Periodic Reporting for period 1 - KIRPAS (Structural characterization of a Kir potassium channel and its involvement in Andersen’s syndrome)
Periodo di rendicontazione: 2022-07-01 al 2024-06-30
Kir2.1 channels are integral membrane proteins that selectively control the permeation of K+ ions across cell membranes. This K+ flow is essential for many physiological processes, including maintaining the electrical stability of cells, especially in the heart and muscles. The opening and closing of these channels, a process known as gating, is modulated by a molecule called PIP2 (phosphatidylinositol 4,5-bisphosphate). However, the description of the way in which PIP2 induces the structural changes in Kir2.1 channels to control the gating mechanism remains poorly understood. This lack of understanding is significant because defects in the Kir2.1 channels are linked to several human diseases, including Andersen-Tawil syndrome (ATS), a genetic disorder that can lead to episodes of muscle weakness and paralysis, and abnormal heart rhythms. Despite the known connection between Kir2.1 mutations and ATS, it is not yet clear how these mutations impact the Kir2.1 channel structure and disrupt the normal function of these channels. The primary objective of this project is to uncover how Kir2.1 channels function at atomic level, both in their normal state and when affected by ATS-related mutations. This project successfully determined two high-resolution structures using cryo-EM of human Kir2.1 in the absence of PIP2, each in a different conformation. The first structure was obtained using a Kir2.1 sample in the presence of DDM detergent and captured the Kir2.1 channel in an extended, closed and non-conductive state and provided insights into its mechanism of function. The second structure, obtained using a Kir2.1 sample in the presence of amphipols, revealed the channel in a compact conformation, but in an inhibited or poorly activated state. Moreover, by combining electrophysiological experiments, cryo-EM analysis and molecular dynamics simulations, this project identified the molecular mechanisms by which two ATS-causing mutations (R312H and C154Y) impact Kir2.1 channel function.
Both Kir2.1-WT and Kir2.1-R312H mutant were successfully obtained as homogeneous samples in DDM detergent and in amphipols A8-35 (a detergent-free environment) in sufficient quantity to perform structural studies using cryo-electron microscopy (cryo-EM). Two high-resolution structures of the human Kir2.1-WT were successfully determined using cryo-EM, in the absence of PIP2, each in a different conformation. The first structure was obtained using a Kir2.1-WT sample in the presence of DDM detergent. This is the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). This structure captured the Kir2.1 channel in an extended, closed and non-conductive state The second structure, obtained using a Kir2.1-WT sample in the presence of amphipols A835 (a detergent-free environment), revealed the channel in a compact conformation, but in an inhibited or poorly activated state. Amphipols A8-35 bind to some residues of the PIP2-binding site of Kir2.1 channels in a manner quite similar to how pyrophosphatidic acid (PPA, a competitive inhibitor of Kir2.2 channels) binds to the Kir2.2 structure. Moreover, Kir2.1-WT was successfully reconstituted in MSP:POPC nanodiscs.
KIRPAS was able to provide novel structural data able to describe the molecular mechanisms by which two ATS-causing mutations (C154Y and R312H) affect the function of these channels. Notably, they hinder the channel function by different mechanisms. The cryo-EM structure of the Kir2.1-R312H mutant was obtained at medium resolution; however, molecular modeling analysis revealed that the residue R312 is involved in a network of intersubunit interactions, which are completely disrupted by the R312H mutation and that impairs the channel gating. Molecular dynamics simulations of the Kir2.1-C154Y mutant showed that the C154Y mutation interrupts the structural connection between the flexible extracellular loops and the selectivity filter, resulting in a loss of structural plasticity of the selectivity filter and impairing the K+ flow.
Unfortunately, any cryo-EM structure in the presence of PIP2 was obtained, which impaired to fully describe the structural mechanisms that govern the Kir2.1 gating. However, alternative computational approaches were performed, which revealed that Kir2.1 channel exhibits an inherent compaction motion that PIP2-binding does not induce the compact conformation but rather stabilizes the compressed state. On the other hand, this project was able to provide a high-resolution cryo-EM structure of the Kir2.1 channel bound to with a specific microRNA called m1R1, which was recently discovered as a novel modulator of the Kir2.1 channel function.
The results concerning the cryo-EM structure of Kir2.1-WT channel obtained with samples in DDM were published in Science Advances (Impact Factor: 13.7; doi: 10.1126/sciadv.abq8489). Moreover, the cryo-EM map of Kir2.1-WT (sample in DDM) is publicly available in the Electron Microscopy Data Bank (EMDB) under the code EMD-14678, and the corresponding atomic model is publicly available in the Protein Data Bank (PDB) under the code 7ZDZ. Additionally, the protocol for performing MD simulations are public available at https://bio-protocol.org/rap30599(si apre in una nuova finestra). These protocols, along with the cryo-EM data processing protocol, were as a book chapter in the book Potassium Channels (Methods in Molecular Biology, volume 2796, doi: 10.1007/978-1-0716-3818-7_10).
The results concerning the cryo-EM structure of Kir-R312H mutant (section 1.2.2.4 above) and the MD simulations of the Kir2.1-C154Y mutant (section 3.1 below) were currently under the second round of peer-reviewing in the Faseb Journal (Impact Factor: 5.9) and are a non-peer-reviewed version is publicly available at bioRxiv (doi: 10.1101/2024.02.09.579451). Moreover, the cryo-EM map of Kir2.1-R312H mutant is publicly available in the EMDB under the code EMD-18595 and its respective atomic model is public available in the Protein Data Bank (PDB) under the code 8QQL.
At least two more papers related to the KIRPAS project are expected to be published in the near future. One will focus on the MD simulations performed in the cryo-EM structure of Kir2.1-WT in amphipols A8-35. The other will cover the cryo-EM structure of Kir2.1-WT bound to m1R1. The results of the KIRPAS project were also presented in conferences and seminars worldwide, including the oral and poster presentation in the 68th Biophysical Society Annual Meeting (Philadelphia, USA) in 2024. Finally, the results of the KIRPAS project were also featured in a report for the general public, produced by the scientific dissemination department from the Sorbonne University: https://shorturl.at/RnGqN(si apre in una nuova finestra).
KIRPAS was able to provide novel structural data able to describe the molecular mechanisms by which two ATS-causing mutations (C154Y and R312H) affect the function of these channels. Notably, they hinder the channel function by different mechanisms. The cryo-EM structure of the Kir2.1-R312H mutant was obtained at medium resolution; however, molecular modeling analysis revealed that the residue R312 is involved in a network of intersubunit interactions, which are completely disrupted by the R312H mutation and that impairs the channel gating. Molecular dynamics simulations of the Kir2.1-C154Y mutant showed that the C154Y mutation interrupts the structural connection between the flexible extracellular loops and the selectivity filter, resulting in a loss of structural plasticity of the selectivity filter and impairing the K+ flow.
Unfortunately, any cryo-EM structure in the presence of PIP2 was obtained, which impaired to fully describe the structural mechanisms that govern the Kir2.1 gating. However, alternative computational approaches were performed, which revealed that Kir2.1 channel exhibits an inherent compaction motion that PIP2-binding does not induce the compact conformation but rather stabilizes the compressed state. On the other hand, this project was able to provide a high-resolution cryo-EM structure of the Kir2.1 channel bound to with a specific microRNA called m1R1, which was recently discovered as a novel modulator of the Kir2.1 channel function.
The results concerning the cryo-EM structure of Kir2.1-WT channel obtained with samples in DDM were published in Science Advances (Impact Factor: 13.7; doi: 10.1126/sciadv.abq8489). Moreover, the cryo-EM map of Kir2.1-WT (sample in DDM) is publicly available in the Electron Microscopy Data Bank (EMDB) under the code EMD-14678, and the corresponding atomic model is publicly available in the Protein Data Bank (PDB) under the code 7ZDZ. Additionally, the protocol for performing MD simulations are public available at https://bio-protocol.org/rap30599(si apre in una nuova finestra). These protocols, along with the cryo-EM data processing protocol, were as a book chapter in the book Potassium Channels (Methods in Molecular Biology, volume 2796, doi: 10.1007/978-1-0716-3818-7_10).
The results concerning the cryo-EM structure of Kir-R312H mutant (section 1.2.2.4 above) and the MD simulations of the Kir2.1-C154Y mutant (section 3.1 below) were currently under the second round of peer-reviewing in the Faseb Journal (Impact Factor: 5.9) and are a non-peer-reviewed version is publicly available at bioRxiv (doi: 10.1101/2024.02.09.579451). Moreover, the cryo-EM map of Kir2.1-R312H mutant is publicly available in the EMDB under the code EMD-18595 and its respective atomic model is public available in the Protein Data Bank (PDB) under the code 8QQL.
At least two more papers related to the KIRPAS project are expected to be published in the near future. One will focus on the MD simulations performed in the cryo-EM structure of Kir2.1-WT in amphipols A8-35. The other will cover the cryo-EM structure of Kir2.1-WT bound to m1R1. The results of the KIRPAS project were also presented in conferences and seminars worldwide, including the oral and poster presentation in the 68th Biophysical Society Annual Meeting (Philadelphia, USA) in 2024. Finally, the results of the KIRPAS project were also featured in a report for the general public, produced by the scientific dissemination department from the Sorbonne University: https://shorturl.at/RnGqN(si apre in una nuova finestra).
The Kir2.1-WT structure in DDM is the first published human Kir2.1 channel structure, containing both transmembrane and cytoplasmic domains, offering insights into the K+ flow mechanism. The Kir2.1-WT in amphipols may reveal how amphipols interact with functional regions of the protein, inducing conformational changes similar to biologically active molecules. KIRPAS also established the structural basis for understanding the impact of Anderson-Tawil syndrome (ATS) mutations on Kir2.1 channel structure and function. These findings could guide therapeutic development, helping design molecules or nanobodies that target critical regions like R312 and C154, potentially restoring disrupted interactions caused by mutations