Biology subjects

Kukreja, R.

Publications and source records attributed to Kukreja, R..

3 recordsLinked to original sources

bioRxiv2026

How a highly acidic SH3 domain binds to its intrinsically disordered partner through the formation of an encounter complex intermediate

Electrostatic interactions often play a role in determining the thermodynamic and kinetic properties of protein-protein interactions. However, the role of long-range electrostatic interactions in intrinsically disordered protein (IDP) binding is less clear, as they often bind in multiple steps including initial formation of a disordered encounter complex, followed by rearrangement into the bound state. We varied the salt concentration to probe the role of long-range electrostatic interactions in the binding of the highly charged AbpSH3 domain and the oppositely charged IDP ArkA. Using isothermal titration calorimetry, we observe that salt enthalpically destabilizes the bound complex. Molecular dynamics and NMR experiments reveal that salt has little effect on the bound state structure. However, simulations show that salt destabilizes the encounter complex intermediate, which primarily affects the association rate as measured by NMR. Consistent with these results, salt has the largest stabilizing effect on the apo SH3 domain, as cations substitute for the transient and long-range electrostatic interactions that can form with ArkA in the complex. We reveal a detailed picture of how a highly charged domain uses long-range, fuzzy, electrostatic interactions to help reach the bound state, a mechanism that is likely common among other highly charged domains that bind IDPs. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/741257v1_ufig1.gif" ALT="Figure 1000"> View larger version (21K): org.highwire.dtl.DTLVardef@dfc7acorg.highwire.dtl.DTLVardef@1ae0438org.highwire.dtl.DTLVardef@19704adorg.highwire.dtl.DTLVardef@1b40b15_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

DNA modulates structural transitions and oligomerization kinetics of the functional amyloid CRES

Functional amyloids play critical roles in diverse physiological processes; however, the molecular mechanisms regulating their assembly remain unknown. The mouse epididymal lumen contains a functional amyloid- and nucleic acid-rich extracellular matrix with roles in host defense and sperm maturation. Cystatin-related epididymal spermatogenic (CRES), a reproductive cystatin and key component of the mammalian epididymal amyloid matrix, assembles into structurally heterogeneous amyloid forms to support the various roles of this extracellular matrix. Here, we show that CRES binds double-stranded DNA with sub-micromolar affinity in a sequence independent manner and that DNA binding accelerates amyloid formation by increasing local protein concentration and promoting specific oligomerization pathways. Using NMR spectroscopy, site-directed mutagenesis, and biophysical analyses, we find that DNA interacts primarily with the CRES loop region, thereby occluding one assembly mechanism and redirecting oligomerization through a pathway involving the L1 loop. These DNA-mediated changes in assembly kinetics and pathway selection suggest a regulatory mechanism for achieving structural and functional diversity in non-pathological amyloid systems. Our findings provide a molecular framework for understanding nucleic acid-guided amyloidogenesis and highlight how functional amyloids may exploit multiple assembly routes to fulfill their physiological roles.

biophysics↗

bioRxiv2025

Alternatively-Spliced CAMKK2 isoforms drive differential metabolic stress response and the regulation of ferroptosis

Calcium/Calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a multifunctional kinase that regulates metabolic processes by phosphorylating downstream targets. CAMKK2 is expressed as several highly similar protein isoforms, although the specific functions of these individual isoforms remain largely unexplored. These isoforms have been shown to display tissue-specific expression patterns, suggesting unique roles in distinct cellular contexts. In this study, we investigated the biochemical and functional relevance of CAMKK2 isoforms, LF and SF, in the context of glucose metabolism. Co-immunoprecipitation experiments revealed that the LF isoform preferentially binds the adaptor protein 14-3-3, while the SF isoform interacts with Calmodulin. Furthermore, the interaction between SF and Calmodulin was enhanced upon glucose starvation, whereas this interaction was not observed for LF. To assess their functional significance, we generated doxycycline-inducible, isoform-specific HeLa cell lines. Under low glucose conditions, cells expressing the LF isoform failed to activate AMPK, while cells expressing the SF isoform exhibited robust increase in AMPK phosphorylation. Moreover, LF-expressing cells accumulated higher levels of reactive oxygen species, upregulated ferroptosis-related genes via BACH1/NRF2, and displayed increased cell death compared to SF-expressing cells. Collectively, these findings demonstrate that CAMKK2 isoforms exhibit differential selectivity for protein partners and mediate distinct downstream signaling pathways, impacting metabolism in cancer.

biochemistry↗