Three-Dimensional Structure of the Triple Mutant DiB3-F53L/F74L/L129M of the Fluorescent Complex of Bacterial Lipocalin with a Chromophore

Россохин, А. В., Горячева, Е. А., Артемьев, И. В. и др.
Russ J Bioorg Chem 50 , 778–783 (2024). https://doi.org/10.1134/S1068162024030373
Objective: In the development of biomarker systems for cell biology, Blc lipocalin proved to be a promising scaffold for binding the fluorogen M739. This work presents the results of the molecular dynamics study of the 3D structure of the Blc lipocaline triple mutant—DiB3-F53L/F74L/L129M with a synthetic GFP-like chromophore M739. The complex is characterized by a higher fluorescence brightness compared to other complexes of genetically engineered variants of lipocalin, which makes it one of the promising biomarkers for cell research. Methods: The starting model of the protein was based on the X-ray 3D structure of DiB1 by introducing the appropriate amino acid substitutions on a stereographic station. The search for the main binding site in the triple mutant DiB3-F53L/F74L/L129M was performed by MD calculations by introducing the chromophore molecule deep into the binding cavity of the Blc protein. The MD calculations were performed in the all-atom approximation at 300 K, based on the CHARMM36 force field using an explicit solvent model. Results and Discussion: The significant brightness increase characteristic of the DiB3-F53L/F74L/L129M complex compared to DiB3 provides stronger binding of the M739 chromophore with increased solvent protection. The three-dimensional structure of the binding protein has the shape of a β-barrel, consisting of eight antiparallel β-segments with an α-helical fragment at the C-terminal region. Four loops at the N-terminus form an oval entrance (8 × 11 Å) into a narrow elongated cavity of the protein with a depth of ~19 Å. The protein cavity provides specific binding of the tricyclic chromophore M739 with increased shielding from solvent. The position of the chromophore is stabilized by three H-bonds with the side chains of Asn76, Trp139 and Gln141. Conclusions: The work presents the results of the molecular dynamics study of the DiB3-F53L/F74L/L129M—Blc lipocalin triple mutant in complex with chromophore. The chromophore-binding site of the protein was found to correspond to the X-ray crystal structure of the related DiB1 complex and is different from the alternative binding site in the parent DiB3 complex.