The X‐ray crystallographic structure of a designed, self‐assembling, and heme‐binding protein
4 affiliations
- 1Department of Chemistry University of Michigan Ann Arbor Michigan USA
- 2Bioénergétique et Ingénierie des Protéines, Unité Mixte de Recherche UMR 7281, CNRS Aix‐Marseille Université Marseille France
- 3Architecture et Fonction des Macromolécules Biologiques, CNRS Aix‐Marseille Université Marseille France
- 4Ingénierie des Systèmes Macromoléculaires (UMR 7255), Institut de Microbiologie de la Méditerranée, CNRS Aix‐Marseille Université Marseille France
Abstract
De novo heme-binding proteins offer powerful minimal scaffolds for elucidating how sequence, oligomerization, and environment rule cofactor coordination, yet high-resolution structural data for these systems remain scarce. Here, we report the high-resolution crystal structure of a designed, selfassembling α-helical and heme-binding protein determined at 1.9 Å resolution, that undergoes a pronounced pH-and cofactor-driven reorganization. The structure crystallized in the orthorhombic P2 1 2 1 2 1 space group (a = 44.8 Å, b = 46.3 Å, c = 101.2 Å), with one tetramer per asymmetric unit and refinement statistics of R fac /R free = 23.5/25.5%. The apo protein folds into a three-stranded coiled coil, whereas heme binding under strongly basic conditions disrupts register-specific interactions and drives a major helix shift. This transition yields a four-stranded coiled coil with an outof-register ABAB arrangement that accommodates two bis-histidine-ligated hemes. Cofactors are His-coordinated via the ε-nitrogen (Fe-N His ≈ 2.0 Å), separated by 38 Å (tetrapyrroles edge-to-edge) and arranged nearly coplanar with a 67°rotation offset relative to one another. Imidazole dihedral angles (99°and 108°) indicate near-orthogonal geometry for the His axial ligands to the heme. Crystallization is promoted by a heme-templated packing in which interactions between macrocycles of neighboring assemblies and several salt-bridges generate an extended supramolecular lattice. These results demonstrate how heme binding and proton concentration reshape oligomeric topology and promote ordered assembly in minimal protein systems, highlighting the varied structural effects of heme binding and providing principles for stabilizing specific coordination and exploiting cofactor-cofactor interactions to guide high-order organization in de novo metalloproteins.
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BibTeX
@article{modenez2026xray,
title = {{The X‐ray crystallographic structure of a designed, self‐assembling, and heme‐binding protein}},
author = {Iago de Assis Modenez and Véronique Roig‐Zamboni and Philippe Leone and Anabella Ivancich and Vincent L. Pecoraro},
journal = {Protein Science},
year = {2026},
month = oct,
doi = {10.1002/pro.70829},
url = {https://hal.science/hal-05840771v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - The X‐ray crystallographic structure of a designed, self‐assembling, and heme‐binding protein
AU - Iago de Assis Modenez
AU - Véronique Roig‐Zamboni
AU - Philippe Leone
AU - Anabella Ivancich
AU - Vincent L. Pecoraro
PY - 2026
DA - 2026/10/27
JO - Protein Science
DO - 10.1002/pro.70829
UR - https://hal.science/hal-05840771v1
AB - De novo heme-binding proteins offer powerful minimal scaffolds for elucidating how sequence, oligomerization, and environment rule cofactor coordination, yet high-resolution structural data for these systems remain scarce. Here, we report the high-resolution crystal structure of a designed, selfassembling α-helical and heme-binding protein determined at 1.9 Å resolution, that undergoes a pronounced pH-and cofactor-driven reorganization. The structure crystallized in the orthorhombic P2 1 2 1 2 1 space group (a = 44.8 Å, b = 46.3 Å, c = 101.2 Å), with one tetramer per asymmetric unit and refinement statistics of R fac /R free = 23.5/25.5%. The apo protein folds into a three-stranded coiled coil, whereas heme binding under strongly basic conditions disrupts register-specific interactions and drives a major helix shift. This transition yields a four-stranded coiled coil with an outof-register ABAB arrangement that accommodates two bis-histidine-ligated hemes. Cofactors are His-coordinated via the ε-nitrogen (Fe-N His ≈ 2.0 Å), separated by 38 Å (tetrapyrroles edge-to-edge) and arranged nearly coplanar with a 67°rotation offset relative to one another. Imidazole dihedral angles (99°and 108°) indicate near-orthogonal geometry for the His axial ligands to the heme. Crystallization is promoted by a heme-templated packing in which interactions between macrocycles of neighboring assemblies and several salt-bridges generate an extended supramolecular lattice. These results demonstrate how heme binding and proton concentration reshape oligomeric topology and promote ordered assembly in minimal protein systems, highlighting the varied structural effects of heme binding and providing principles for stabilizing specific coordination and exploiting cofactor-cofactor interactions to guide high-order organization in de novo metalloproteins.
SN - 0961-8368
SN - 1469-896X
ER -