPL4 (red) and OCPL5 (black) in the dark and under high intensities of blue-green light (dashed line). OCP-like proteins were added at 50 sec (indicated by an arrow). (B) PBS fluorescence quenching induced by OCPL5 in the absence (black closed circles) and in the presence of FRP under two conditions: 1) OCP and FRP were premixed and added to PBS at 50 sec (black open circles). 2) OCP and FRP were added at 50 sec without being premixed (blue open squares). (C) OCPL5 and OCPL2 were added at 15 sec, the PBS fluorescence quenching was induced and at 600 sec FRP was added in two samples to observe the effect of FRP on attached OCPLs. OCPL5 alone (open black circles), OCPL5 +FRP (closed black circles), OCPL2 alone (open red squares), OCPL2 + FRP (closed red squares). (D) Absorbance spectra of OCPL5 before (black) and after (red) 5 min incubation with FRP (ratio 5 FRP per 1 OCPL5).
<figure><img src="image_35.png" /><figcaption>**Fig. S11: HCP4-CTDH interaction superimposed to OCP<sup>R</sup> and OCP<sup>O</sup>.** Cartoon representation of the model of the HCP4-CTDH structure (azure), OCP<sup>R</sup> (grey), OCP<sup>O</sup> (yellow) showing the different conformation of the HCP4-CTDH in comparison to OCP NTD/CTD in active OCP<sup>R</sup> and inactive OCP<sup>O</sup> forms.</figcaption></figure>
<figure><img src="image_36.png" /><figcaption>**Fig. S12: CAN docking into apo-CTDH face-to-face dimer.** (A) Apo-CTDH face to face dimer docked with CAN molecule inside potential ligand cavity. (B) Close-up view of the CAN interacting residues showing the potential clashes between the ligand cavity residues and the CAN molecule.</figcaption></figure>

**References**

[1] M. A. Domínguez-Martín *et al.*, “Structures of a phycobilisome in light-harvesting and photoprotected states,” *Nat.* 2022 *609*7928, vol. 609, no. 7928, pp. 835–845, Aug. 2022, doi: 10.1038/s41586-022-05156-4.