Figures and data
Structure of full-length human PIEZO1 channel
a, 38-TM topology model of a single hPIEZO1 subunit. The 9 THUs, a long beam helix, an anchor domain, and two pore module helices (OH and IH, respectively). Each THU contains four transmembrane helices. THU1 and THU2 are likely disordered and therefore not visible in the hPIEZO1 cryo-EM density map.
b, The 3.3 Å cryo-EM density map of hPIEZO1, viewed from the top. The density of each single subunit is colored in cyan, wheat and pink, respectively. Around 30 nm digitonin disk is shown as grey density by low pass filtered.
c, The 3.3 Å cryo-EM density map of hPIEZO1, viewed from the side. The density of each single subunit is colored in cyan, wheat and pink, respectively. The potential PTM at the THUs region is indicated. A flexible density binds to the cytosolic plug, which may stand for an additional hPIEZO1 auxiliary.
d, The 3.3 Å cryo-EM density map of hPIEZO1, viewed from the bottom. The density of each single subunit is colored in cyan, wheat and pink, respectively.
e, The cartoon model of hPIEZO1, viewed from the top. Each single subunit is colored in cyan, wheat and pink, respectively.
f, The cartoon model of hPIEZO1, viewed from the side. Each single subunit is colored in cyan, wheat and pink, respectively.
g, The cartoon model of hPIEZO1, viewed from the bottom. Each single subunit is colored in cyan, wheat and pink, respectively.
Structural comparison of hPIEZO1 and mPIEZO1
a, Structural comparison of hPIEZO1 (this study) and curved mPIEZO1 (5Z10), viewed from the side. The distance of the distal blade between curved mPIEZO1 and hPIEZO1 is measured as 5 Å, and the distance of the cap between curved mPIEZO1 and hPIEZO1 is measured as 2 Å.
b, Structural comparison of hPIEZO1(this study) and curved mPIEZO1 (5Z10), viewed from the top. The distance between the V650 residue in hPIEZO1 and the corresponding residue in curved mPIEZO1 is around 22 Å; therefore, the blades of hPIEZO1 present a contracted state from the top view. The blade center lines are shown as red dashed lines.
c, Structural comparison of hPIEZO1 (this study) and flattened mPIEZO1 (7WLU), viewed from the side. Compared to the flattened mPIEZO1, the mimic membrane curvature of hPIEZO1 is between the curved and flattened mPIEZO1.
d, Structural comparison of hPIEZO1(this study) and curved mPIEZO1 (5Z10), viewed from the top. hPIEZO1 presents a similar extended state to the flattened mPIEZO1.
e, The cartoon model of the hPIEZO1 pore module with calculated pore. The restricted residues are labeled.
f, The cartoon model of curved mPIEZO1 pore module with calculated pore. The restricted residues are labeled.
g, The cartoon model of flattened mPIEZO1 pore module with calculated pore.
h, The calculated pore diameter of hPIEZO1 (cyan), curved mPIEZO1 (wheat) and flattened mPIEZO1(grey) along the z axis.
Structure of full-length human PIEZO1-MDFIC complex
a, The 3.0 Å cryo-EM density map of hPIEZO1-MDFIC, viewed from the top. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C-terminal helices of MDFIC is colored in red. Around 30 nm digitonin disk is shown as grey density by low pass filtered.
b, The 3.0 Å cryo-EM density map of hPIEZO1-MDFIC, viewed from the side. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C-terminal helices of MDFIC is colored in red. The potential PTM at the THUs region is indicated. A flexible density binds to the cytosolic plug, which may stand for an additional hPIEZO1 auxiliary subunit. c, The 3.0 Å cryo-EM density map of hPIEZO1-MDFIC, viewed from the bottom. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of three C-terminal helices of MDFIC is colored in red.
d, The cartoon model of hPIEZO1-MDFIC, viewed from the top. Each single subunit is colored in cyan, wheat and pink, respectively. Three C-terminal helices of MDFIC are colored in red.
e, The cartoon model of hPIEZO1-MDFIC, viewed from the side. Each single subunit is colored cyan, wheat and pink, respectively. Three C-terminal helices of MDFIC are colored in red.
f, The cartoon model of hPIEZO1-MDFIC, viewed from the bottom. Each single subunit is colored in cyan, wheat and pink, respectively. Three C-terminal helices of MDFIC are colored in red.
g, Structural comparison of hPIEZO1 and hPIEZO1-MDFIC, viewed from the side. The motion of the distal blade between hPIEZO1 and hPIEZO1-MDFIC is around 2 Å from the side view.
h, Structural comparison of hPIEZO1 and hPIEZO1-MDFIC, viewed from top. The motion of the distal blade between hPIEZO1 and hPIEZO1-MDFIC is around 2 Å from the top view.
Structure of human PIEZO1-A1988V-MDFIC and hPIEZO1-E756del-MDFIC complex a,
38-TM topology model of a single hPIEZO1 subunit. The E756del mutation is located at the THU4 and THU5 linker, the A1988V mutation is located at the THU9 linker and the R2456H mutation is located at the IH of the pore module.
b, The 3.1 Å cryo-EM density map of hPIEZO1-A1988V-MDFIC, viewed from the top. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C-terminal helices of MDFIC is colored in red. Around 30 nm digitonin disk is shown as grey density by low pass filtered.
c, The 3.1 Å cryo-EM density map of hPIEZO1-A1988V-MDFIC, viewed from the side. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C-terminal helices of MDFIC is colored in red. The potential PTM at the THUs region is indicated. A flexible density binds to the cytosolic plug, which may represent an additional hPIEZO1 auxiliary.
d, The 3.1 Å cryo-EM density map of hPIEZO1-A1988V-MDFIC, viewed from the bottom. The density of each single subunit is colored in cyan, wheat and pink, respectively.
e, The 2.8 Å cryo-EM density map of hPIEZO1-E756del-MDFIC, viewed from the top. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C- terminal helices of MDFIC is colored in red. Around 30 nm digitonin disk is shown as grey density by low pass filtered.
f, The 2.8 Å cryo-EM density map of hPIEZO1-E756del-MDFIC, viewed from the side. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C- terminal helices of MDFIC is colored in red. The potential PTM at the THUs region is indicated. A flexible density binds to the cytosolic plug, which may represent an additional hPIEZO1 auxiliary. g, The 2.8 Å cryo-EM density map of hPIEZO1-E756del-MDFIC, viewed from the bottom. The density of each single subunit is colored in cyan, wheat and pink, respectively.
h, Superimposed cartoon models of hPIEZO1-MDFIC (cyan), hPIEZO1-A1988V-MDFIC (wheat) and hPIEZO1-E756del-MDFIC (pink), viewed from the top. The RSMD is around 0.001, indicating that they are almost identical.
i, Superimposed cartoon models of hPIEZO1-MDFIC (cyan), hPIEZO1-A1988V-MDFIC (wheat) and hPIEZO1-E756del-MDFIC (pink), viewed from the side.
j, Superimposed cartoon models of hPIEZO1-MDFIC (cyan), hPIEZO1-A1988V-MDFIC (wheat) and hPIEZO1-E756del-MDFIC (pink), viewed from the bottom.
Structure of human PIEZO1-R2456H-MDFIC complex
a, The 4.5 Å cryo-EM density map of hPIEZO1-R2456H-MDFIC, viewed from the top. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C- terminal helices of MDFIC is colored in red. Around 25 nm digitonin disk of hPIEZO1-R2456H- MDFIC is shown as grey density by low pass filtered, smaller than the wildtype hPIEZO1-MDFIC. b, The 4.5 Å cryo-EM density map of hPIEZO1-R2456H-MDFIC, viewed from the side. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C-terminal helices of MDFIC is colored in red. The potential PTM at the THUs region is indicated. A flexible density binds to the cytosolic plug, which may represent an additional hPIEZO1 auxiliary.
c, The 4.5 Å cryo-EM density map of hPIEZO1-R2456H-MDFIC, viewed from the bottom. The density of each single subunit is colored in cyan, wheat and pink, respectively. The density of the three C-terminal helices of MDFIC is colored in red.
d, The cartoon model of hPIEZO1-R2456H-MDFIC, viewed from the top. Each single subunit is colored in cyan, wheat and pink, respectively. Three C-terminal helices of MDFIC are colored in red.
e, The cartoon model of hPIEZO1-R2456H-MDFIC, viewed from the side. Each single subunit is colored in cyan, wheat and pink, respectively. Three C-terminal helices of MDFIC are colored in red.
f, The cartoon model of hPIEZO1-R2456H-MDFIC, viewed from the bottom. Each single subunit is colored in cyan, wheat and pink, respectively. Three C-terminal helices of MDFIC are colored in red.
g, Structural comparison of hPIEZO1-R2456H-MDFIC and hPIEZO1-MDFIC, viewed from the side. The motion of the distal blade between hPIEZO1-R2456H-MDFIC and hPIEZO1-MDFIC is around 7 Å from the side view.
h, Structural comparison of hPIEZO1-R2456H-MDFIC and hPIEZO1-MDFIC, viewed from the top. The motion of the distal blade between hPIEZO1-R2456H-MDFIC and hPIEZO1-MDFIC is around 7 Å from the top view.
i, The cartoon model of hPIEZO1 pore module with calculated pore. The restricted residues are labeled.
j, The cartoon model of hPIEZO1-MDFIC pore module with calculated pore.
k, The cartoon model of hPIEZO1-R2456H-MDFIC pore module with calculated pore.
l, The calculated pore diameter of hPIEZO1 (cyan), hPIEZO1-MDFIC (pink) and flattened hPIEZO1-R2456H-MDFIC (wheat) along the z-axis. The hPIEZO1-R2456H-MDFIC presents a more extended extracellular side pore.
The pore module, multi-lipidated MDFIC and pore lipid interaction and the pore remodeling of the HX pathogenesis R2456H mutant
a, The cartoon model of hPIEZO1-MDFIC/hPIEZO1-A1988V-MDFIC/hPIEZO1-E756del- MDFIC pore module, viewed from the top. The marine spheres present the pore lipids.
b, The cartoon model of hPIEZO1-MDFIC/hPIEZO1-A1988V-MDFIC/hPIEZO1-E756del- MDFIC pore module, viewed from the side. The hydrophobic pore region formed by I2447, V2450 and F2454 is marked by red. The R2456 on the lateral side of the inner helix of the pore is marked by yellow.
c, The cartoon model of hPIEZO1-MDFIC/hPIEZO1-A1988V-MDFIC/hPIEZO1-E756del-MDFIC pore module, viewed from the bottom. The multi-lipidated MDFIC subunits are colored in green and marked. One hydrophobic fatty acid chain of the pore lipid occupies the hydrophobic pore region. Another hydrophobic fatty acid chain of the pore lipid interacts with the fatty acid chains of the MDFIC-covalently-linked lipids. In contrast, the hydrophilic head of the pore lipid directly interacts with the R2456 on the lateral side of the inner helix of the pore, thus forming a stable hPIEZO1- multi-lipidated MDFIC-pore lipid complex. The pore lipids seal the pore and prevent ion flow.
d, The cartoon model of hPIEZO1-MDFIC/hPIEZO1-A1988V-MDFIC/hPIEZO1-E756del- MDFIC IH, pore lipid and multi-lipidated MDFIC interactions. The multi-lipidated MDFIC subunits are colored in green. The marine spheres present the pore lipids. The hydrophobic pore region formed by I2447, V2450 and F2454 is marked by red. The R2456 on the lateral side of the inner helix of the pore is marked by yellow.
e, The cartoon model of twisted IH and MDFIC in hPIEZO1-R2456H-MDFIC.
f, The overlay cartoon model of IH and MDFIC in hPIEZO1-MDFIC/ hPIEZO1-A1988V-MDFIC/hPIEZO1-E756del-MDFIC and hPIEZO1-R2456H-MDFIC.
g, The cartoon model of TM pore in hPIEZO1-MDFIC/ hPIEZO1-A1988V-MDFIC/ hPIEZO1- E756del-MDFIC and hPIEZO1-R2456H-MDFIC, viewed from the top. The TM pore of hPIEZO1- R2456H-MDFIC presents a twisted and extended state.
h, The cartoon model of TM pore in hPIEZO1-MDFIC/ hPIEZO1-A1988V-MDFIC/ hPIEZO1- E756del-MDFIC and hPIEZO1-R2456H-MDFIC, viewed from the side. The TM pore of hPIEZO1- R2456H-MDFIC presents a twisted and extended state.
Occupancy pattern of pore lipids in hPIEZO1 underlying fast inactivation
a, Top view of hPIEZO1 pore domain. The pore helices are shown as cylinders, and the pore lipid sealing the hydrophobic pore region is shown as spheres. The residue R2456s in inner helices, interacting with pore lipids, are labeled.
b, Top view of hPIEZO1-A1988V pore domain. Pore lipids are retracted from the hydrophobic pore region.
c, Top view of hPIEZO1-MDFIC pore domain. Lipidated-MDFICs are shown as sticks.
d, Top view of curved mPIEZO1 (PDB: 7WLT). The residues R2482s, responding to R2456 in hPIEZO1, are labeled. Pore lipids are modeled in the latency side of the IH pore.
e, Top view of flattened mPIEZO1 (PDB: 7WLU). Pore lipids are also located in the latency side of the IH pore.
f, Cryo-EM density of pore lipids in WT hPIEZO1 (cyan), hPIEZO1-A1988V (pink) and hPIEZO1- MDFIC (brown). The cryo-EM density is shown as blue mesh.
The proposed putative fast inactivation model of PIEZO1 channel.
a, Cartoon representation of the fast inactivation model of the hPIEZO1 channel. Two of the three subunits are shown for clarity. The cap (purple semicircle), pore helices (pink rectangle), THUs (blue arc), beam (brown rectangle), and pore lipids (red) are present. The pore lipids interacting with R2456 (yellow star) seal the TM pore. The membrane force can reduce the membrane curvature and transduce the force to the pore region, removing the barrier created by the pore lipids, which might induce the channel pore to open. The pore lipids then rapidly return to the hydrophobic pore, exhibiting a fast-inactivation pattern.
b, Cartoon diagram of the slow inactivation model of the hPIEZO1-MDFIC complex. Multi- lipidated MDFICs are shown as green rectangles. The fatty acid chains of the covalently linked MDFIC lipids can stabilize the pore lipids. Therefore, the hPIEZO1-MDFIC complex exhibits a very stable deep resting state. Once a higher threshold of mechanical force removes the pore lipids, it is also tricky for them to return to the hydrophobic pore region and reform the stable non- conducting pore module, thus exhibiting the prolonged slow inactivation phenotype.
c, Cartoon diagram of the slow inactivation model of hPIEZO1 channel mild channelopathy or mPIEZO1. Mild channelopathy mutants or mPIEZO1 show the pore lipids retracted from the pore and thus exhibit a slow inactivation pattern.
d, Cartoon representation of the slow inactivation model of the hPIEZO1 channel HX pathogenesis mutant R2456H with MDFIC auxiliary subunit. The R2456H channelopathy mutant probably disrupts the IH pore and the pore lipid interaction. Due to the stabilizing effect of the MDFIC auxiliary subunit, the pore has a twisted shape and the overall architecture of hPIEZO1-R2456H- MDFIC has a more curved and contracted blow-like shape.
Purification procedures of hPIEZO1, the channelopathy mutants with or without MDFIC proteins.
a, A schematic diagram of the hPIEZO1-GFP-3×FLAG constructs for protein purifications and electrophysiological studies.
b-e, Representative size-exclusion chromatography (SEC) trances and Coomassie-staining SDS- PAGE analysis of purified WT hPIEZO1 (b), hPIEZO1-R2456H (c), hPIEZO1-A1988V (d) and hPIEZO1-E756del (e) protein samples. Red arrows denote the peak position and bands of hPIEZO1 and its mutant. Green arrows denote the peak position of the cleaved GFP tag.
f-g, Representative size-exclusion chromatography (SEC) trances and Coomassie-staining SDS- PAGE analysis of purified WT hPIEZO1 co hMDFIC (b), hPIEZO1-R2456H co hMDFIC (c), hPIEZO1-A1988V co hMDFIC (d) and hPIEZO1-E756del co hMDFIC (e) protein samples. The peak positions and bands of hPIEZO1 and its mutant are denoted by red arrows. Green arrows indicate the peak position of the cleaved GFP tag. Blue arrows represent the bands of hMDFIC.
The whole-cell poking evoked currents of wild-type hPIEZO1, the GOF channelopathy mutants with or without MDFIC.
a, The representative traces of whole-cell poking evoked currents of wild-type hPIEZO1, hPIEZO1- A1988V, hPIEZO1-E756del and hPIEZO1-R2456H subjected to a series of mechanical steps in 1 µm increments from 5 μm at a holding potential of −60 mV in HEK293T-PIEZO1-KO cells. The hPIEZO1 shows a very fast inactivation behavior with a half inactivation time of 10.1 ± 2.1 ms. At the same time, the channelopathy mutants A1988V, E756del and R2456H present a slow inactivation rate with half inactivation time of 13.4 ± 3.8 ms, 18.5 ± 4.1 ms and 23.2 ± 2.9 ms, respectively.
b, The representative traces of whole-cell poking evoked currents of wild-type hPIEZO1, hPIEZO1- A1988V, hPIEZO1-E756del and hPIEZO1-R2456H co-expressed with the auxiliary subunit MDFIC subjected to a series of mechanical steps in 1 µm increments from 8 μm at a holding potential of −60 mV in HEK293T-PIEZO1-KO cells. The evoking MS currents were negligible even with large increments, presenting the significant slow inactivation behavior. Therefore, the MDFIC may attenuate the mechanosensitivity of wild-type hPIEZO1 and GOF channelopathy mutants. All data are replicated at least three times and the statistic is based on the mean ± SEM.
Cryo-EM data processing procedure of WT hPIEZO1.
a, Representative raw micrograph of hPIEZO1.
b, Representative 2D class averages of the cryo-EM particles of hPIEZO1.
c, Flowchart of the image processing procedure for hPIEZO1. Resolution estimation for the tight masked map is based on the criterion of an FSC cutoff of 0.143.
d-e, Gold-standard Fourier shell correlation (FSC) curves of the final refined maps (d) and angular distribution histogram (e) of the final hPIEZO1 reconstruction. This is a standard output from cryoSPARC v4.
The EM density of WT hPIEZO1.
The EM density segments of the transmembrane helices domain (TH3-TH9), cap domain, pore domain, anchor and beam of WT hPIEZO1.
Cryo-EM data processing procedure of hPIEZO1-E756del mutant.
a, Representative raw micrograph of hPIEZO1-E756del.
b, Representative 2D class averages of the cryo-EM particles of hPIEZO1- E756del.
c, Flowchart of the image processing procedure for hPIEZO1- E756del. This is a standard output from cryoSPARC v4.
Cryo-EM data processing procedure of hPIEZO1-R2456H mutant.
a, Representative raw micrograph of hPIEZO1-R2456H.
b, Representative 2D class averages of the cryo-EM particles of hPIEZO1-R2456H.
c, Flowchart of the image processing procedure for hPIEZO1-R2456H. This is a standard output from cryoSPARC v4.
Cryo-EM data processing procedure of hPIEZO1-A1988V mutant. a, Representative raw micrograph of hPIEZO1-A1988V.
b, Representative 2D class averages of the cryo-EM particles of hPIEZO1- A1988V.
c, Flowchart of the image processing procedure for hPIEZO1- A1988V. Resolution estimation for the tight masked map is based on the criterion of an FSC cutoff of 0.143.
d-e, Gold-standard Fourier shell correlation (FSC) curves of the final refined maps (d) and angular distribution histogram (e) of the final hPIEZO1- A1988V reconstruction. This is a standard output from cryoSPARC v4.
Structural comparison of hPIEZO1 and hPIEZO1-A1988V a,
Overall structure of hPIEZO1 viewed from the top (left) and side (right).
b, Overall structure of hPIEZO1-A1998V viewed from the top (left) and side (right).
c, Overall structure comparison of hPIEZO1 (cyan) and hPIEZO1-A1998V (brown) viewed from the top (left) and side (right).
Cryo-EM data processing procedure of hPIEZO1 Co MDFIC.
a, Representative raw micrograph of hPIEZO1 co MDFIC.
b, Representative 2D class averages of the cryo-EM particles of hPIEZO1 co MDFIC.
c, Flowchart of the image processing procedure for hPIEZO1 co MDFIC. Resolution estimation for the tight masked map is based on the criterion of an FSC cutoff of 0.143.
d-e, Gold-standard Fourier shell correlation (FSC) curves of the final refined maps (d) and angular distribution histogram (e) of the final hPIEZO1 co MDFIC reconstruction. This is a standard output from cryoSPARC v4.
Cryo-EM data processing procedure of hPIEZO1-A1988V Co MDFIC. a, Representative raw micrograph of hPIEZO1-A1988V co MDFIC.
b, Representative 2D class averages of the cryo-EM particles of hPIEZO1-A1988V co MDFIC.
c, Flowchart of the image processing procedure for hPIEZO1-A1988V co MDFIC. Resolution estimation for the tight masked map is based on the criterion of an FSC cutoff of 0.143.
d-e, Gold-standard Fourier shell correlation (FSC) curves of the final refined maps (d) and angular distribution histogram (e) of the final hPIEZO1-A1988V co MDFIC reconstruction. This is a standard output from cryoSPARC v4.
Cryo-EM data processing procedure of hPIEZO1-E756del Co MDFIC.
a, Representative raw micrograph of hPIEZO1-E756del co MDFIC.
b, Representative 2D class averages of the cryo-EM particles of hPIEZO1-E756del co MDFIC.
c, Flowchart of the image processing procedure for hPIEZO1-E756del co MDFIC. Resolution estimation for the tight masked map is based on the criterion of an FSC cutoff of 0.143.
d-e, Gold-standard Fourier shell correlation (FSC) curves of the final refined maps (d) and angular distribution histogram (e) of the final hPIEZO1-E756del co MDFIC reconstruction. This is a standard output from cryoSPARC v4.
The EM density of hPIEZO1-E756del-MDFIC.
The EM density segments of the transmembrane helices domain (TH3-TH9), cap domain, pore domain, anchor and beam of hPIEZO1-E756del. The EM density of the auxiliary subunit MDFIC and the bound lipids is also shown.
Cryo-EM data processing procedure of hPIEZO1-R2456H Co MDFIC. a, Representative raw micrograph of hPIEZO1-R2456H co MDFIC.
b, Representative 2D class averages of the cryo-EM particles of hPIEZO1-R2456H co MDFIC.
c, Flowchart of the image processing procedure for hPIEZO1-R2456H co MDFIC. Resolution estimation for the tight masked map is based on the criterion of an FSC cutoff of 0.143.
d-e, Gold-standard Fourier shell correlation (FSC) curves of the final refined maps (d) and angular distribution histogram (e) of the final hPIEZO1-R2456H co MDFIC reconstruction. This is a standard output from cryoSPARC v4.
The EM density of hPIEZO1-R2456H Co MDFIC.
The EM density segments of the transmembrane helices domain (TH3-TH9), cap domain, pore domain, anchor and beam of hPIEZO1-R2456H Co MDFIC. The EM density of the auxiliary subunit MDFIC and the bound lipids is also shown.
