The β-hairpin from the Thermus thermophilus HB27 laccase works as a pH-dependent switch to regulate laccase activity

• Published in: Journal of structural biology Vol. 213; no. 2; p. 107740
• Main Authors: Miranda-Blancas, R., Avelar, M., Rodriguez-Arteaga, A., Sinicropi, A., Rudiño-Piñera, E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2021
• Subjects: Amino Acid Motifs; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Crystallography, X-Ray; Hydrazones - metabolism; Hydrogen-Ion Concentration; Laccase; Laccase - chemistry; Laccase - genetics; Laccase - metabolism; Methionine; Molecular dynamics; Molecular Dynamics Simulation; Multi-copper oxidase; Oxidation-Reduction; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Phylogeny; Protein Conformation; Temperature; Thermus thermophilus; Thermus thermophilus - metabolism; β-Hairpin; β-Hairpin; APS; Multi-copper oxidase; SGZ; Molecular dynamics; TNC; T1Cu; PDB; Thermus thermophilus; T2Cu; MD; T3Cu; Tth-MCO; RCSB-PDB; MrLac; MGy; Laccase
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2021.107740

[Display omitted] •Two new conformations of the β-hairpin from Tth-MCO are described.•The β-hairpin in Tth-MCO has crystallographic and dynamical conformations driven by pH.•The laccase activity, from the Tth-MCO, can be regulated by its β-hairpin conformations.•Laccases from Thermus and Meiothermus genus share a Methionine-rich sequence insertion similar to the β-hairpin from Tth-MCO. The multi-copper oxidase from the hyper-thermophilic bacteria Thermus thermophilus (Tth-MCO), has been previously characterized and described as an example of a laccase with low catalytic properties, especially when it is compared with the activity of fungal laccases, but it is active at high temperatures. Structurally, Tth-MCO has a unique feature: a β-hairpin near the T1Cu site, which is not present in any other laccases deposited at the PDB. This β-hairpin has an expected crystallographic behavior in solvent-exposed areas of a crystallized protein: lack of electron density, high B-values and several crystalline contacts with neighboring crystallographic copies; however, its dynamical behavior in solution and its biological implications have not been described. Here, we describe four new Tth-MCO crystallographic structures, and the β-hairpin behavior has been analyzed by molecular dynamics simulations, considering the effect of pH and temperature. The β-hairpin new crystallographic conformations described here, together with their dynamics, were used to understand the pH-restrained laccase activity of Tth-MCO against substrates as syringaldazine. Remarkably, there are insertions in laccases from Thermus and Meiothermus genus, sharing the same position and a methionine-rich composition of the Tth-MCO β-hairpin. This unique high methionine content of the Tth-MCO β-hairpin is responsible to coordinate, Ag+1 and Hg+1 in oxidative conditions, but Cu+1 and Cu+2 are not coordinated in crystallographic experiments, regardless of the redox conditions; however, Ag+1 addition does not affect Tth-MCO laccase activity against syringaldazine. Here, we propose that the pH-dependent β-hairpin dynamical behavior could explain, at least in part, the inefficient laccase activity displayed by Tth-MCO in acidic pH values.