bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.06.02.543453

Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states

Lin, D. Y.-w.·Kueffer, L. E.·Juneja, P.·Wales, T. E.·Engen, J. R.·Andreotti, A. H.
View original source ↗Export metadata ↓

Abstract

Full-length BTK has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology (PHTH) domain and proline-rich regions (PRR) contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveals only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. CryoEM data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with PIP 3. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

Source connections

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Lin, D. Y.-w., Kueffer, L. E., Juneja, P., Wales, T. E., Engen, J. R., Andreotti, A. H.. 2023-06-03. Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states. https://doi.org/10.1101/2023.06.02.543453

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A quantum state of mitochondria in the living cell

The high energy-efficiency of life is hard to understand only with classical physics. Many efforts have been made to study its mechanism based on quantum mechanics; the progress is nevertheless slow due to lack of experimental evidence with living cells. Here, combining experiments on cells, tissues and mitochondria with a theoretical model, we demonstrate a quantum state of mitochondria, which can be employed to modulate ATP production in living cells. We found an anomalous 71.0-THz oscillation mode only in living cells and tissues, which is highly determined by intact structure of mitochondria, and cannot be assigned to any specific molecules. Based on experimental data, a quantum model of light-matter coupling was introduced to trace the origin of this mode. Our calculations suggest a quantum superposition state of functional mitochondrion that forms by the coupling of light and lipid CH2 bonds in functional cristae, and induces a splitting of the intrinsic CH2 vibration mode of 87 THz to two levels at 71 THz and 103 THz, respectively. The former can be observed only in living cells and tissues; whereas the latter falls in the range (90-110 THz) of biomolecular and water vibrations, thus indistinguishable. Additional experiments revealed this mitochondrial quantum state able to serve as an efficient channel to modulate ATP production. Our findings provide a quantum mechanics view for understanding living cells, and it will be interesting to further explore whether such quantum state could act as a channel for energy metabolism, and even information transmission in life.

biophysics↗
bioRxiv2026

Mechanistical and structural basis of Kv channel inhibition by 4 aminopyridine

Inhibition of Kv channels by 4-aminopyridine (4AP) improves motor function in multiple sclerosis by enhancing neuronal excitability. The mechanism of inhibition and the structural basis of 4AP binding to Kv channels remain unclear. Here, we determined the structure of the Shaker V369I-I372L-S376T (ILT) mutant bound to 4AP at 3.3 [A], demonstrating that 4AP binds to the closed state of the channel. This structure is inconsistent with an open channel block mechanism. Electrophysiology experiments show that 4AP binds even when intracellular pore access is constitutively blocked, suggesting that 4AP enters the pore through membrane-facing fenestrations. MD simulations and mutational analysis agree with the proposed fenestration pathway and suggest that 4AP binds in its neutral form. These results support a mechanism where 4AP binds to a partially activated closed state that prevents complete activation of Kv channels, explaining its pharmacological activity.

biophysics↗
bioRxiv2026

Substrate binding reorganizes the energetic landscape of Plasmodium falciparum hexose transporter PfHT1

Malaria parasites depend on the Plasmodium falciparum hexose transporter PfHT1 for sugar uptake, yet how substrate binding reshapes transporter energetics and kinetics of sugar transport remains poorly understood. Here, we investigate how glucose reorganizes the conformational landscape, transition pathways, and residue interaction network underlying membrane transport. Using over 800 s of adaptive molecular dynamics simulations combined with Markov state models, transition-path theory, residue-contact analysis, and graph attention learning, we reconstruct the apo and glucose-bound conformational cycles. We show that glucose selectively stabilizes productive outward-facing, occluded, and inward-facing conformations, reshapes transition kinetics, and channels reactive flux through the occluded state. We identify TM7b helix cracking as a local structural transition coupled to extracellular-gate closure and substrate progression, providing a flexible connection between the binding pocket and global alternating access. Experimental testing of mechanistically critical residues validated their functional importance in PfHT1-dependent sugar utilization. Together, these results show how substrate binding reorganizes the energetic, kinetic, and interaction landscape of membrane transport and establish a transferable framework for studying transporter mechanisms.

biophysics↗