Olewniczak, M. | Search bioRxiv

bioRxiv2025

Mechanism and energetics of JDP induced Hsp70's conformational transition towards catalytically active state

Hsp70 chaperones are crucial for maintaining protein homeostasis by regulating the stability and conformational states of client polypeptides through cycles of their binding and release. These cycles require conformational transitions of Hsp70 driven by ATP binding and hydrolysis. The ATPase activity of Hsp70 is controlled by J-domain protein (JDP) cochaperones, which allosterically stimulate ATP hydrolysis via interactions between their J-domains and Hsp70. The J-domain binds at the interface between the nucleotide (NBD) and substrate (SBD) binding domains of ATP bound Hsp70. Although, it was established that the JD interaction involves residues of helices II and III, and the interhelical loop critical for ATPase stimulation, the mechanism by which the allosteric signal induced by J-domain binding is transmitted to the distal nucleotide-binding pocket of Hsp70 remains unclear, as do the conformational changes leading to the ATP hydrolysis. Here, we addressed these questions by means of all-atom free energy simulations and dynamic network analysis, starting from the crystal structures of ATP-bound Hsp70 DnaK alone and in complex with the J-domain of DnaJ. We demonstrated that the presence of the J-domain results in the rearrangement of the nucleotide-binding pocket into a hydrolysis competent state, characterized by close contact between universally conserved T199 of NBD and {gamma}-phosphate of ATP. With network analysis we revealed that the allosteric signal for this rearrangement is transmitted along the {beta}-strand containing T199. Finally, we provide rationale for the signal transmission, where steric repulsion between the J-domains helix III and SBD induces a push of the T199 containing {beta}-strand. Overall, our study provides mechanistic insights into allosteric signal transmission within Hsp70, bridging the gap between J-domain binding and ATPase stimulation. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/655504v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ff0f30org.highwire.dtl.DTLVardef@3e4157org.highwire.dtl.DTLVardef@133f159org.highwire.dtl.DTLVardef@12a35c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Large-scale conformational analysis explains G-quadruplex topological landscape

G-quadruplexes (G4) are four-stranded nucleic acid structures formed within sequences containing repeated guanine tracts separated by loop regions. Abundant in the human genome, they play crucial roles in transcription regulation and genome maintenance. Although theoretically capable to adopt 26 different folding topologies--primarily differing in loop arrangements--only 14 of these have been observed experimentally. This raises fundamental questions about whether the remaining topologies are energetically inaccessible and what molecular factors shape the conformational landscape of G-quadruplexes. To address these questions, we systematically explored the conformational space of G-quadruplexes using a set of 128 G4-forming DNA sequences with varying loop lengths. Evaluation of nearly 20,000 unique G4 conformations revealed significant foldability differences across the 26 theoretical topologies. Crucially, we demonstrated that the presence of long-distance propeller loops in 12 of these topologies imposes strict loop length constraints, hindering their formation, especially in sequences with shorter loops. Additionally, we found that the occurrence of long-distance propeller loops is governed by G4 helicity, resulting in opposite folding preferences in right-handed and left-handed G4s. By providing geometric explanation for G4 folding patterns, our study advances the understanding of the G-quadruplex conformational landscape and offers valuable insights for the rational design of G4 structures. Author summaryDNA sequences enriched in guanines have the remarkable ability to form helical, four-stranded structures called G-quadruplexes (G4s). These structures have been found across the human genome, where they play a vital role in the regulation of various cellular processes, such as gene expression, replication, or genome maintenance. Moreover, designed G4 structures can be utilized as versatile building blocks in a variety of nanodevices. G4s are characterized by extensive structural diversity, arising from the multiple ways of arranging a DNA strand into a G4. Among the 26 geometrically valid arrangements--called looping topologies--only 14 have been proven experimentally, posing the question of whether the remaining topologies are energetically restricted and, if so, what molecular factors shape the topological landscape of G-quadruplexes. To address this question, in this work, we systematically searched the G4 conformational space for a set of 128 DNA sequences. We used an MD-based de novo folding procedure to evaluate foldability on nearly 20,000 unique G4 conformations. An analysis of the obtained data revealed that the topological landscape of G4s is predominantly restricted by long-distance propeller loops, whose occurrence among topologies is governed by G4s helicity.

bioinformatics↗