Dataset Information

A conserved Trp residue in HwBR contributes to its unique tolerance toward acidic environments.

ABSTRACT: Bacteriorhodopsin (BR) is a light-driven outward proton pump found mainly in halophilic archaea. A BR from an archaeon Haloquadratum walsbyi (HwBR) was found to pump protons under more acidic conditions compared with most known BR proteins. The atomic structural study on HwBR unveiled that a pair of hydrogen bonds between the BC and FG loop in its periplasmic region may be a factor in such improved pumping capability. Here, we further investigated the retinal-binding pocket of HwBR and found that Trp94 contributes to the higher acid tolerance. Through single mutations in a BR from Halobacterium salinarum and HwBR, we examined the conserved tryptophan residues in the retinal-binding pocket. Among these residues of HwBR, mutagenesis at Trp94 facing the periplasmic region caused the most significant disruption to optical stability and proton-pumping capability under acidic conditions. The other tryptophan residues of HwBR exerted little impact on both maximum absorption wavelength and pH-dependent proton pumping. Our findings suggest that the residues from Trp94 to the hydrogen bonds at the BC loop confer both optical stability and functionality on the overall protein in low-pH environments.

SUBMITTER: Yu CH

PROVIDER: S-EPMC9463644 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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A conserved Trp residue in HwBR contributes to its unique tolerance toward acidic environments.

Yu Cheng-Han CH Wu Hsiang-Yu HY Lin Hong-Syuan HS Yang Chii-Shen CS

Biophysical journal 20220708 16

Bacteriorhodopsin (BR) is a light-driven outward proton pump found mainly in halophilic archaea. A BR from an archaeon Haloquadratum walsbyi (HwBR) was found to pump protons under more acidic conditions compared with most known BR proteins. The atomic structural study on HwBR unveiled that a pair of hydrogen bonds between the BC and FG loop in its periplasmic region may be a factor in such improved pumping capability. Here, we further investigated the retinal-binding pocket of HwBR and found tha ...[more]

PMID: 35808832

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Project description:The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of phenylalanine. It thus appears that the second-coordination sphere Asp residue in cPAH, and, by extrapolation, the equivalent residue in other AAAHs, plays a role in fine-tuning pterin affinity in the ground state via deformable interactions with bridging waters and assumes a more significant role in the transition state by aligning the cofactor through direct hydrogen bonding.

Dataset Information

A conserved Trp residue in HwBR contributes to its unique tolerance toward acidic environments.

Publications

A conserved Trp residue in HwBR contributes to its unique tolerance toward acidic environments.

Similar Datasets

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