Functional dissection of velvet DNA-binding region in Aspergillus nidulans VelB uncovers an essential conserved arginine cluster
Abstract Velvet regulators, characterized by a conserved velvet domain, function as central hubs that coordinately govern fungal development, secondary metabolism, stress adaptation, and pathogenicity. The velvet domain is organized into an N-terminal DNA-binding region of approximately 30 amino acids and a C-terminal dimerization region of approximately 100 amino acids. In this study, Aspergillus nidulans VelB was used as a paradigm to systematically dissect the velvet DNA-binding region. The three arginine residues R71, R80, and R81 in the N-terminal velvet domain that are indispensable for VelB function were identified through alanine-scanning mutagenesis of 15 conserved residues. Alanine substitutions at these positions caused severe defects in long-term spore viability, sexual development, and secondary metabolism. Further comparative characterization of electrostatic-potential dynamics pre- and post-mutation revealed that the three individual substitutions markedly attenuated local electrostatic potential across the DNA-binding interface. Notably, these mutations drove comprehensive remodeling of the protein’s electrostatic properties, whereby electrostatic perturbations propagated across the entire protein exterior. Analysis of 4,999 velvet-domain sequences across the fungal kingdom revealed extraordinary conservation of these positions: arginine was present at position 71 in 85% of sequences, at position 80 in 91%, and at position 81 in 84%. Cross-kingdom complementation experiments further demonstrated that the wild-type velvet DNA-binding region from Capsaspora owczarzaki, a unicellular holozoan lacking the equivalent of R71, failed to rescue the A. nidulans velB deletion phenotype, whereas the introduction of arginine at this position conferred substantial functional restoration. These findings establish that a cluster of conserved arginine residues generates the positive electrostatic surface potential required for velvet–DNA interaction and define the molecular basis of DNA recognition by this ancient family of fungal transcription factors.