Biology subjects

Shinobu, A.

Publications and source records attributed to Shinobu, A..

3 recordsLinked to original sources

bioRxiv2026

Trans-Allosteric Activation Releases Distinct Conformational Traps in Kinase Heterodimers

Protein kinases function as dynamic, mechanically coupled nodes, yet the conformational drivers of multimeric activation remain unclear. Here, we present AlloQuant, a computational suite that translates AlphaFold3 structural ensembles into quantitative metrics of kinase regulation, including internal network rigidity, metastable-state populations, and sub-angstrom conformational drivers. Applying AlloQuant to CDK1, we demonstrate that binding of the Cyclin B1 (CCNB1) cofactor mechanically decouples a hyper-rigid inactive kinase core, allowing activating phosphorylation (pT161) to subsequently re-impose localized tension on the catalytic machinery. Conversely, the C-terminal Src kinase (CSK) faces a distinct conformational trap. While nucleotide-free monomeric CSK spontaneously samples a pre-active geometry, ATP binding excludes the active C-In conformation in all but 1 of 225 models. We show that docking partner engagement overcomes this blockade. Autophosphorylation of SRC at the activation loop (Y419) redistributes SRC conformational states without altering bulk rigidity. This redistribution is structurally coupled to the conformational state of CSK via the regulatory spine, not the catalytic machinery. Rather than mechanically deforming CSK, SRC engagement acts by conformational selection, committing roughly a quarter of CSK molecules to a fully active state. Thus, trans-allosteric kinase activation operates by defining the accessible conformational landscape of the receiver kinase. That control is exerted through mechanical remodeling in cofactor-dependent complexes and through conformational selection in transient kinase-kinase heterodimers. These findings establish AlloQuant as a general framework for quantifying how a binding partner reshapes a kinase's conformational landscape, applicable across the kinome because it assigns landmarks by profile-HMM alignment.

biophysics↗

bioRxiv2026

Protein-peptide binding pathways revealed by two-dimensional replica-exchange molecular dynamics

Protein kinases regulate signaling by recognizing short sequence motifs, and how these motifs bind influences both specificity and therapeutic strategies that target kinase pathways. Peptide-based inhibitors that engage substrate-recognition regions are attracting interest, but designing them requires an understanding of how a flexible peptide approaches and settles into the bound pose. Traditional studies have focused on the bound pose and affinities, whereas the steps that link the initial encounter with the bound pose have been explored less thoroughly because the relevant intermediates are too short-lived to capture experimentally and evolve on timescales that standard molecular dynamics cannot readily access. Here, we focused on Abl kinase and Abltide, the experimentally identified optimal substrate peptide for Abl kinase, and examined the sequence of events linking initial encounter to the bound pose using two-dimensional replica exchange (gREST/REUS), which selectively enhances flexibility in the peptide and its binding interface while also sampling progression along a distance coordinate. The resulting simulations yielded a detailed binding landscape, revealing five distinct encounter regions outside the substrate-binding site and six intermediate states that may connect the initial approach to the bound pose. Some encounter regions and intermediate states participate in the dominant binding pathways. During this process, EF/G/{beta}11 hydrophobic patch, together with G helix negative patch, plays a central role in guiding Abltide toward the substrate-binding site. These findings provide mechanistic insight into substrate recognition by protein kinases and offer a foundation for the rational design of peptide-based inhibitors.

biophysics↗

bioRxiv2022

The impact of inhibitor size and flexibility on the binding pathways to c-Src kinase

Considering dynamical aspects of protein-drug binding processes is inevitable in current drug compound design. Conformational plasticity of protein kinases poses a challenge for the design of their inhibitors, and therefore, atomistic molecular dynamics (MD) simulations have often been utilized. While protein conformational changes have been increasingly discussed, a fundamental yet non-trivial question remains for the effect of drug compound flexibility, which is hardly detectable from experiments. In this study, we apply two-dimensional replica-exchange MD simulations as enhanced sampling to investigate how c-Src kinase can bind PP1, a small inhibitor, and dasatinib, a larger inhibitor with greater flexibility. 600 microseconds simulations in total sample binding and unbinding events of these inhibitors much more frequently than conventional MD simulation, resulting in statistically converged binding pathways. While the two inhibitors adopt a similar mechanism of multiple binding pathways, the non-canonical binding poses become less feasible for dasatinib. A notable difference is apparent in their energetics where dasatinib stabilizes at intermediate states more than PP1 to raise the barrier toward the canonical pose. Conformational analysis shows that dasatinib adopts linear and bent forms for which relative populations are altered upon binding. We further find hidden conformations of dasatinib at intermediate regions, and unexpectedly one of them could efficiently bypasses the intermediate-to-bound state transition. The results demonstrate that inhibitor size and flexibility impact the binding mechanism, which could potentially modulate inhibitor residence time.

biophysics↗