Structural insights into 3Fe-4S ferredoxins diversity in M. tuberculosis highlighted by a first redox complex with P450

Abstract

Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis-Fdx alone and the fusion FdxE-CYP143. Our SPR analysis demonstrated a high-affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 and modeled Fdx-CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.

Keywords: 3Fe–4S ferredoxins; crystal structure; cytochrome P450; protein–protein interactions; redox complex.

FIGURE 1

(A) Overall structure of Fdx ferredoxin of M. tuberculosis. (B) Structural alignment of Fdx (salmon) with other structurally characterized 3Fe–4S ferredoxins from R. palustris HaA2 (PDB: 4OV1; blue) and P. furiosus (PDB: 1SJ1; green). The Fe–S clusters are shown in stick representation (Fe, orange; S, gold).

FIGURE 2

Multiple sequence alignment of ferredoxins taken from the genome context of Rv1786 (mtb_1786, NP_216302.1) and Rv0763c (mtb_0763c, WP_003403890.1) of Mtb (Supplementary Figure S3). Description for abbreviation of proteins is given in Supplementary Figure S2. Alignment was prepared using ClustalW algorithm. Sequence logos were prepared using WebLogo (https://weblogo.berkeley.edu/). Violet color indicates identical residues, turquoise color—residues with 75% of homology, and gray color—residues with 35% of homology. Numbers at the beginning and end of each row correspond to the first and the last amino acids of each sequence taken for alignment, respectively. Numbers in brackets indicate the total number of amino acids in the corresponding proteins. Amino acids important for the interaction with redox partner are shown as triangles.

FIGURE 3

(A) Overall structure of the FdxE–CYP143 fusion protein. (B,C) Zoomed-in view of redox cofactors. (B) 2Fo-Fc maps are countered at 7σ, donor–acceptor edge to the edge distance is shown as gray dashed lines. (C) Anomalous difference Fourier maps contoured at 4 σ. FdxE binds (raspberry) at the proximal face of CYP143 (green). Cyan spheres are Ni ions from crystallization conditions bound to His-tag. The N-term His-tag of the FdxE introduced to facilitate purification by metal affinity chromatography is visible and chelated by the Ni ions from crystallization conditions, while the electron density for the part of the linker between two proteins and the first eight residues of CYP143 cannot be resolved.

FIGURE 4

(A,B) Views of the interactions between FdxE and CYP143. (C) Residues important for mediating electron transfer coupling between the donor and acceptor. Coupled atoms were computed separately by selecting either Fe1 or Fe4 atoms of the [3Fe-4S] cluster as the donor using HARLEM. FdxE is colored raspberry and CYP143—green.

FIGURE 5

(A) Superposition of the CYP143 structure alone (green ribbon) with the structure of the FdxE−CYP143 complex (raspberry) detailing the meander region. (B) Superposition of Fdx (cyan) and FdxE (raspberry).

FIGURE 6

Results of SAXS data analysis of the fusion FdxE−CYP143 complex. (A) SAXS I(q) profile (orange circles) and their approximations corresponding to the PDB models obtained by using CORAL (purple dashed line) and EOM (blue solid line); (B) CORAL model of the fusion FdxE-CYP143 complex; (C) Rg distributions for the initial pool (gray histogram) and for a set of selected conformations (blue histogram) in the EOM (Supplementary Figure S6).

FIGURE 7

Comparison of two complexes of CYP with 3Fe–4S ferredoxins. (A) FdxE−CYP143 complex obtained in this work, (B) CYP51 – Fdx modeled with AlphaFold2. CYP143, and CYP51 are colored by secondary structures in cyan, magenta, and salmon for the helix, sheet, and loop, respectively; FdxE and Fdx are colored in red, yellow, and green for helix, sheet, and loops, respectively.