Unraveling the roles of aromatic cluster side-chain interactions on the structural stability and functional significance of psychrophilic Sphingomonas sp. glutaredoxin 3

• Published in: PloS one Vol. 18; no. 8; p. e0290686
• Main Authors: Tran, Trang Van, Hoang, Trang, Jang, Sei-Heon, Lee, ChangWoo
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 31.08.2023; Public Library of Science (PLoS)
• Subjects: Amino acids; Biology and Life Sciences; Clusters; E coli; Ethylenediaminetetraacetic acid; Evaluation; Glutaredoxin; High temperature; Hydrogen bonds; Hydrophobicity; Medicine and Health Sciences; Molecular weight; Mutagenesis; Oxidoreductase; Physical Sciences; Plasmids; Polar environments; Properties; Protein interaction; Protein structure; Proteins; Redox potential; Research and Analysis Methods; Spectrum analysis; Sphingolipids; Sphingomonas; Structural stability; Structure-function relationships; South Korea; Arctic Ocean; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0290686

This study investigates the impact of aromatic cluster side-chain interactions in Grx3 (SpGrx3) from the psychrophilic Arctic bacterium Sphingomonas sp. Grx3 is a class I oxidoreductase with a unique parallel arrangement of aromatic residues in its aromatic cluster, unlike the tetrahedral geometry observed in Trxs. Hydrophilic-to-hydrophobic substitutions were made in the aromatic cluster, in [beta]1 (E5V and Y7F), adjacent [beta]2 (Y32F and Y32L), both [beta]1 and [beta]2 (E5V/Y32L), and short [alpha]2 (R47F). The hydrophobic substitutions, particularly those at or near Tyr7 (E5V, Y7F, Y32F, and R47F), increased melting temperatures and conformational stability, whereas disrupting [beta]1-[beta]2 interactions (Y32L and E5V/Y32L) led to structural instability of SpGrx3. However, excessive hydrophobic interactions (Y7F and E5V/Y32L) caused protein aggregation at elevated temperatures. All mutations resulted in a reduction in [alpha]-helical content and an increase in [beta]-strand content. The R47F mutant, which formed dimers and exhibited the highest [beta]-strand content, showed increased conformational flexibility and a significant decrease in catalytic rate due to the disturbance of [beta]1-[alpha]2 interactions. In summary, the configuration of the aromatic cluster, especially Tyr7 in the buried [beta]1 and Arg47 in the short [alpha]2, played crucial roles in maintaining the active conformation of SpGrx3 and preventing its protein aggregation. These modifications, reducing hydrophobicity in the central [beta]-sheet, distinguish Grx3 from other Trx-fold proteins, highlighting evolutionary divergence within the Trx-fold superfamily and its functional versatility.