Solution NMR investigation on the structure and function of the isolated J-domain from Sis1: Evidence of transient inter-domain interactions in the full-length protein

• Published in: Archives of biochemistry and biophysics 2019-07, Vol.669, p.71-79
• Main Authors: Pinheiro, Glaucia M.S., Amorim, Gisele C., Iqbal, Anwar, Almeida, Fabio C.L., Ramos, C.H.I.
• Format: Article
• Language: English
• Subjects: Binding Sites; Chaperone; Escherichia coli - genetics; Heat shock protein; HSP40 Heat-Shock Proteins - chemistry; HSP40 Heat-Shock Proteins - genetics; HSP40 Heat-Shock Proteins - metabolism; Hsp70; HSP70 Heat-Shock Proteins - metabolism; Humans; J-domain; Mutation; NMR; Protein Binding; Protein Domains; Protein Structure, Tertiary; Protein-protein interaction; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2019.05.020

J-domain/Hsp40 proteins cooperate in aiding with folding in the cell by binding partially folded client proteins and delivering them to be folded by Hsp70. The delivery occurs concomitantly to the stimulation of the ATPase activity of Hsp70 via the N-terminally located J-domain. Although several lines of investigation have been used to study J-domain proteins, the presence of highly flexible domains (G/F- and G/M-rich) hold up obtaining a detailed full-length structure. In this work, we present the high-resolution structure of the J-domain and the N-terminal part of the G/F domain of Sis1, solved by NMR, and used chemical-shift perturbation approaches to further study the structure/function relationship of the Sis1/Hsp70 interaction. When the J-domain was compared to the full-length protein and to a G/M domain deletion mutant, an internal interaction patch formed by hydrophobic and positively charged residues (V2, D9, R27, T39, F52 and R73) was identified. Curiously, the same patch is protected by internal contacts in the full-length protein and, in combination with the loop containing the conserved HPD motif, participates in the interaction with Hsp70. Combined, these results suggest that the J-domain in the full-length Sis1 is in a transient intermediate conformation, in which its interacting patch is protected and, at the same time, also in a favorable condition to bind Hsp70, facilitating the interaction between the two proteins. Finally, 1D NMR experiments showed that the addition of ATP is followed by the disruption of the J-domain/Hsp70 complex, a necessary step for aiding the folding of the client protein. [Display omitted] •Six residues in the J-domain change conformation when in the full-length Sis1 (a class B DnaJ).•These residues are part of a hydrophobic and positively charged patch.•This patch is protected by internal contacts and interacts with Hsp70.•Disruption of the J-domain/Hsp70 complex by ATP was validated by NMR.