Biology subjects

Nierzwicki, L.

Publications and source records attributed to Nierzwicki, L..

4 recordsLinked to original sources

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↗

bioRxiv2024

Mechanism of expanded DNA recognition in xCas9

xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9s expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.

biophysics↗

bioRxiv2021

Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9

CRISPR-Cas9 is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, solution NMR is combined with multi-microsecond molecular dynamics and graph theory-derived models to probe the allosteric role of key enhancement specificity mutations. We show that the mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the HNH domain allosteric structure, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric intercommunication. This differential perturbation of the allosteric signaling reflects the different capabilities of the single mutants to increase Cas9 specificity, with the mutation achieving the highest specificity also strongly perturbing the signaling transfer. These outcomes reveal that the allosteric regulation is critical for the specificity enhancement of the Cas9 enzyme, and are valuable to harness the signaling network to improve the systems specificity.

biophysics↗

bioRxiv2020

Two-step mechanism of J-domain action in driving Hsp70 function

J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface - thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition - via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/901538v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@ba0d3aorg.highwire.dtl.DTLVardef@ba9193org.highwire.dtl.DTLVardef@ab7974org.highwire.dtl.DTLVardef@1b2de33_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗