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The conformational wave in capsaicin activation of Transient Receptor Potential Vanilloid 1 ion channel
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Fan Yang1,3# ¶, Xian Xiao2,3#, Bo Hyun Lee3#§, Simon Vu3, Wei Yang1, Vladimir YarovYarovoy3 and Jie Zheng3¶
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1Department
of Biophysics and Kidney Disease Center, First Affiliated Hospital, Institute of Neuroscience, National Health Commission and Chinese Academy of Medical Sciences Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou 310058, Zhejiang Province, China. 2Institute for Basic Medical Sciences, Westlake Institute for Advanced Study, Westlake University, Shilongshan Road No. 18, Xihu District, Hangzhou 310024, Zhejiang Province, China. 3Department of Physiology and Membrane Biology, University of California, Davis, California 95616, USA.
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#
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§
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¶
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These authors contributed equally to this work.
Current address: University of Washington, Department of Physiology and Biophysics, Seattle WA 98195 Correspondence and requests for materials should be addressed to F.Y. (email:
[email protected]) or to J.Z. (email:
[email protected])
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Supplementary Figure 1. Functional confirmation of ANAP-incorporated TRPV1 channels. (a) Location of ANAP-incorporated sites. (b to e) In calcium imaging, 10 µM capsaicin was used to test the function of channels expressed in HEK293T cells. 3 mM ionomycin was perfused at the end of imaging as a positive control. In whole-cell recordings, 10 µM capsaicin and 3 mM 2-APB were perfused. Channels with ANAP incorporated at C579, T651 and Y654 site were activated by capsaicin in both calcium imaging and whole-cell recording. The scale bar: 100 μm.
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Supplementary Figure 2. Clusters of the top 10 open state models. (a) Top 10 models of capsaicin-induced open state channel. The selectivity filter was boxed in dashed line. (b to d) Cluster 1 to 3 were shown, respectively. (e) Distribution of pore radii in Cluster 1 to 3. (f) Predicted conductance of the models in different Clusters.
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Supplementary Figure 3. Comparison between our refined open state model and cryoEM structure models. (a and b) Side and top view of our refined open state model fitted into the cryo-EM density map (capsaicin-bound state, EMD ID: 5777). (c and d) Side of top view of our refined open state model (blue) aligned with the open state model before refinement (red).
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Supplementary Figure 4. Incorporation of ANAP into TRPV1 models. (a) A representative backbone dependent rotamer library of ANAP (phi and psi angle: -60° and -30°, respectively). Atoms in the backbone are colored in gray. (b) The full backbone dependent rotamer library. Phi and psi angle are binned from -180° to 180° with a 10° interval, respectively. (c and d) ANAP residue (red) was incorporated into TRPV1 models in the closed and open states, respectively. (e) SASA of ANAP at 651 site was significantly increased in the open state (*, p < 0.05; n = 4) (f) Correlation between shift in ANAP emission peak (y axis) and changes in SASA measured from cryo-EM structures (x axis) for the 651 site. When ANAP was incorporated at this site, it showed an increase in SASA similar to the increase in SASA measured for a Threonine residue in Fig. 3c. n = 5-to-7. All statistical data are given as mean ± s.e.m.
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Supplementary Figure 5. Φ-analysis for additional sites on TRPV1. (a and b) Representative single-channel recordings of multiple mutations at the T551 and M548 sites, respectively. (c) M548 site (red) locates within the capsaicin binding pocket. (d) Brønsted plot to determine the Φ value for the 548 site. (e and f) Location and Brønsted plot to determine the Φ value for the 643 site. (g and h) Location and Brønsted plot to determine the Φ value for the 647 site. (i and j) Location and Brønsted plot to determine the Φ value for the 648 site. (k and l) Location and Brønsted plot to determine the Φ value for the 649 site. (m and n) Location and Brønsted plot to determine the Φ value for the 682 site. (o and p) Location and Brønsted plot to determine the Φ value for the 685 site.
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Supplementary Table 1. Assessments of model quality. TRPV1 (3J5P) TRPV1 (5IRZ) TRPV1 Open Model
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ERRAT Overall quality 86.213
VERIFY_3D Compatibility with sequence 67.47%
WHAT_CHECK PROVE 67 Pass Warning Error Outlier atoms 68
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7.2%
69 70
63.375
35.79%
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7.4%
71 72
86.301
56.83%
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6.0%
73 74
TRPV1 structures determined from cryo-EM, as well as open model of TRPV1 we computed with the Rosetta suite (shaded in green), were assessed by four independent protein structure analysis and verification methods (Green: higher in number is better; Orange and Red: lower in number is better).
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Supplementary Table 2. Primers used in this study to generate point mutations. Name D647A D647E D647Q D647V E571A E571C E571D E571Q E571R E649A E649D E685A E685D E685Q F650A F650A F650I F650I F650L F650M F650M F650Y G684A G684S G684V I574A I643A I643L I643V I680A I680F I680G I680L I680S L648A
Primer-Forward GACACTGATCGAGTGTGGGAAGAATAACTC CACCATCGGCATGGGTGCCCTGGAGTTCAC CACCATCGGCATGGGTGTCCTGGAGTTCAC CTATCGGCCTGTGTGTGGCTTGCCCCCCTA TGCTGTCATGATTTGCAAGATGATCCTCAG TGCTGTCATGATTGACAAGATGATCCTCAG TGCTGTCATGATTCAGAAGATGATCCTCAG GTCATGATTAGGAAGATGATCCTCAGAGACC GTGACACTGATCTGCGATGGGAAGAATAA TGGGTGACCTGGAGGCCACCGAGAACTATGA TGGGTGACCTGGAGATCACCGAGAACTATGA TGGGTGACCTGGAGATGACCGAGAACTATGA TCGGCCTGTGGAATGTTTGCCCCCCTATA CACTGATCGAGGATTGCAAGAATAACTCAC GCTGTCTTCATCGCCCTGTTACTGGCCTATG TGCTCAACATGCTCGCTGCTCTCATGGG TGCTCAACATGCTCTTTGCTCTCATGGG TGCTCAACATGCTCGGTGCTCTCATGGG TGCTCAACATGCTCCTTGCTCTCATGGG TGCTCAACATGCTCAGTGCTCTCATGGG CTGATCGAGGATGGGTGCAATAACTCACTGC CTGGAGCTGTTCGAGTTCACCATCGGCATG GGACAGCTACAGTGAGATAGCTTTCTTTGTA CAG GCATGGGTGACGCGGAGTTCACCGAGAA GGCATGGGTGACTTCGAGTTCACCGAGAA TGGCCTATGTGATTGCCACCTACATCCTC CTACATCCTCCTGGCCAACATGCTCATTG CTACATCCTCCTGTGCAACATGCTCATTG CTACATCCTCCTGTTCAACATGCTCATTG CATGCTCATTGCTGCCATGGGCGAGACTG CATGCTCATTGCTTTCATGGGCGAGACTG CATGCTCATTGCTGGAATGGGCGAGACTG CATGCTCATTGCTAGCATGGGCGAGACTG TTCTCCCTGGCCTTCGGCTGGACCAACATG GTTTGTGGACAGCTACACTGAGATACTTTTC TTTG
Primer-Reverse GAGTTATTCTTCCCACACTCGATCAGTGTC GTGAACTCCAGGGCACCCATGCCGATGGTG GTGAACTCCAGGACACCCATGCCGATGGTG TAGGGGGGCAAGCCACACACAGGCCGATAG CTGAGGATCATCTTGCAAATCATGACAGCA CTGAGGATCATCTTGTCAATCATGACAGCA CTGAGGATCATCTTCTGAATCATGACAGCA GGTCTCTGAGGATCATCTTCCTAATCATGAC TTATTCTTCCCATCGCAGATCAGTGTCAC TCATAGTTCTCGGTGGCCTCCAGGTCACCCA TCATAGTTCTCGGTGATCTCCAGGTCACCCA TCATAGTTCTCGGTCATCTCCAGGTCACCCA TATAGGGGGGCAAACATTCCACAGGCCGA GTGAGTTATTCTTGCAATCCTCGATCAGTG CATAGGCCAGTAACAGGGCGATGAAGACAGC CCCATGAGAGCAGCGAGCATGTTGAGCA CCCATGAGAGCAAAGAGCATGTTGAGCA CCCATGAGAGCACCGAGCATGTTGAGCA CCCATGAGAGCAAGGAGCATGTTGAGCA CCCATGAGAGCACTGAGCATGTTGAGCA GCAGTGAGTTATTGCACCCATCCTCGATCAG CATGCCGATGGTGAACTCGAACAGCTCCAG CTGTACAAAGAAAGCTATCTCACTGTAGCTG TCC TTCTCGGTGAACTCCGCGTCACCCATGC TTCTCGGTGAACTCGAAGTCACCCATGCC GAGGATGTAGGTGGCAATCACATAGGCCA CAATGAGCATGTTGGCCAGGAGGATGTAG CAATGAGCATGTTGCACAGGAGGATGTAG CAATGAGCATGTTGAACAGGAGGATGTAG CAGTCTCGCCCATGGCAGCAATGAGCATG CAGTCTCGCCCATGAAAGCAATGAGCATG CAGTCTCGCCCATTCCAGCAATGAGCATG CAGTCTCGCCCATGCTAGCAATGAGCATG CATGTTGGTCCAGCCGAAGGCCAGGGAGAA CAAAGAAAAGTATCTCAGTGTAGCTGTCCAC AAAC 8
L648A L648F L648F L648G L648G L648I L648V L676C L676F L676G L682A L682F L682G L682S M548A M548F M548L M548Q M645I M645L M645V M683F M683L M683T T551A T551S T551V T642A T642G T642L T642M T642N T642S T651L T651Q T651S
GTTTGTGGACAGCTACTATGAGATACTTTTC CAAGTGTCGGGGACCTGCCTGCAGGCCAG GTAACTCTTACAACCCACTGTATTCCACATG GCCATGGGCTGGGCCAACATGCTCTACTAC GCCATGGGCTGGTCCAACATGCTCTACTAC GCCATGGGCTGGGTCAACATGCTCTACTAC CAGCCTGTATTCCCCATGTCTGGAGCTGTTC GTTTGTGGACAGCTGCAGTGAGATACTTTTC GTTTGTGGACAGCTTCAGTGAGATACTTTTC CATGGGTGACCTGGCGTTCACCGAGAACTA CATGGGTGACCTGGACTTCACCGAGAACTA GTGACCTGGAGTTCTCCGAGAACTATGACTT CA GTGACCTGGAGTTCCAGGAGAACTATGACTT CA GTGACCTGGAGTTCTTGGAGAACTATGACTT CA GCATGGGTGACGGGGAGTTCACCGAGAA GGCATGGGTGACTTCGAGTTCACCGAGAA GGCATGGGTGACGTCGAGTTCACCGAGAA GGCATGGGTGACATCGAGTTCACCGAGAA TGGGTGACCTGGAGGCCACCGAGAACTATGA TGGGTGACCTGGAGATCACCGAGAACTATGA TGGGTGACCTGGAGATGACCGAGAACTATGA TGGGTGACCTGGAGTACACCGAGAACTATGA TGGGTGACCTGGAGCTGACCGAGAACTATGA CATCGGCATGGGTGAGCTGGAGTTCACCGA GTTCACCATCGGCATCGGTGACCTGGAGTTC A CTCATTGCTCTCTTCGGCGAGACTGTCAA CTCATTGCTCTCCTGGGCGAGACTGTCAA CATTGCTCTCATGGCCGAGACTGTCAACAA CATTGCTCTCATGGTCGAGACTGTCAACAA CATTGCTCTCATGAGCGAGACTGTCAACAA TTGCTCTCATGGGCGACACTGTCAACAAGA TTGCTCTCATGGGCCAGACTGTCAACAAGA TTGCTCTCATGGGCGCGACTGTCAACAAGA CTCTCATGGGCGAGTCTGTCAACAAGATTG CTCTCATGGGCGAGCTTGTCAACAAGATTG CTCTCATGGGCGAGAATGTCAACAAGATTG
GAAAAGTATCTCATAGTAGCTGTCCACAAAC CTGGCCTGCAGGCAGGTCCCCGACACTTG CATGTGGAATACAGTGGGTTGTAAGAGTTAC GTAGTAGAGCATGTTGGCCCAGCCCATGGC GTAGTAGAGCATGTTGGACCAGCCCATGGC GTAGTAGAGCATGTTGACCCAGCCCATGGC GAACAGCTCCAGACATGGGGAATACAGGCTG GAAAAGTATCTCACTGCAGCTGTCCACAAAC GAAAAGTATCTCACTGAAGCTGTCCACAAAC TAGTTCTCGGTGAACGCCAGGTCACCCATG TAGTTCTCGGTGAAGTCCAGGTCACCCATG TGAAGTCATAGTTCTCGGAGAACTCCAGGTC AC TGAAGTCATAGTTCTCCTGGAACTCCAGGTC AC TGAAGTCATAGTTCTCCAAGAACTCCAGGTC AC TTCTCGGTGAACTCCCCGTCACCCATGC TTCTCGGTGAACTCGAAGTCACCCATGCC TTCTCGGTGAACTCGACGTCACCCATGCC TTCTCGGTGAACTCGATGTCACCCATGCC TCATAGTTCTCGGTGGCCTCCAGGTCACCCA TCATAGTTCTCGGTGATCTCCAGGTCACCCA TCATAGTTCTCGGTCATCTCCAGGTCACCCA TCATAGTTCTCGGTGTACTCCAGGTCACCCA TCATAGTTCTCGGTCAGCTCCAGGTCACCCA TCGGTGAACTCCAGCTCACCCATGCCGATG TGAACTCCAGGTCACCGATGCCGATGGTGAA C TTGACAGTCTCGCCGAAGAGAGCAATGAG TTGACAGTCTCGCCCAGGAGAGCAATGAG TTGTTGACAGTCTCGGCCATGAGAGCAATG TTGTTGACAGTCTCGACCATGAGAGCAATG TTGTTGACAGTCTCGCTCATGAGAGCAATG TCTTGTTGACAGTGTCGCCCATGAGAGCAA TCTTGTTGACAGTCTGGCCCATGAGAGCAA TCTTGTTGACAGTCGCGCCCATGAGAGCAA CAATCTTGTTGACAGACTCGCCCATGAGAG CAATCTTGTTGACAAGCTCGCCCATGAGAG CAATCTTGTTGACATTCTCGCCCATGAGAG 9
T686L T686N T686S C579_TAG T651_TAG Y654_TAG
CATGCTCTACTACACCGAAGGATTCCAGCAG A CATGCTCTACTACACCGACGGATTCCAGCAG A CATGCTCTACTACACCAACGGATTCCAGCAG A AGACGAACATAAACCGCTACAGGTCTCTGAG GATCATCTTC GCCTTGAAGTCATAGTTCTCCTAGAACTCCA GGTCACCCATGC GACAGCCTTGAAGTCCTAGTTCTCGGTGAAC TCC
TCTGCTGGAATCCTTCGGTGTAGTAGAGCAT G TCTGCTGGAATCCGTCGGTGTAGTAGAGCAT G TCTGCTGGAATCCGTTGGTGTAGTAGAGCAT G GAAGATGATCCTCAGAGACCTGTAGCGGTTT ATGTTCGTCT GCATGGGTGACCTGGAGTTCTAGGAGAACTA TGACTTCAAGGC GGAGTTCACCGAGAACTAGGACTTCAAGGCT GTC
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Supplementary Methods
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Commands in Rosetta to perform loop modeling:
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/home/fanyang/rosetta/main/source/bin/loopmodel.linuxgccrelease \
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-in:path:database /home/fanyang/rosetta2015.25/main/database \
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-score:weights membrane_highres_Menv_smooth.wts \
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-in:file:fullatom \
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-membrane:normal_cycles 100 \
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-membrane:normal_mag 15 \
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-membrane:center_search \
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-ignore_unrecognized_res \
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-symmetry:symmetry_definition /home/fanyang/projects/input_files/3J5R_2017/4D_ABCD_r_4D_after_ccd_relaxed_r.symm \
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-symmetry:initialize_rigid_body_dofs \
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-in:file:spanfile /home/fanyang/projects/input_files/3J5R_2017/3J5R_FL.span \
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-in:file:s /share/work/fanyang/work/3J5R_FL_KIC5-3J5R_2017_/top20_score_filtered/_1D/${SLURM_ARRAY_TASK_ID}.pdb \
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-loops:loop_file /home/fanyang/projects/input_files/3J5R_2017/3J5R_FL_SF.loop \
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-loops:remodel perturb_kic \
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-loops:refine refine_kic \
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-loops:relax no \
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-loops:strict_loops \
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-loops:build_attempts 20 \
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-relax:bb_move false \
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-max_inner_cycles 30 \
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-nstruct 51 \
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-out:prefix msymm-loop-kic- \
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-out:file:silent /share/work/fanyang/work/3J5R_FL_KIC6-3J5R_2017_/${SLURM_ARRAY_TASK_ID}/msymm-relax-_3J5R_2017_${SLURM_ARRAY_TASK_ID}.silent \
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-out:file:silent_struct_type binary \
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-mute all
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Commands in Rosetta to perform SASA calculation and filtering:
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/home/fanyang/rosetta/main/source/bin/rosetta_scripts.linuxgccrelease \
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-database /home/fan/rosetta_2016.20/main/database
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-membrane:normal_cycles 100
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-membrane:normal_mag 15
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-membrane:center_search
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-in:file:spanfile /media/fan/Data/Rosetta/Project_3J5R_loopmodeling2017/inputs_fullLength/3J5R_FL.span
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-score:weights membrane_highres_Menv_smooth.wts
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-parser:protocol /media/fan/Data/OneDrive/Academic/Academic_Computation/Rosetta_script/xml_scripts_Fan /filter_sasa_FY_T651.xml
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-symmetry:symmetry_definition /media/fan/Data/Rosetta/Project_3J5R_loopmodeling2017/inputs_fullLength/4D_ABCD_r_4D_ 3J5R_FL_r.symm
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-symmetry:initialize_rigid_body_dofs
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-ignore_unrecognized_res
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-in:file:silent_struct_type binary
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-in:file:silent /media/fan/Data/Rosetta/Project_3J5R_loopmodeling2017/FL_after_KIC4/3J5R_FL_KIC33J5R_2017-_SILENT.out
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-nstruct 1
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-out:file:silent /media/fan/Data/Rosetta/Project_3J5R_loopmodeling2017/FL_after_KIC4/Filtered_T651_aft erKIC4.silent
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-out:file:silent_struct_type binary
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-overwrite
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Rosetta scripts to perform SASA calculation and filtering:
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Commands in Rosetta to perform global refinement and FSC calculation:
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-database /home/fan/rosetta/main/database
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-in::file::s /media/fan/Data1/Rosetta/Project_3J5R_loopmodeling2017/relax_cryoEM/3J5R.pdb
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-parser::protocol /media/fan/Data1/Rosetta/Project_3J5R_loopmodeling2017/relax_cryoEM/B_relax_density.x ml
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-parser::script_vars reso=4.2 map=/media/fan/Data1/Rosetta/Project_3J5R_loopmodeling2017/relax_cryoEM/EMD5777_CAP_3j5r.map testmap=/media/fan/Data1/Rosetta/Project_3J5R_loopmodeling2017/relax_cryoEM/EMD5777_CAP_3j5r.map
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-ignore_unrecognized_res
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-default_max_cycles 200
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-edensity::cryoem_scatterers
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-out::suffix cryoEM_Relax_
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-out:file:silent /media/fan/Data1/Rosetta/Project_3J5R_loopmodeling2017/relax_cryoEM/CAP_OpenModel_cry oEM_Relaxed.silent
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-out:file:silent_struct_type binary
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-nstruct 1
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-overwrite
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Rosetta scripts to perform global refinement and FSC calculation:
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Commands in Rosetta to incorporate ANAP into TRPV1 models
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-database /home/fan/rosetta/main/database
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-in:file:fullatom
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-ignore_unrecognized_res
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-s /home/fan/Rosetta/Project_ANAP_Parameterization/3J5P.pdb
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-backrub:ntrials 10
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-nstruct 1
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-mc_kt 0.6
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-resfile /home/fan/Rosetta/Project_ANAP_Parameterization/T651ANP_3J5P.resfile
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-overwrite
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T651ANP_3J5P.resfile:
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NATRO
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start
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651 A EMPTY NC ANP
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651 B EMPTY NC ANP
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651 C EMPTY NC ANP
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651 D EMPTY NC ANP
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