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Accurate prediction by AlphaFold2 for ligand binding in a reductive dehalogenase and implications for PFAS (per-and polyfluoroalkyl substance) biodegradation

Guo, Hao-Bo, Varaljay, Vanessa A., Kedziora, Gary, Taylor, Kimberly, Farajollahi, Sanaz, Lombardo, Nina, Harper, Eric, Hung, Chia, Gross, Marie, Perminov, Alexander, Dennis, Patrick, Kelley-Loughnane, Nancy, Berry, Rajiv·2023·Scientific Reports
DOI 10.1038/s41598-023-30310-xPMC PMC10008544

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Abstract

Despite the success of AlphaFold2 (AF2), it is unclear how AF2 models accommodate for ligand binding. Here, we start with a protein sequence from Acidimicrobiaceae TMED77 (T7RdhA) with potential for catalyzing the degradation of per- and polyfluoroalkyl substances (PFASs). AF2 models and experiments identified T7RdhA as a corrinoid iron-sulfur protein (CoFeSP) which uses a norpseudo-cobalamin (BVQ) cofactor and two Fe 4 S 4 iron-sulfur clusters for catalysis. Docking and molecular dynamics simulations suggest that T7RdhA uses perfluorooctanoic acetate (PFOA) as a substrate, supporting the reported defluorination activity of its homolog, A6RdhA. We showed that AF2 provides processual (dynamic) predictions for the binding pockets of ligands (cofactors and/or substrates). Because the pLDDT scores provided by AF2 reflect the protein native states in complex with ligands as the evolutionary constraints, the Evoformer network of AF2 predicts protein structures and residue flexibility in complex with the ligands, i.e., in their native states. Therefore, an apo-protein predicted by AF2 is actually a holo-protein awaiting ligands.

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Figures

Figure 1 T7RdhA structure. (a) The structure of T7RdhA in complex with BVQ (green) cofactor, two Fe4S4 (purple) clusters and the PFOA (purple) substrate. In the protein cartoon α-helices are in red, β-sheets are in yellow and coils in white. (b) A closer view of the BVQ cofactor, Fe4S4 clusters and the binding Cys residues, and the PFOA substrate. Fe in purple, S in yellow, Co in green, F in pink, C in cyan, N in blue and O in red. All structures are plotted using VMD66. (c) A wire presentation of the secondary structures in T7RdhA plotted by PDBsum37. Note that the red dots on top of the amino acids indicate that the residue is involved in cofactor/substrate binding. The β-strands form three sheets (A, B and C). Positions of β- and γ-turns in the loop regions are labeled.
Figure 1 T7RdhA structure. (a) The structure of T7RdhA in complex with BVQ (green) cofactor, two Fe4S4 (purple) clusters and the PFOA (purple) substrate. In the protein cartoon α-helices are in red, β-sheets are in yellow and coils in white. (b) A closer view of the BVQ cofactor, Fe4S4 clusters and the binding Cys residues, and the PFOA substrate. Fe in purple, S in yellow, Co in green, F in pink, C in cyan, N in blue and O in red. All structures are plotted using VMD66. (c) A wire presentation of the secondary structures in T7RdhA plotted by PDBsum37. Note that the red dots on top of the amino acids indicate that the residue is involved in cofactor/substrate binding. The β-strands form three sheets (A, B and C). Positions of β- and γ-turns in the loop regions are labeled.CC BY · PubMed Central
Figure 2 Root-mean square fluctuation profiles (RMSF) of five systems in comparison with the AF2 scores. The dots correspond to the Cys residues that are covalently bonded to the Fe atoms in the Fe4S4 clusters. AF2 (0, black solid) is a reverse normalization of the per-residue pLDDT scores of the AF2 protein model. The protein in complex with the PFOA substrate, BVQ cofactor and two Fe4S4 clusters (1, red solid) shows the best match with the AF2 scores with Pearson’s correlation coefficient (PCC) of 0.561. The other systems include apo-T7RdhA (2, light blue dashed), T7RdhA with BVQ (3, dark blue dashed), T7RdhA with two Fe4S4 clusters (4, orange dashed) and T7RdhA with both BVQ and two Fe4S4 clusters (5, purple dashed) are also plotted with PCC in parentheses. The RMSF calculated from 320 models (6, black dashed) matches well with the AF2-scores (PCC = 0.719).
Figure 2 Root-mean square fluctuation profiles (RMSF) of five systems in comparison with the AF2 scores. The dots correspond to the Cys residues that are covalently bonded to the Fe atoms in the Fe4S4 clusters. AF2 (0, black solid) is a reverse normalization of the per-residue pLDDT scores of the AF2 protein model. The protein in complex with the PFOA substrate, BVQ cofactor and two Fe4S4 clusters (1, red solid) shows the best match with the AF2 scores with Pearson’s correlation coefficient (PCC) of 0.561. The other systems include apo-T7RdhA (2, light blue dashed), T7RdhA with BVQ (3, dark blue dashed), T7RdhA with two Fe4S4 clusters (4, orange dashed) and T7RdhA with both BVQ and two Fe4S4 clusters (5, purple dashed) are also plotted with PCC in parentheses. The RMSF calculated from 320 models (6, black dashed) matches well with the AF2-scores (PCC = 0.719).CC BY · PubMed Central
Figure 3 T7RdhA distance map, PAE map and RIN. (a) Distance map of T7RdhA at 100 ns compared with (b) the PAE map from AF2. (c) The RIN at 100 ns constructed from the contact map. The BVQ cofactor in sphere (dark green), the Fe4S4 clusters in square (purple) and PFOA in rectangle (pink). Blue nodes are positively charged residues (Lys and Arg) and red are negatively charged residues (Glu and Asp); black nodes are aromatic residues (His, Tyr, Phe and Trp); and all other residues in yellow. The PIN is plotted using the R package igraph53.
Figure 3 T7RdhA distance map, PAE map and RIN. (a) Distance map of T7RdhA at 100 ns compared with (b) the PAE map from AF2. (c) The RIN at 100 ns constructed from the contact map. The BVQ cofactor in sphere (dark green), the Fe4S4 clusters in square (purple) and PFOA in rectangle (pink). Blue nodes are positively charged residues (Lys and Arg) and red are negatively charged residues (Glu and Asp); black nodes are aromatic residues (His, Tyr, Phe and Trp); and all other residues in yellow. The PIN is plotted using the R package igraph53.CC BY · PubMed Central
Figure 4 The protein-cofactor/ligand interactions. Distribution of interaction residues from 100 RINs (constructed from snapshots of a 100 ns MD) to (a) BVQ cofactor, (b) Fe4S4-A cluster, (c) Fe4S4-B cluster and (d) PFOA substrate. Number of interactions (mean ± standard deviation) detected in all RINs are indicated. All residues are ranked by the percentage of interactions observed in all RINs. The y-axis indicates the percentage of the interactions in all RINs. Representative clusters are shown for the interactions centering (e) BVQ, (f) Fe4S4-A, (g) Fe4S4-B and (h) PFOA. Motifs (sequences in Fig. 1c) that contain interacting residues and some of the important residues are labeled. Cobalt is colored in silver, iron in purple, fluorine in pink, carbon in cyan, nitrogen in blue and oxygen in red. All structures in (e)–(h) are plotted using VMD66.
Figure 4 The protein-cofactor/ligand interactions. Distribution of interaction residues from 100 RINs (constructed from snapshots of a 100 ns MD) to (a) BVQ cofactor, (b) Fe4S4-A cluster, (c) Fe4S4-B cluster and (d) PFOA substrate. Number of interactions (mean ± standard deviation) detected in all RINs are indicated. All residues are ranked by the percentage of interactions observed in all RINs. The y-axis indicates the percentage of the interactions in all RINs. Representative clusters are shown for the interactions centering (e) BVQ, (f) Fe4S4-A, (g) Fe4S4-B and (h) PFOA. Motifs (sequences in Fig. 1c) that contain interacting residues and some of the important residues are labeled. Cobalt is colored in silver, iron in purple, fluorine in pink, carbon in cyan, nitrogen in blue and oxygen in red. All structures in (e)–(h) are plotted using VMD66.CC BY · PubMed Central
Figure 5 Comparisons of protein structures during MD and AF2 models. (a) A structure-based phylogenetic tree using the protein structure snapshots during the MD at different simulation time (100 ns, 200 ns and 300 ns for snapshots 1–3) for the MD systems 1–5 and AF2 structures (system 6), see Fig. 2. The initial MD structure is sys6-1, and sys6-2 and sys6-3 has the RMSD values 0.391 and 0.937 Å to sys6-1, respectively. (b) Superimposed structures of systems1-6 colored by RMSD to the initial MD structure (sys6-1). Systems1-5 were collected from the MD simulations after 300 ns. A BWR color scheme was applied with blue for low, red for high and white for in-between RMSD values. The overall RMSD for all pairs of structures plotted in the phylogenetic tree is 2.45 ± 0.57 Å. The superimposed structures are plotted using VMD66.
Figure 5 Comparisons of protein structures during MD and AF2 models. (a) A structure-based phylogenetic tree using the protein structure snapshots during the MD at different simulation time (100 ns, 200 ns and 300 ns for snapshots 1–3) for the MD systems 1–5 and AF2 structures (system 6), see Fig. 2. The initial MD structure is sys6-1, and sys6-2 and sys6-3 has the RMSD values 0.391 and 0.937 Å to sys6-1, respectively. (b) Superimposed structures of systems1-6 colored by RMSD to the initial MD structure (sys6-1). Systems1-5 were collected from the MD simulations after 300 ns. A BWR color scheme was applied with blue for low, red for high and white for in-between RMSD values. The overall RMSD for all pairs of structures plotted in the phylogenetic tree is 2.45 ± 0.57 Å. The superimposed structures are plotted using VMD66.CC BY · PubMed Central

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Metadata

Authors
Guo, H.-B., Varaljay, V. A., Kedziora, G., Taylor, K., Farajollahi, S., Lombardo, N., Harper, E., Hung, C., Gross, M., Perminov, A., Dennis, P., Kelley-Loughnane, N., & Berry, R.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2023-03-11
Volume
13
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-023-30310-x
PMC
PMC10008544
Cited by
22
References
10
Language
en
Licence
Funders
OUSD (R&E) ARAP Program

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