Comparative structural and functional analysis of the glycine‐rich regions of Class A and B J‐domain protein cochaperones of Hsp70

Abstract

J‐domain proteins are critical Hsp70 co‐chaperones. A and B types have a poorly understood glycine‐rich region (Grich) adjacent to their N‐terminal J‐domain (Jdom). We analyzed the ability of Jdom/Grich segments of yeast Class B Sis1 and a suppressor variant of Class A, Ydj1, to rescue the inviability of sis1‐∆. In each, we identified a cluster of Grich residues required for rescue. Both contain conserved hydrophobic and acidic residues and are predicted to form helices. While, as expected, the Sis1 segment docks on its J‐domain, that of Ydj1 does not. However, data suggest both interact with Hsp70. We speculate that the Grich–Hsp70 interaction of Classes A and B J‐domain proteins can fine tune the activity of Hsp70, thus being particularly important for the function of Class B.

Unique characteristics of the J-domain proximal regions of Hsp70 cochaperone Apj1 in prion propagation/elimination and its overlap with Sis1 function

“…(C) AlphaFold predictions for Sis1-121 (left) and Ydj1-101 (right). In Sis1-121, Helix V interacts with Helices II and III and sterically blocks the HPD motif; this location for Helix V is congruent with NMR data for Sis1 ( Ciesielski, et al, 2024 ) and similar to that observed in NMR structures of the human Sis1 ortholog DNAJB1 ( Faust et al, 2020 ). In Ydj1-101, Helix V is in the foreground on the right side, away from Helix II and III and the HPD motif.…”

“…Sis1 has DAFNIFSQFF, containing residues important for prion maintenance and the ability of Sis1-121 to rescue the lethality of sis1 -∆ ( Lopez et al, 2003 ). Residues in this region form a helix in Ydj1, Sis1, and its human ortholog, DNAJB1, referred to as “Helix V” in the literature as it is typically the predominant helix after the four helices of the J-domain ( Ciesielski, et al, 2024 ). The remaining 40 residues comprise another low complexity region highly enriched in S, N, and F residues (38% S, 18% N, 13% F, and 8% G).…”

“…A recent analysis of the GF region of Sis1 identified residues that were critical for cell viability within the sequence DAFNIFSQFF ( Ciesielski, et al, 2024 ). The F two positions preceding the QFF was identified as a key residue; when Sis1-121 is expressed as the sole version of Sis1, mutation of this phenylalanine to alanine was lethal.…”

“…The similarity between the Apj1 GF sequence (DLFAQFF) and the important Ydj1 sequence (DIFSQFF) is more striking than that with the Sis1 region ( Figure 4B ), particularly regarding the spacing of negatively charged and hydrophobic residues ( Ciesielski, et al, 2024 ). In all three, formation of a helix is predicted—generally referred to as Helix V ( Figure 4C )—and confirmed by biophysics analysis for Sis1 and Ydj1 ( Ciesielski, et al, 2024 ). However, what Helix V is doing in the context of the short N-terminal segments of Class A/B JDPs is not well understood.…”

“…Although Ydj1 cannot normally substitute for Sis1, a single residue substitution at the junction of the J-domain and the adjacent GF region (G 70 →N) results in a gain in function, allowing the growth of sis1 -∆ cells ( Schilke et al, 2017 ). The Ydj1 J-domain-GF fragment bearing the same mutation but lacking the C-terminal domains (Ydj1-109 G70N ) actually supports better growth, suggesting that perhaps these domains sterically interfere ( Schilke et al, 2017 ; Ciesielski, et al, 2024 ). Such interference may also explain the lack of rescue by full-length Apj1.…”