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PreprintCited by 4Photosynthetic Processes and MechanismsProtein Structure and DynamicsEnzyme Structure and Function

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Exploring the structural diversity and evolution of the D1 subunit of photosystem II using AlphaFold and Foldtree

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Kim, Tom Dongmin, Pretorius, Daniella, Murray, James W., Cardona, Tanai·2025·Physiologia Plantarum
DOI 10.1101/2025.03.06.641835PMC PMC12096807

Preprint — published as 10.1111/ppl.70284 · Paper page

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Abstract

While our knowledge of photosystem II has expanded to time-resolved atomic details, the diversity of experimental structures of the enzyme remains limited. Recent advances in protein structure prediction with AlphaFold offer a promising approach to fill this gap in structural diversity in non-model systems. This study used AlphaFold to predict the structures of the D1 protein, the core subunit of photosystem II, across a broad range of photosynthetic organisms. The prediction produced high-confidence structures, and structural alignment analyses highlighted conserved regions across the different D1 groups, which were in line with high pLDDT scoring regions. In contrast, varying pLDDT in the DE loop and terminal regions appear to correlate with different degrees of structural flexibility or disorder. Subsequent structural phylogenetic analysis provided a phylogeny that is in good agreement with previous sequence-based studies. Moreover, the phylogeny supports a parsimonious scenario in which far-red D1 and D1 INT evolved from the ancestral form of G4 D1. This study demonstrates the potential of AlphaFold in studies on structural diversity and the evolution of photosynthesis.

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FIGURE 1 Representative structures from AlphaFold predictions and average pLDDT scores by amino acid position. (A) The predicted AF structures from different D1 groups with pLDDT colouring scheme indicative of the pLDDT score by position. Every group shows an alignment of 3 representative structures except G0, which is comprised of two structures. (B) The recent CryoEM structure of the G4 D1 of Synechocystis sp. PCC 6803 (PDB ID 7N8O) as a reference. (C) The average pLDDT score by position (black line) overlaid on the sequence alignment of the same D1 dataset (light turquoise) to indicate corresponding amino acid positions. Standard deviation of pLDDT scores is shown in grey. The secondary structure annotation is depicted on top of the plot to illustrate the locational context in D1 and the colour scheme corresponds to that of (B). Helix A to E denote transmembrane helices, Na denotes the N‐terminal helix, eC denotes C‐terminal helix, and ab, cd and de denote α helices in between the transmembrane helices. The D1 grouping in the sequence alignment is indicated with dotted lines on the right side of the plot.
FIGURE 1 Representative structures from AlphaFold predictions and average pLDDT scores by amino acid position. (A) The predicted AF structures from different D1 groups with pLDDT colouring scheme indicative of the pLDDT score by position. Every group shows an alignment of 3 representative structures except G0, which is comprised of two structures. (B) The recent CryoEM structure of the G4 D1 of Synechocystis sp. PCC 6803 (PDB ID 7N8O) as a reference. (C) The average pLDDT score by position (black line) overlaid on the sequence alignment of the same D1 dataset (light turquoise) to indicate corresponding amino acid positions. Standard deviation of pLDDT scores is shown in grey. The secondary structure annotation is depicted on top of the plot to illustrate the locational context in D1 and the colour scheme corresponds to that of (B). Helix A to E denote transmembrane helices, Na denotes the N‐terminal helix, eC denotes C‐terminal helix, and ab, cd and de denote α helices in between the transmembrane helices. The D1 grouping in the sequence alignment is indicated with dotted lines on the right side of the plot.CC BY · PubMed Central
FIGURE 2 Structural alignment analysis with mTM‐Align comparing global similarity of the full‐length proteins. (A) The global alignment of 23 representative D1 AF structures from different D1 groups in pLDDT colouring, aligned to the reference G4 D1 (PDB ID:7N8O). The reference structure is depicted alone in (B) for comparison, and (C) depicts the common core illustrating the structurally conserved regions.
FIGURE 2 Structural alignment analysis with mTM‐Align comparing global similarity of the full‐length proteins. (A) The global alignment of 23 representative D1 AF structures from different D1 groups in pLDDT colouring, aligned to the reference G4 D1 (PDB ID:7N8O). The reference structure is depicted alone in (B) for comparison, and (C) depicts the common core illustrating the structurally conserved regions.CC BY · PubMed Central
FIGURE 3 Diversity of DE loops in D1. The reference structure is shown in pink at the bottom left. Sections with high pLDDT scores are shown in magenta and their AA positions are indicated next to them. AF structures are aligned by group, and the reference structure is included in all the alignments for comparison. AF structures are depicted using the pLDDT colouring scheme. QB is shown in green and the lipidic tail is omitted for clarity. The non‐heme iron is shown in red coordinated by a bicarbonate molecule in grey.
FIGURE 3 Diversity of DE loops in D1. The reference structure is shown in pink at the bottom left. Sections with high pLDDT scores are shown in magenta and their AA positions are indicated next to them. AF structures are aligned by group, and the reference structure is included in all the alignments for comparison. AF structures are depicted using the pLDDT colouring scheme. QB is shown in green and the lipidic tail is omitted for clarity. The non‐heme iron is shown in red coordinated by a bicarbonate molecule in grey.CC BY · PubMed Central
FIGURE 4 Details of the surrounding region of Mn4CaO5 cluster and C‐terminus, aligned by group. The reference structure is shown in pink on the top left and the ligands for Mn4CaO5 cluster are labelled next to their atomic depiction. AF structures are aligned with the reference structure for comparison, using pLDDT colouring. Residues after A344 are hidden in G3 and G4 to account for the cleavage by CtpA protease.
FIGURE 4 Details of the surrounding region of Mn4CaO5 cluster and C‐terminus, aligned by group. The reference structure is shown in pink on the top left and the ligands for Mn4CaO5 cluster are labelled next to their atomic depiction. AF structures are aligned with the reference structure for comparison, using pLDDT colouring. Residues after A344 are hidden in G3 and G4 to account for the cleavage by CtpA protease.CC BY · PubMed Central
FIGURE 5 Phylogenetic trees of D1 proteins constructed from amino acid sequences and AF D1 structures. For comparison, SeqML shows the Maximum Likelihood tree of cyanobacterial D1 by Cardona et al. (2015), with G.k. indicating G0 from Gloeobacter kilaueensis. 3Di + AA shows the phylogenetic tree constructed with AF D1 structures by Foldtree (Moi et al. 2023). SeqNJ shows the neighbour‐joining tree based on the D1 sequence dataset used in this study. Scale bars represent AA substitutions per site. 3Di + AA G4 shows the G4 D1 topology of the structural tree in more detail, with D1INT indicating ‘intermediate D1’ as described in Sheridan et al. (2020). G.I. in SeqNJ and 3Di + AA G4 indicates the only D1 sequence found in the genome of Gloeomargarita lithophora, which is considered as the closest living cyanobacterium to the ancestor of the chloroplast. Green and yellow circles indicate the most recent common ancestor (MRCA) of cyanobacteria and primary plastid, respectively. Glaucophyta are shown in magenta.
FIGURE 5 Phylogenetic trees of D1 proteins constructed from amino acid sequences and AF D1 structures. For comparison, SeqML shows the Maximum Likelihood tree of cyanobacterial D1 by Cardona et al. (2015), with G.k. indicating G0 from Gloeobacter kilaueensis. 3Di + AA shows the phylogenetic tree constructed with AF D1 structures by Foldtree (Moi et al. 2023). SeqNJ shows the neighbour‐joining tree based on the D1 sequence dataset used in this study. Scale bars represent AA substitutions per site. 3Di + AA G4 shows the G4 D1 topology of the structural tree in more detail, with D1INT indicating ‘intermediate D1’ as described in Sheridan et al. (2020). G.I. in SeqNJ and 3Di + AA G4 indicates the only D1 sequence found in the genome of Gloeomargarita lithophora, which is considered as the closest living cyanobacterium to the ancestor of the chloroplast. Green and yellow circles indicate the most recent common ancestor (MRCA) of cyanobacteria and primary plastid, respectively. Glaucophyta are shown in magenta.CC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Kim, T. D., Pretorius, D., Murray, J. W., & Cardona, T.
Affiliations
  • Queen Mary University of London (GB)
  • Imperial College London (GB)
ORCID
Venue
Physiologia Plantarum
Publisher
openRxiv
Published
2025-03-07
Volume
177
Issue
3
Pages
e70284-e70284
ISSN
0031-9317 (ISSN-L)
DOI
10.1101/2025.03.06.641835
PMC
PMC12096807
OpenAlex
W4410589506
Cited by
4
References
1
Citations by year
2026: 3 · 2025: 1
FWCI
1.20
Type
journal article
Language
en
Open access
Licence
Locations
  • Physiologia Plantarum (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Spiral (Imperial College London) (submittedVersion)
Funders
  • UK Research and Innovation (MR/Y011635/1, MR/T017546/1, MR/T017546/2)
  • Engineering and Physical Sciences Research Council (EP/S022856/1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
photosystem II, protein structure prediction, structural diversity, DE loop, structural flexibility, intrinsically disordered regions, photosynthetic organisms
MeSH terms
Models, Molecular, Photosynthesis, Phylogeny, Evolution, Molecular, Photosystem II Protein Complex

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