In signaling cascades, signaling proteins often encode multiple domains or motifs, which presents the possibility for avidity-where multivalent binding drastically increases interaction strength and duration. However, predicting and validating multivalent interactions that interact with avidity is a challenge. Here, we integrate mechanistic modeling, structure-based analysis, and experimental approaches as a framework for defining the conditions under which avidity plays a role. We explore the tandem SH2 domain family of interactions with bisphosphorylated partners as a multivalent archetype, which encompasses key secondary messengers in tyrosine kinase signaling networks. Theoretical modeling suggests that maximum avidity occurs with closely spaced tyrosine phosphorylation sites combined with moderate monovalent affinities-exactly around the innate range of SH2 domain affinity-or with phosphorylation sites separated by sufficiently flexible linkers. Surprisingly, despite sequence diversity, structure-based analysis showed relatively conserved three-dimensional spacing between SH2 domains across all tandem SH2 families, which we corroborate experimentally, suggesting evolutionary optimization for avidity interactions. The combination of structure-based analysis of domain spacing with available monovalent experimental data, along with iterative experimental refinement of biophysical parameters, appears able to identify high-affinity interactions of tandem SH2 domain recruitment to the epidermal growth factor receptor C-terminal tail. Using these principles, we extended bivalent predictions into the full phosphoproteome space and structural parameterization of other partners of SH2 domain binding, providing resources and methods for more rapid expansion of bivalent analysis. These approaches lay the groundwork for larger utility in multivalent prediction and testing to help better understand protein interactions that drive cell signaling.
Calcium-binding protein 2 (CaBP2), a member of the calmodulin (CaM) superfamily, modulates the L-type calcium channel CaV1.3 in the cochlear inner hair cells by suppressing its voltage-and Ca2+-dependent inactivation. Like CaM, CaBP2 contains four EF-hand motifs, although only three are functional, yet the precise nature and physiological relevance of its Ca2+binding remain poorly understood. In this study, we present the binding properties of the individual EF-hand motifs of CaBP2L for Ca2+ and Mg2+ using isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. The measured dissociation constants of the individual EF-hand motifs span several orders of magnitude, suggesting a hierarchical mode of Ca2+sensing. Our data demonstrate that the CaBP2L structure is stabilized upon Ca2+ rather than Mg2+ binding. While the N-lobe of CaBP2L significantly contributes to structural fluctuation in the absence of Ca2+, this fluctuation is reduced upon Ca2+binding. Furthermore, our electrophysiology experiments show that upon disruption of all three Ca2+ binding EF hands of CaBP2 its ability to attenuate Ca2+-dependent inactivation is reduced. Therefore, we propose that CaBP2L displays a distinct Ca2+ binding property compared to other CaBPs, and that Ca2+ binding influences the stability of its structure and consequently channel regulation.
Ion channels are transmembrane proteins that regulate cellular functions by facilitating ion movement across membranes. G-protein–gated inwardly rectifying potassium channel 1 (GIRK1) is expressed in the brain and heart. Interestingly, GIRK1 has been associated with estrogen receptor-positive (ER+) breast cancer. Elevated GIRK1 expression is correlated with shorter survival times and increased metastatic propensity, highlighting its potential as a cancer biomarker. Herein, we developed approximately 4 nm GAT1508-coated poly(ethylene glycol) (PEG)-functionalized gold nanoparticles (AuNPs) for optical detection of ER+ breast cancer cells. GAT1508 is a urea-based small molecule that preferentially binds GIRK1 subunits and activates GIRK1/2 channels. Two derivatives, GAT1508-EA and GAT1508-PA, were synthesized by extending the molecular structure with ethylamine and propylamine chains from the benzene and pyrazole rings, respectively. Electrophysiological experiments, including two-electrode voltage clamp and whole-cell patch clamp recordings, alongside fluorescent studies (thallium assay), demonstrated that only GAT1508-PA inhibited GIRK1/2-mediated K+ currents in transfected HEK293 cells. Molecular docking further supported strong binding of GAT1508-PA and its PEG-conjugated amide derivative, GAT1508-PA-EG2. Subsequently, GAT1508-PEG-AuNPs were synthesized with approximately 65 wt% metal loading. UV-Visible confirmed ligand conjugation (260 nm peak). Flow cytometry studies demonstrated binding of GAT1508-NPs in ER+ MCF-7 breast cancer cells. Furthermore, incubation of fixed MCF-7 cells with GAT1508-NPs enabled optical detection without fluorescent dyes or signal amplification. In contrast, detection was unsuccessful in triple (−) MDA-MB-231 cells, which lack GIRK1 expression. This study introduces a novel approach integrating small-molecule design, electrophysiology, and nanotechnology to develop ion channel-targeted molecular probes for screening ER+ breast cancer cells.
Small cell lung cancer (SCLC) remains a highly lethal malignancy with limited therapeutic advances. Although high-dose vitamin C (Vc) exhibits anti-tumor activity in various cancers, its therapeutic potential and underlying resistance mechanisms in SCLC remain poorly understood. Here, we identified heterogeneous responses to Vc among SCLC cell lines and found that Vc-resistant cells acquire enhanced antioxidant capacity to tolerate Vc-induced oxidative stress. Through transcriptomic, multi-omics, and functional analyses, we identified glucose-6-phosphate dehydrogenase (G6PD) as a critical determinant of Vc resistance. Mechanistically, Vc activates the p-AKT/p-GSK3β/NRF2 signaling axis in resistant cells, leading to NRF2-dependent transcriptional upregulation of G6PD and activation of antioxidant adaptation programs. Integrated analyses of transcriptomic, proteomic, single-cell, and spatial transcriptomic datasets further supported the involvement of the NRF2-G6PD axis in oxidative stress adaptation in SCLC. Importantly, pharmacological inhibition of G6PD with RRx-001 restored Vc sensitivity and synergistically suppressed SCLC growth in vitro and in vivo. Collectively, our findings reveal a previously unrecognized mechanism underlying Vc resistance in SCLC and demonstrate that co-targeting G6PD provides a potential strategy to enhance the therapeutic efficacy of Vc.
