Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti

Abstract High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein Aa HMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, Aa HMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for Aa HMGB and Aa HMGB1 during embryogenesis. Immunolocalization in the midgut revealed that Aa HMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that Aa HMGB exhibits common features of α-helical structures and is more stably folded than Aa HMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between Aa HMGB and Aa HMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of Aa HMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.