Biology subjects

Ramos, C. H. I.

Publications and source records attributed to Ramos, C. H. I..

2 recordsLinked to original sources

bioRxiv2022

Structural and dynamical basis for the interaction of HSP70-EEVD with JDP Sis1

We employed NMR spectroscopy to investigate the structure and dynamics of the class B J domain protein (JDP) of S. cerevisiae (Sis1) complexed with an EEVD peptide of HSP70. It is widely recognized that the interactions between the EEVD motif and Sis1 play a crucial role in the chaperone activity. Notably, the deletion of the EEVD impairs the ability of Sis1 to bind with HSP70, while leaving the interaction between the class A JDP Ydj1 and HSP70 unaffected. Leveraging the advantages of NMR, which is particularly suitable for studying transient interactions, we provide compelling evidence that the EEVD motif transiently engages multiple sites on Sis1. Our findings revealed that EEVD binds to two distinct sites within the C-terminal domain I (CTDI) of Sis1. The interaction at these sites plays a crucial role in anchoring HSP70 to Sis1 at site I, as well as displacing the client protein at site II. Notably, site II is also the binding site for the client protein, and its displacement occurs through competition with the binding to site II. In addition to these interactions, we observed that EEVD, as a transient electrostatic binder, also interacts with the J domain and the GF-rich loop located between the J domain and -helix 6. We propose that the interaction between EEVD and Sis1 facilitates the dissociation of -helix 6, promoting a conformational state that is more favorable for interaction with HSP70 at the nucleotide-binding domain (NBD) and substrate-binding domain (SBD) interface. We also employed -synuclein as a substrate to investigate the competitive nature between EEVD and the client protein. Our experimental findings provided evidence supporting the interaction of EEVD with the client protein at multiple sites. Our findings contribute essential insights into the mechanistic cycle of class B JDPs, paving the way toward a more complete understanding of the primary function of Sis1, which is the transfer of the client protein to HSP70, where multiple site transient interactions play a collective role.

molecular biology↗

bioRxiv2018

Insights into the effect of the J-domain on the substrate binding domain (SBD) of the 70 kDa heat shock protein, Hsp70, from a chimeric human J-SBD polypeptide

DnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain, a crucial event for the function that this system has in assisting protein folding. The interaction between Hsp40 and Hsp70 is transient and thus difficult to study, since mixing the binding partners can lead to quick dissociation due to their low affinity, creating a challenge for detailed analysis. As a consequence, knowledge of many important aspects of the mechanism of interaction is still lacking, for instance, the effect that J-domain binding has on Hsp70. In this study, we investigated whether it would be possible to gain understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale for this is that an increase in the proximity between the interacting partners in this engineered chimera will promote the natural interaction and facilitate the characterization of the protein- protein interaction, which is a requirement to gain further understanding of many biological processes. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the SBD alone. J-SBD behaved primarily as a monomer in all conditions tested and exhibited chaperone activity, as shown by aggregation protection and substrate binding assays, which revealed decreased binding to bis-ANS, a probe for hydrophobic patches. Collectively, our results suggest that Hsp40 binding to Hsp70 via the J-domain shifts the Hsp70 equilibrium towards the monomer state to expose hydrophobic sites prone to substrate accommodation.\n\nAbbreviationsBis-ANS (4,4-Dianilino-1,1-Binaphthyl-5,5-Disulfonic Acid; CD, circular dichroism; Hsp, heat shock protein; J-SBD, chimeric polypeptide in which the J-domain of Hsp40 (at the N-terminus) is covalently attached to the substrate binding domain of Hsp70 (at the C-terminus) by a flexible linker; SBD: substrate binding domain of Hsp70.

biochemistry↗