The hydrophobic cluster on the surface of protein is the key structural basis for the SDS-resistance of chondroitinase VhChlABC

Abstract

The application of chondroitinase requires consideration of the complex microenvironment of the target. Our previous research reported a marine-derived sodium dodecyl sulfate (SDS)-resistant chondroitinase VhChlABC. This study further investigated the mechanism of VhChlABC resistance to SDS. Focusing on the hydrophobic cluster on its strong hydrophilic surface, it was found that the reduction of hydrophobicity of surface residues Ala¹⁸¹, Met¹⁸², Met¹⁸³, Ala¹⁸⁴, Val¹⁸⁵, and Ile³⁰⁵ significantly reduced the SDS resistance and stability. Molecular dynamics (MD) simulation and molecular docking analysis showed that I305G had more conformational flexibility around residue 305 than wild type (WT), which was more conducive to SDS insertion and binding. The affinity of A181G, M182A, M183A, V185A and I305G to SDS was significantly higher than that of WT. In conclusion, the surface hydrophobic microenvironment composed of six residues was the structural basis for SDS resistance. This feature could prevent the binding of SDS and the destruction of hydrophobic packaging by increasing the rigid conformation of protein and reducing the binding force of SDS-protein. The study provides a new idea for the rational design of SDS-resistant proteins and may further promote chondroitinase research in the targeted therapy of lung diseases under the pressure of pulmonary surfactant.

Supplementary information: The online version contains supplementary material available at 10.1007/s42995-023-00201-1.

Keywords: Chondroitinase; Hydrophobic cluster; Protein surface; SDS-resistance.

Fig. 1

Hydrophilicity/hydrophobicity analysis of VhChlABC. A Hydrophilic/hydrophobic map. The vertical axis represents the hydrophobic fraction, and the horizontal axis represents the amino acid residues at the corresponding positions. The online tool ProtScale was used to map the hydrophilicity and hydrophobicity of VhChlABC as described in the “Materials and Methods”. B 3D-distribution map of strongly hydrophilic/hydrophobic amino acids. Amino acids with hydrophobicity index higher than 1 and less than − 1.5 were defined as strongly hydrophobic (red) and strongly hydrophilic (green), respectively. C The hydrophilic/hydrophobic surface, the surface hydrophobic cluster and the critical catalytic residues. PyMOL was used for visualization of hydrophilic/hydrophobic surface of VhChlABC. The darker the red, the more hydrophobic the amino acid. The six amino acids that form the hydrophobic cluster were represented as spheres. The critical catalytic residues were shown as mesh

Fig. 2

Relative specific activity of WT and mutants. Error bars indicated standard deviation (n = 3). **Represents an extremely significant difference, p < 0.01

Fig. 3

Effects of SDS on activity and stability of WT and its mutants. A Relative activity of enzymes in different concentrations of SDS. B Stability of enzymes pre-incubated with 0.1% (w/v) SDS for 1 h. Error bars indicated standard deviation (n = 3). **Represents an extremely significant difference, p < 0.01. Enzyme activity without SDS pre-incubation was defined as 100%

Fig. 4

Calculation RMSF (Å) for VhChlABC (WT) and its mutants with a 40–50 ns trajectory. WT is shown in gray; A181G (A), M182A (B), M183A (C), A184G (D), V185A (E) and I305G (F) are shown in light blue, green, cyan, dark blue, orange and pink, respectively. The corresponding mutant sites are highlighted with red dots in the local RMSF map, and the two loops formed by Gly³⁰⁴-Asp³¹⁶ and Ser³³⁸-Glu³⁴² are marked by dashed box (F)

Fig. 5

Binding conformation of SDS on the hydrophobic surface of enzyme molecules. A WT, B I305G

Fig. 6

Average binding free energy (ΔG_bind) analysis of SDS to WT and its mutants. Error bars indicated standard deviation (n = 3). **Represents an extremely significant difference, p < 0.01