Biology subjects

Mim, C.

Publications and source records attributed to Mim, C..

4 recordsLinked to original sources

bioRxiv2025

Structural basis of ATP release and ion selectivity in Pannexin1 channels

This study investigated the structural basis of adenosine triphosphate (ATP) release and ion selectivity by Pannexin1 (Panx1). The microinjection of active SRC tyrosine kinase increased Panx1-mediated currents in Panx1-expressing oocytes. Furthermore, a mutation mimicking phosphorylation at Y308 turned Panx1 into a constitutively open ATP-releasing channel with broad ion permeability. Cryogenic electron microscopy showed that for most maps, the channel alternated between wide and narrow conformations. However, in the map of the phosphomimetic Y308E mutation, Panx1 remains in a wide ATP-releasing conformation. The pore-lining residues, W74 and R75, are flexible and create, in concert with the sequestered N-terminal helix, a path for ATP. The data point to phosphorylation as a reversible switch that activates ATP permeability by stabilizing Panx1 in the ATP-releasing conformation, which is crucial for developing modulators for diseases such as cancer and chronic pain.

biophysics↗

bioRxiv2025

In silico classification and identification co-purified protein complexes yield new structures and multiple MSP assembly states

Native protein complexes have garnered interest as targets for structural dissemination. Cryogenic electron microscopy (cryoEM) with its ability to image protein mixtures is the most promising tool to enable structural proteomics. Additionally, image processing has evolved to deal with conformational and compositional heterogeneity. Integrative approaches, namely mass spectrometry in conjunction with cryoEM have made it possible to characterize and identify complex mixtures. However, this comes at a cost of generating models and interpreting mass spectra. Here we present a modified approach that only requires electron micrographs and a computer for unsupervised model building and protein identification. We were able to identify co-purified membrane proteins, resulting in a novel structure and unexpected nanodisc assemblies, which imply direct interaction between membrane proteins and membrane scaffolding proteins.

biochemistry↗

bioRxiv2024

A metastasis-associated Pannexin1 mutant (Panx11-89) forms a minimalist ATP release channel

A truncated form of the ATP release channel pannexin 1 (Panx1), Panx11-89, is enriched in metastatic breast cancer cells and has been proposed to mediate metastatic cell survival by increasing ATP release through mechanosensitive Panx1 channels. However, whether Panx11-89 on its own (without the presence of wtPanx1) mediates ATP release has not been tested. Here, we show that Panx11-89 by itself can form a constitutively active membrane channel, capable of releasing ATP even in the absence of wild type Panx1. Our biophysical characterization reveals that most basic structure-function features of the channel pore are conserved in the truncated Panx11-89 peptide. Thus, augmenting extracellular potassium ion concentrations enhances Panx11-89-mediated conductance. Moreover, despite the severe truncation, Panx11-89 retains the sensitivity to most of wtPanx1 channel inhibitors and can thus be targeted. Therefore, Panx1 blockers have the potential to be of therapeutic value to combat metastatic cell survival. Our study not only elucidates a mechanism for ATP release from cancer cells, but it also supports that the Panx11-89 mutant should facilitate structure-function analysis of Panx1 channels.

biophysics↗

bioRxiv2020

The Structural Basis for SARM1 Inhibition, and Activation Under Energetic Stress

SARM1 is a central executor of axonal degeneration (1). Mechanistically, SARM1 contains NADase activity, which, in response to nerve injury, depletes the key cellular metabolite, NAD+ (2-5). Interestingly, SARM1 knockout mouse models do not present any apparent physiological impairment. Yet, the lack of SARM1 protects against various neuropathies (6, 7), thereby highlighting SARM1 as a likely safe and effective drug target (8). However, the absence of a SARM1 structure, in its active or inhibited form, makes it impossible to understand the molecular basis of SARM1 inhibition, and its activation under stress conditions. In this study we present two cryo-EM maps of SARM1 (at 2.6 [A] and 2.9 [A] resolution). We show that the inhibited SARM1 homo-octamer assumes a packed conformation with well-ordered inner and peripheral rings. Here the catalytic TIR domains are held apart from each other and are unable to form dimers, which is a prerequisite for NADase activity. More importantly, after screening several cellular metabolites we discovered that the inactive conformation is stabilized by the binding of SARM1s own substrate: NAD+. The NAD+ inhibitory allosteric site is located away from the NAD+ catalytic site of the TIR domain. Site-directed mutagenesis of the allosteric site leads to constitutive active SARM1. Based on our data we propose that a reduction of cellular NAD+ concentrations (an early indication of disease-associated and age-related neurodegeneration (9)) disassemble SARM1s peripheral ring, which allows NADase activity. This leads to an energetic catastrophe and eventually cell death. The discovery of the allosteric inhibitory site opens the door for the development of effective drugs that will prevent SARM1 activation, rather than compete for binding to the NADase catalytic site. Brief descriptionIt is not known how NAD+ depletion brings about neurodegeneration. Here, we show that the intrinsic NADase activity of SARM1 is allosterically inhibited by physiological concentrations of NAD+. NAD+ stabilizes a compact, auto-inhibited conformation of the SARM1 octamer. Once NAD+ levels are depleted, the allosteric inhibition is released, enabling SARM1s NADase activity, which eventually leads to energetic catastrophe and cell death.

biochemistry↗