28FS
Cryo-EM structure of hydrogenase-2 in complex with L1 (left)
Summary for 28FS
| Entry DOI | 10.2210/pdb28fs/pdb |
| EMDB information | 81712 |
| Descriptor | binder L1, Hydrogenase-2 small chain, Hydrogenase-2 large chain, ... (7 entities in total) |
| Functional Keywords | complex, binder, oxidoreductase |
| Biological source | synthetic construct More |
| Total number of polymer chains | 5 |
| Total formula weight | 200602.47 |
| Authors | |
| Primary citation | Sun, X.,Xiao, Q.,Zhang, N.,Li, W.,Luo, H.,Gao, Y.,Zhang, L.,Fan, Y.,Jiang, P.,Huang, Y.,Wu, G.,Zhang, L. De Novo Design of a Protein Binder to Probe Gas Channel and Enhance the Oxygen Tolerance of [NiFe]-Hydrogenase. Angew.Chem.Int.Ed.Engl., :e6395554-e6395554, 2026 Cited by PubMed Abstract: [NiFe]-Hydrogenases display remarkable catalytic efficiency for H production and oxidation but are sensitive to O, which severely restricts their biotechnological applications. The identities and functional relevance of gas channels leading to the buried [NiFe] active site remain elusive, hindering rational enzyme engineering. Here, we introduce an artificial intelligence-guided de novo protein binder design strategy to map hydrophobic O diffusion pathways in Escherichia coli hydrogenase-2 (Hyd-2). By integrating RFdiffusion, ProteinMPNN, and AlphaFold, ∼100,000 candidate binders were computationally screened, yielding two high-affinity binders, L1 and L2. Biophysical and electrochemical analyses show that L1 selectively occludes the primary O ingress channel, enhancing the enzyme's oxygen tolerance by more than threefold, whereas L2, which targets another putative channel, has a negligible effect, indicating that this pathway contributes minimally to O ingress under the tested conditions. Furthermore, we determined the L1-Hyd-2 complex structure by cryo-electron microscopy and revealed the key interaction residues and interface conformation. Integrated structural and computational analyses provide mechanistic insights into the system's oxygen tolerance and high-affinity L1-Hyd-2 interaction. All findings provide the first direct experimental evidence for hierarchical O diffusion channels in [NiFe]-hydrogenases and establish binder-mediated channel occlusion as a generalizable, mutation-free strategy for elucidating and modulating gas transport in metalloenzymes. PubMed: 42655993DOI: 10.1002/anie.6395554 PDB entries with the same primary citation |
| Experimental method | ELECTRON MICROSCOPY (2.49 Å) |
Structure validation
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