Biology subjects

Blachowicz, L.

Publications and source records attributed to Blachowicz, L..

3 recordsLinked to original sources

bioRxiv2025

The Closed State of the Shaker Potassium Channel and the Mechanism of Voltage Activation

To understand the process of voltage activation in Kv channels, we determined the cryo-EM structure of the Shaker channel ILT mutant (V369I, I372L, S376T), known to partially decouple the gating charge movement of the voltage sensing domain (VSD) and the opening of the pore domain (PD). The structure captures a previously unobserved intermediate state in which the VSD is only partially activated while the PD remains closed. Combined with computational modeling based on AlphaFold2 predictions and molecular dynamics simulations, it is shown that VSD activation does not mechanically pull the gate open, but instead induces a dynamic shift in the population equilibrium of the VSD-PD linker, providing the basis for electromechanical coupling between the two domains.

biophysics↗

bioRxiv2025

Molecular Dynamics-Guided Design and Chemoproteomic Profiling of Covalent Kinase Activity Probes

Covalent small molecule activity probes can be powerful tools to interrogate protein function in native cellular environments. The design of family-wide activity probes requires an understanding of the molecular sources of general targeting potential and specificity to enable broad targeting of protein family members. Here, we developed and applied a multifaceted docking and molecular dynamics (MD) simulation pipeline to design and test cell-permeable covalent kinase activity probes from a set of hinge-binding pharmacophores. This computationally-guided approach yielded a new cell-active probe, K60P, which targets around 114 kinases across distinct kinase classes in live cells. Chemoproteomic profiling of this probe and a clinical candidate sharing the same indazole core, KW-2449, identified kinase and non-kinase target profiles that differ from recombinant protein assay profiles, underscoring the utility of native kinase profiling in situ. Biochemical studies with a model target kinase, ABL1, confirmed covalent labeling of the active site lysine across several kinase probes with distinct kinetics, as well as covalent labeling of key tyrosines in trans between ABL1 monomers. Finally, focused proteomics, kinetic modeling, and molecular dynamics simulations revealed that K60P, as well as the comparator probe XO44, preferentially engage with target kinases in their active, DFG-in conformations, which is driven by increasing population of reaction-ready small molecule conformation. These results together establish a computational and kinetic modeling framework for designing covalent activity probes and highlight the balance of target selectivity and kinetic efficiency as a key factor in determining their proteome-wide reactivity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/683178v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1ea3e5forg.highwire.dtl.DTLVardef@1a4e999org.highwire.dtl.DTLVardef@1e468c3org.highwire.dtl.DTLVardef@99e04e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

A different mechanism of C-type inactivation in the Kv-like KcsA mutant E71V

A large class of K+ channels display a time-dependent phenomenon called C-type inactivation whereby prolonged activation by an external stimulus leads to a non-conductive conformation of the selectivity filter. C-type inactivation is of great physiological importance particularly in voltage-activated K+ channels (Kv), affecting the firing patterns of neurons and shaping cardiac action potentials. While understanding the molecular basis of inactivation has a direct impact on human health, its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the pH-activated bacterial K+ channel KcsA, whose selectivity filter under inactivating conditions adopts a constricted conformation at the level of the central glycine (TTVGYGD) that is stabilized by tightly bound water molecules. However, C-type inactivation is highly sensitive to the molecular environment surrounding the selectivity filter in the pore domain, which is different in Kv channels than in the model KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is consistently substituted by a nonpolar valine in most Kv channels, suggesting that this side chain is an important molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and molecular dynamics simulations of the E71V mutant of KcsA is undertaken to explore the features associated with this Kv-like construct. In both X-ray and ssNMR data, it is observed that the filter of the Kv-like KcsA mutant does not adopt the familiar constricted conformation under inactivating conditions. Rather, the filter appears to adopt a conformation that is slightly narrowed and rigidified over its entire length. No structural inactivation water molecules are present. On the other hand, molecular dynamics simulations indicate that the familiar constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like E71V mutation in the KcsA channel may be associated with different modes of C-type inactivation, showing that distinct selectivity filter environments entail distinct C-type inactivation mechanisms.

molecular biology↗