STRUCTURAL AND FUNCTIONAL ROLES OF THE ZINC-BINDING DOMAINS IN HUMAN DNAJA1

HSP40s, also known as DNAJs, constitute a diverse group of proteins that function as co-chaperones within the HSP70 chaperone system. These proteins bind to and stabilize client proteins, facilitating their delivery to HSP70 while also stimulating the chaperone's ATPase activity and promoting its folding cycle. Eukaryotic DNAJs are subdivided into three classes based on their domain architecture. The DNAJA subclass is characterized by the presence of a Zinc Finger-Like Domain (ZFLD), which comprises four conserved CXXCXGXG motifs that coordinate two zinc ions, forming two Zinc-Binding Domains (ZBDs). Previous studies from our group have demonstrated that this domain plays critical structural and functional roles in the DNAJA1 protein. The conserved motifs stabilize the protein’s folding through zinc coordination and also contribute to the stabilization of client interactions. Furthermore, the ZFLD is implicated in modulating DNAJA1 conformation upon interaction with HSP70. To further elucidate the role of each ZBDs in DNAJA1, we disrupted the conserved zinc-binding motifs obtaining three mutants. All variants were recombinantly expressed and obtained in soluble form. Our objective is to perform structural and functional characterization of these mutants in comparison to the wild-type human DNAJA1, aiming to clarify the specific contributions of each ZBD to the protein’s structural integrity, client binding and stabilization, and its interaction with the HSP70 system.