Figures and data in TMEM120A is a coenzyme A-binding membrane protein with structural similarities to ELOVL fatty acid elongase

Figures
Tables
Additional files

5 figures, 1 table and 1 additional file

Figures

Figure 1

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Electrophysiology of TMEM120A.

(a) Sample traces of patch-clamp recordings of HEK293 cells with and without TMEM120A expression. (b) Recordings of Piezo1 knockout HEK293 cells with and without TMEM120A expression. (c) Recordings of CHO cells with and without TMEM120A expression. (d) Sample traces of giant liposome patching using proteoliposomes with 1:500 (left) or 1:100 (right) protein-to-lipid (w/w) ratio.

Figure 2 with 3 supplements

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Overall structure of TMEM120A.

(a) Side view of 3D reconstruction of TMEM120A. Channel subunits are colored individually with bound substrate density in purple and lipid density in gray. (b) Side and bottom views of cartoon representation of TMEM120A structure. Coenzyme A (CoA) molecules are rendered as sticks. (c) Topology and domain arrangement in a single TMEM120A subunit. (d) Side and bottom views of a single subunit in a similar orientation as the green-colored subunit in (b). (e) Transmembrane domain (TMD)-enclosed pocket (colored in salmon) analyzed using the program CAVER (Jurcik et al., 2018). (f) Zoomed-in view of CoA-binding site with its density (blue mesh). (g) Schematic diagram detailing the interactions between TMEM120A residues and CoA. Toothed wheels mark the hydrophobic contacts between protein residues and CoA. Dotted lines mark the salt bridges and hydrogen bonds.

Figure 2—source data 1 Cryo-EM data collection and model statistics.: https://cdn.elifesciences.org/articles/71220/elife-71220-fig2-data1-v2.doc
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Figure 2—figure supplement 1

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Cryo-EM data processing scheme of human TMEM120A.

(a) A representative micrograph. Scale bar is 20 nm. (b) Flow chart of the cryogenic electron microscopy (cryo-EM) data processing procedure. Selected 2D class averages are shown. The particle numbers are indicated under the corresponding 3D classes. (c) Euler angle distribution of particles used in the final 3D reconstruction. (d) Fourier Shell Correlation (FSC) curves showing the overall resolution of 3.24 Å at FSC = 0.143.

Figure 2—figure supplement 2

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Sample density maps of human TMEM120A at various regions.

Maps of CC1 and CC2 are contoured at 4.5 σ. All other maps are contoured at 6.0 σ.

Figure 2—figure supplement 3

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Sequence alignment of vertebrate TMEM120A.

Secondary structure assignments are based on the structure of human TMEM120A.

Figure 3

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Dimerization of TMEM120A.

(a) Extensive dimerization interactions occur in three boxed regions: the coiled coil domain (CCD) (box 1), the re-entrant loop (box 2), and the external side of transmembrane domain (TMD) (box 3). (b) Zoomed-in view of dimerization interactions at CCD. Residues that participate in the inter-subunit contact are W16, L19, F23, I26, H30, Y33, L37, L40, L43, I51, L58, and L61 in CC1, and L83, M87, L93, F94, M97, Y100, and L101 in CC2. (c) Zoomed-in view of dimerization at the re-entrant loop. Shown in the left panel are the inter-subunit hydrogen bonding interactions between R178 side chain and the carbonyl oxygen atoms of S110, L111, and V112, between Y129 side chain and the carbonyl oxygen of L113, and between the side chains of E132 and N115. Shown in the right panel are the inter-subunit hydrophobic contacts between the two re-entrant loops and between the re-entrant loop and transmembrane helices (TMs) 1-2 of the neighboring subunit. (d) Zoomed-in view of the inter-subunit hydrophobic contacts between the two TMDs.

Figure 4

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Structural comparison between TMEM120A and ELOVL7 elongase.

(a) Structures of the 6-TM α-barrel transmembrane domains (TMDs) from TMEM120A (TMs 1–6, left) and ELOVL7 elongase (TMs 2–7, right). Coenzyme A (CoA) in TMEM120A and 3-keto acyl-CoA in ELOVL7 are rendered as sticks. (b) Structural comparison between the 6-TM barrels from TMEM120A (orange) and ELOVL7 (blue) in side view (left) and bottom view (right). HxxHH motif in ELOVL7 is colored in cyan. The WVFHH sequence of TMEM120A at the equivalent location is colored in yellow.

Figure 5

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Biochemical and mass spectrometry assay of CoA in TMEM120A.

(a) Coenzyme A (CoA) assay. A blue standard curve is obtained from fluorometric measurements of various amounts of pure CoA provided in the assay kit. Red dots mark the measured CoA contents in 1, 3, 4, 5, and 7 μl of protein samples. The measure of CoA concentration in 4 mg/ml protein sample is 0.1564 ± 0.0027 mM (mean ± SEM, n = 5). (b) Liquid chromatography (LC) separation of extracted substrates from TMEM120A protein sample in liquid chromatography-tandem mass spectrometry (LC-MS/MS). (c) Precursor ion scan of peak 1 effluent using 303 Da (left) and 428 Da (right) fragments. (d) Precursor ion scan of peak 2 effluent using 428 Da fragments. (e) Fragmentation (product ion scan) of the 768 Da mass peak.

Figure 5—source data 1 CoA assay.: https://cdn.elifesciences.org/articles/71220/elife-71220-fig5-data1-v2.xlsx
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Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers
Strain, strain background (Escherichia coli)	TOP10	Thermo Fisher Scientific	Cat# 18258012
Strain, strain background (E. coli)	DH10bac	Thermo Fisher Scientific	Cat# 10361012
Cell line (Spodoptera frugiperda)	Sf9 cells	Thermo Fisher Scientific	Cat# 11496015; RRID:CVCL_0549
Cell line (Homo sapiens)	FreeStyle 293 F cells	Thermo Fisher Scientific	Cat# R79007; RRID:CVCL_D603
Transfected construct (H. sapiens)	pEZT-BM-TMEM120A-N_flag	This paper	N/A
Recombinant DNA reagent	pEZT-BM	DOI:10.1016/j.str.2016.03.004	Addgene:74099
Sequence-based reagent	TMEM120A_F_primer: gatataGCTAGCCAACCGCCACCACCCGGGCCATTG	This paper	N/A
Sequence-based reagent	TMEM120A_R_primer: gatataGCGGCCGCTCAATCTTTTTTTGAGCCATG	This paper	N/A
Peptide, recombinant protein	Flag peptide	Sigma-Aldrich	Cat# F3290
Commercial assay or kit	Coenzyme A Assay Kit	Sigma-Aldrich	Cat# MAK034
Chemical compound, drug	Sodium Butyrate	Sigma-Aldrich	Cat# 303410
Chemical compound, drug	Lauryl Maltose Neopentyl Glycol	Anatrace	Cat# NG310
Chemical compound, drug	Digitonin	Acros Organics	Cat# 11024-24-1
Software, algorithm	MotionCor2	Zheng et al., 2017	http://msg.ucsf.edu/em/software/motioncor2.html
Software, algorithm	GCTF	Zhang, 2016	https://www.mrc-lmb.cam.ac.uk/kzhang/Gctf
Software, algorithm	RELION	Scheres, 2012	http://www2.mrc-lmb.cam.ac.uk/relion
Software, algorithm	Chimera	Pettersen et al., 2004	https://www.cgl.ucsf.edu/chimera; RRID:SCR_004097
Software, algorithm	PyMol	Schrödinger	https://pymol.org/2; RRID:SCR_000305
Software, algorithm	COOT	Emsley et al., 2010	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot; RRID:SCR_014222
Software, algorithm	MolProbity	Chen et al., 2010	http://molprobity.biochem.duke.edu/
Software, algorithm	PHENIX	Adams et al., 2010	https://www.phenix-online.org
Other	Superose 6 Increase10/300 GL	GE Healthcare	Cat# 29091596
Other	Anti-DYKDDDDK G1 Affinity Resin	GeneScript	Cat# L00432
Other	Amicon Ultra-15 Centrifugal Filter Units	Milliporesigma	Cat# UFC9100
Other	Quantifoil R 1.2/1.3 grid Au300	Quantifoil	Cat# Q37572
Other	Cellfectin	Thermo Fisher Scientific	Cat# 10362100
Other	Sf-900 II SFM medium	Thermo Fisher Scientific	Cat# 10902088
Other	FreeStyle 293 Expression Medium	Thermo Fisher Scientific	Cat# 12338018
Other	Antibiotic Antimycotic Solution	Sigma-Aldrich	Cat# A5955
Other	Proteinase K	Thermo Fisher Scientific	Cat# EO0491