Weak Interactions in Molecular Machinery Volume II

Editorial

15 September 2023

Editorial: Weak interactions in molecular machinery volume II

Laura Corrales-Guerrero

Filippo Prischi

and

Irene Díaz-Moreno

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0 citations

Editors

Irene Diaz-Moreno

Sevilla University

Filippo Prischi

King's College London

Laura Corrales-Guerrero

Sevilla University

Impact

$Combination of spectroscopic and XFEL data to understand ligand binding [d-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) and O2] to the enzyme isopenicillin N-synthase (Rabe et al., 2021). Initial XFEL data were obtained for the enzyme with the substrate ACV, a precursor to for all natural cephalosporins and penicillins. Reactivity within microcrystals was initiated by passing them through an O2 chamber and SFX data were measured in a time-dependent manner. X-ray emission spectroscopy (XES) data (A) was obtained using the same X-ray pulse that generated the diffraction patterns allowing a two-step transition between the resting states Fe(II) and intermediate Fe(III)-O2•−states to be characterised over a wide range of reaction times (B) SFX data were obtained at a time point of 1,600 ms such that the Fe(III)-O2•−intermediate had fully formed (C) Figure adapted from (Rabe et al., 2021).$

Perspective

23 January 2023

Perspective: Structure determination of protein-ligand complexes at room temperature using X-ray diffraction approaches

Michael A. Hough

, 1 more and

Jonathan A. R. Worrall

4,753 views

6 citations

Influence of FMN source in the recovery of the aPNPOx activity to transform PNP into PLP. (A) Scheme of the PNPOx steady-state enzymatic assay: upon PNP transformation into the PLP product, PLP accumulation is detected by formation of the aldimine PLP-Tris adduct that absorbs maximally at 414 nm. (B) Michaelis-Menten plots for the formation of the PLP-Tris adduct upon PNP transformation by PNPOx, as well as by aPNPOx reconstituted by free FMN, by hRFK (RFK pre-incubated with the products of its reaction, RFK:FMN:ADP:Mg2+) and by a just prepared mixture of RFK with its RF and ATP:Mg2+ substrates (RFK:RF + ATP:Mg2+, sRFK). Dependence of aPNPOx activation on FMN concentration when measuring PLP-adduct formation (C) upon mixing and (D) after 5 min pre-incubation with the FMN source while keeping PNP substrate at saturating concentrations. All measurements were performed in 50 mM Tris/HCl pH 7.6, 0.3 mM MgCl2, 1 mM DTT at 37°C. (E) Fast kinetics evolution for the formation of the PLP-Tris adduct as followed in the stopped-flow equipment upon the mixing of PNPOx (syringe 1) with PNP (syringe 2). In all cases syringe 2 also contained the source of FMN (either as FMN, hRFK, sRFK or RF substrate). Note that for sRFK samples two different reactions mixtures were prepared; one contained in syringe 1 RFK together with aPNPOx while substrates of both enzymes where placed in syringe 2 (equivalent to sRFK), in the other aPNPOx was alone in syringe 1 and RFK was placed in syringe 2 together its substrates and PNP (sRFK2). Final concentrations in the stopped-flow mixture chamber were [PNPOx homodimer] = 0.05 µM, [PNP] = 25 μM, [FLV] = 0.25 µM. Measurements were performed in 50 mM Tris/HCl pH 7.6, 0.3 mM MgCl2, 1 mM DTT at 25°C. Data for control holo PNPOx are shown in black, while those for FMN sources (FLV) being free FMN, hRFK, sRFK and sRFK2 are respectively shown in red, green, blue and dark purple.

Original Research

28 March 2023

Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery

Maribel Rivero

, 4 more and

Milagros Medina

3,957 views

7 citations

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.

Original Research

09 January 2023

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

Andrei Gilep

, 21 more and

Natallia Strushkevich

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.

4,665 views

10 citations

$Crowder packing. The receptor (1s78 chain A) is in red, the native pose of the ligand (1s78 chains CD) is in green, and the albumin crowders at a 0.06 volume fraction (shown for clarity only within 50 Å of the receptor surface) are in cyan.$