Complex structures of Rsu1 and PINCH1 reveal a regulatory mechanism of the ILK/PINCH/Parvin complex for F-actin dynamics
- Department of Biology, Southern University of Science and Technology, China
- Faculty of Health Sciences, University of Macau, China
- Academy for Advanced Interdisciplinary Studies, Southern University of Science and Technology, China
- Department of Pathology, School of Medicine and University of Pittsburgh Cancer Institute, University of Pittsburgh, United States
- Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, and Shenzhen Key Laboratory of Cell Microenvironment, China
Figures
Figure 1 with 4 supplements
Biochemical characterization of the Rsu1/PINCH1 interaction.
(A and B) Schematic domain organization of Rsu1 (A) and PINCH1 (B). The fragments used in this study are indicated. The color coding of the regions is applied in all figures as otherwise indicated. …
Figure 1—figure supplement 1
Multiple sequence alignment of Rsu1 proteins from different species.
‘XENTR’, ‘DANRE’, ‘DROME’, and ‘CAEEL’ indicate Xenopus tropicalis, Danio rerio, Drosophila melanogaster, and C. elegans, respectively. The secondary structure elements of Rsu1 are labeled above the …
Figure 1—figure supplement 2
Multiple sequence alignment of PINCH1_LIM45C from different species.
The secondary structure elements are labeled above the alignment using the same color coding as used in Figure 1. Residues interacting with Rsu1 are indicated by triangles. Residues chelating Zn2+ …
Figure 1—figure supplement 3
Analytical gel filtration analysis of the interaction between the variants of Rsu1 and PINCH.
(A - C) Rsu1 or Rsu1ΔC mixed with PINCH1_LIM45C, or LIM5C. (D) Rsu1 mixed with PINCH2_LIM5C. (E) Rsu1 mutant mixed with PINCH1_LIM45C. (F) Rsu1 mixed with PINCH1_LIM45C mutant. The concentration of …
Figure 1—figure supplement 4
Binding of LIM5C from PINCH2 or PINCH1 to Rsu1 analyzed by isothermal titration calorimetry (ITC).
The curves were captured by titrating 200 μM Rsu1 to 20 μM PINCH2_LIM5C or PINCH1_LIM5C on a PEAQ-ITC Microcal calorimeter (Malvern). The binding affinity between PINCH2 and Rsu1 was largely …
Figure 2 with 1 supplement
Structural analysis of the Rsu1/PINCH1_LIM45C complex.
(A and B) Ribbon representations of Rsu1/PINCH1-LIM45C complex structure with two different views. Four Zn2+ ions are indicated by blue spheres. (C and D) Molecular details of the C-terminal (C) and …
Figure 2—figure supplement 1
Structural comparison of the Rsu1/PINCH1 complexes solved in this study.
(A) Structural superposition of the Rsu1/PINCH1_LIM45C complex in different crystal forms. The structural difference is highlighted by a red dashed circle. (B) Structural superposition of the …
Figure 3 with 2 supplements
The Rsu1/PINCH1 interface.
(A) Surface representations of Rsu1. The protein surface of Rsu1 is rendered with the amino acid conservation (Figure 1—figure supplement 1). (B) Structural superposition of the PINCH1_LIM5C …
Figure 3—figure supplement 1
Electron density map of two buried water molecules in the Rsu1/PINCH1 complex structures.
The 2Fo-Fc densities are contoured at 2.0σ with the structures superimposed. The black arrows indicate the densities of the water molecules.
Figure 3—figure supplement 2
Isothermal titration calorimetry (ITC)-based binding affinity measurements showing the impaired interaction between PINCH1 and Rsu1 with the interface mutations on Rsu1.
The Rsu1 proteins used in the ITC experiments were checked by SDS-PAGE analysis.
Figure 4 with 3 supplements
Rsu1 disrupts F-actin bundles induced by the ILK/PINCH/Parvin (IPP) complex.
(A) Surfaces on PINCH1_LIM5C that are involved in the Rsu1-binding or actin-binding. (B) Schematic cartoon showing the inhibitory role of Rsu1 in the IPP-mediated F-actin bundling. (C) Rsu1 but not …
Figure 4—figure supplement 1
Purification of ILK/PINCH/Parvin (IPP) complex and F-actin bundling assay of Rsu1 proteins.
(A) Analytical gel filtration of IPP complex, and the multiangle static light scattering analysis showed that the molecular weight of IPP is 134 kDa (the theoretical molecular weight of IPP is 132 …
Figure 4—figure supplement 2
Quantification of actin bundles.
(A) Represented images of bundled F-actins by ILK/PINCH/Parvin (IPP) complex or IPP mixed with different Rsu1 proteins. The images here were about one quarter of the images used for the quantitative …
Figure 4—figure supplement 3
Electron microscopic analysis of F-actin in the presence of ILK/PINCH/Parvin (IPP) or the IPP/Rsu1 mixture.
The white arrowhead in the middle panel indicated the actin bundle.
Figure 5 with 1 supplement
Overexpression of Rsu1 in HeLa cells regulates actin-related cellular processes.
(A) Confocal images of HeLa cells transiently expressed of GFP, Rsu1-GFP, or Rsu1 mutations with a GFP tag. Focal adhesions (FAs) and stress fibers were stained by paxillin and phalloidin, …
Figure 5—figure supplement 1
The expression level of wild-type Rsu1 and different mutants in HeLa cells as detected by western blot.
Figure 6 with 1 supplement
The inhibition of Rsu1 on the ILK/PINCH/Parvin (IPP) complex is released by the fusion of PINCH1-WH2.
(A) Design of the Rsu1-WH2 chimera, in which the WH2 motif (308–325) of PINCH1 was fused to the C-terminus of Rsu1. This chimera provides an additional WH2 motif for F-actin bundling as indicated by …
Figure 6—figure supplement 1
Conservation analysis of the convex surface on Rsu1.
Left panel: surface representations of Rsu1 to show the convex surface. The view is related to that in Figure 3A by rotating 180° along the y-axis. Right panel: cartoon representations of Rsu1 to …
Author response image 1
Depletion of Rsu1 in HeLa cells and rescue effects of wild type Rsu1 and its mutants.
(A) Confocal images of HeLa cells transfected with control siRNA and GFP-vector, or Rsu1-siRNA with GFP-vector, Rsu1-GFP or Rsu1 mutants. FAs and stress fibers were stained by paxillin and …
Author response image 2
Rescue effect of wild type Rsu1 and its mutants in Rsu1-KO HT1080 cells.
(A) Confocal images of normal HT1080 cells or Rsu1-KO cells transfected with GFP-vector, Rsu1-GFP or Rsu1 mutants. FAs and stress fibers were stained by paxillin and phalloidin, respectively. (B) …
Author response image 3
SDS-PAGE analysis of the purified PINCH proteins.
Tables
Table 1
Statistics of data collection and model refinement.
| Rsu1ΔC/PINCH1_LIM5 (PDB id: 7D2S) | Rsu1/PINCH1_LIM45 (7D2T/7D2U) | ||
|---|---|---|---|
| Data collection | |||
| Space group | I 4 | P 21 | I 2 2 2 |
| Cell dimensions | |||
| a, b, c (Å) | 124.6, 124.6, 50.5 | 114.6, 51.3, 119.6 | 51.4, 144.4, 185.0 |
| α, β, γ (°) | 90, 90, 90 | 90, 101.6, 90 | 90, 90, 90 |
| Resolution (Å) | 50–1.65 (1.68–1.65) | 50–2.20 (2.24–2.20) | 50–3.15 (3.20–3.15) |
| Rmerge* | 0.089 (0.931) | 0.152 (1.204) | 0.131 (0.967) |
| I/σI | 31.2 (2.8) | 14.1 (1.4) | 26.0 (1.9) |
| CC1/2† | (0.823) | (0.687) | (0.885) |
| Completeness (%) | 100 (100) | 100 (100) | 99.9 (100) |
| Redundancy | 13.4 (12.9) | 6.7 (6.9) | 12.9 (12.4) |
| Refinement | |||
| Resolution (Å) | 50–1.65 (1.69–1.65) | 50–2.20 (2.25–2.20) | 50–3.15 (3.44–3.15) |
| No. reflections | 46570 (2695) | 70228 (4880) | 12487 (3033) |
| Rwork/Rfree‡ | 0.166 (0.241) / 0.185 (0.271) | 0.170 (0.281) / 0.198 (0.308) | 0.192 (0.262) / 0.216 (0.316) |
| No. atoms | |||
| Protein | 2236 | 6055 | 2980 |
| Ligand/ion | 14 | 74 | 17 |
| Water | 187 | 358 | 0 |
| Mean B (Å) | |||
| Protein | 32.4 | 53.1 | 134.2 |
| Ligand/ion | 32.2 | 78.1 | 151.2 |
| Water | 38.8 | 52.0 | - |
| r.m.s. deviations | |||
| Bond lengths (Å) | 0.006 | 0.003 | 0.002 |
| Bond angles (°) | 1.01 | 0.74 | 0.55 |
| Ramachandran analysis | |||
| Favored region (%) | 96.1 | 96.7 | 95.4 |
| Allowed region (%) | 3.9 | 3.3 | 4.6 |
| Outliers (%) | 0 | 0 | 0 |
-
The numbers in parentheses represent values for the highest resolution shell.
*Rmerge = ∑|Ii− Im|/∑Ii, where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry related reflections.
-
†CC1/2 is the correlation coefficient of the half data sets.
‡Rwork = Σ||Fobs| − |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are observed and calculated structure factors.
-
Rfree = ΣT||Fobs| − |Fcalc||/ΣT|Fobs|, where T is the test data set of about 4–5% of the total reflections randomly chosen and set aside prior to refinement.
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | RSU1 | GenBank | NP_036557.1 | |
| Gene (Homo sapiens) | PINCH1 | GenBank | NP_001180417.1 | |
| Gene (Homo sapiens) | PINCH2 | GenBank | NP_001154875.1 | |
| Gene (Homo sapiens) | ILK | GenBank | NP_001014794.1 | |
| Gene (Homo sapiens) | Parvinα | GenBank | NP_060692.3 | |
| Recombinant DNA reagent | pFastBac-HTB-RSU1 | This paper | (1-277) / Full-length | BamHI/XhoI |
| Recombinant DNA reagent | pFastBac-HTB-RSU1 | This paper | (1-215) / LRR | BamHI/XhoI |
| Recombinant DNA reagent | pFastBac-HTB-RSU1 | This paper | /N69A/F71Q/R137E/Y140K/D143K/R165A/D115K-Y140K/D115K-D143K/F71Q-D115K-Y140K/WH2(308–325)/ | Kinds of Mutations in full-length gene |
| Recombinant DNA reagent | pEGFP-N3-hRSU1 | This paper | (1-277) / Full-length | HindIII / BamHI |
| Recombinant DNA reagent | pEGFP-N3-hRSU1 | This paper | /Y140K/D115K-Y140K/WH2/ | Kinds of Mutations in full-length gene |
| Recombinant DNA reagent | pET.32M.3C | PMID:19665975 | Dr. Mingjie Zhang (SUSTech, China) | |
| Recombinant DNA reagent | pET.32M.3C-hPINCH1 | This paper | LIM5C(249-325) | BamHI/XhoI |
| Recombinant DNA reagent | pET.32M.3C-hPINCH1 | This paper | LIM45C(188-325) | BamHI/XhoI |
| Recombinant DNA reagent | pET.32M.3C-hPINCH1 | This paper | LIM45(188-307) | BamHI/XhoI |
| Recombinant DNA reagent | pET.32M.3C-hPINCH1 | This paper | LIM45(188-325)-/D295K/K297D/F253Y-H254N/ | BamHI/XhoI |
| Recombinant DNA reagent | pET.32M.3C-hPINCH2 | This paper | LIM5C(276-363) | BamHI/XhoI |
| Recombinant DNA reagent | pRSF-SUMO-hPINCH1 | This paper | (1-325) / Full-length | NdeI/XhoI |
| Recombinant DNA reagent | pETDuet-SUMO-hILK(C346S-C422S) / His-hParvinα | This paper | hILK(1-452)C346S-C422S / hParvinα(1-372) | (EcoRI/HindIII) for hILK (Recombinational method) for hParvinα |
| Peptide, recombinant protein | actin | Cytoskeleton, Inc | Cat. # AKL99 | Rabbit Skeletal Muscle |
| Strain, strain background (Escherichia coli) | BL21(DE3) | Kangti Health | Cat. # KTSM104L | |
| Strain, strain background (Escherichia coli) | Rosseta(DE3) | PMID:28966017 | Dr. Mingjie Zhang (SUSTech, China) | |
| Cell line (Spodoptera frugiperda) | IPLB‐SF21‐AE | Gibco/Thermo Fisher | Cat. # 11496015 | Maintained in Sf-900 II SFM, large scale in ESF 921 |
| Cell line (Homo sapiens) | HeLa | National Collection of Authenticated Cell Cultures | Cat. # TCHu187 | |
| Chemical compound, drug | Sf-900 II SFM | Gibco/Thermo Fisher | Cat. # 10-902-096 | Medium for Sf9 cell |
| Chemical compound, drug | ESF 921 | Expression Systems | Cat. # 96-001-01 | Medium for Sf9 cell |
| Chemical compound, drug | MEM | CORNING | Cat. # 10–010-CV | |
| Chemical compound, drug | DMEM | CORNING | Cat. # 10–013-CVRC | |
| Chemical compound, drug | Fetal Bovine Serum | PAN BIOTECH | Cat. # P30-3302 | |
| Chemical compound, drug | Fibronectin | Millipore | Cat. # FC020-5MG | |
| Chemical compound, drug | Cellfectin II Reagent | Thermo Fisher | Cat. # 10362100 | Transfection reagent for Sf9 cell |
| Chemical compound, drug | Lipofectamine 2000 reagent | Invitrogen | Cat. # 11668–019 | Transfection reagent for HeLa cell |
| Chemical compound, drug | Lipofectamine 3000 reagent | Invitrogen | Cat. # L3000-015 | Transfection reagent for HeLa cell |
| Chemical compound, drug | Alexa Fluor 488 Phalloidin | Invitrogen/THermo Fisher | Cat. # A12379 | F-actin staining (1:100, v/v) |
| Chemical compound, drug | Alexa Fluor 594 Phalloidin | Invitrogen/THermo Fisher | Cat. # A12381 RRID: AB_2315633 | IF (1:200, v/v) |
| Chemical compound, drug | Alexa Fluor 647 Phalloidin | Invitrogen/THermo Fisher | Cat. # A22287 RRID: AB_2620155 | IF (1:100, v/v) |
| Chemical compound, drug | DAPI stain | SIGMA | D9542 | 1 µg/mL |
| Antibody | Anti-Paxillin (mouse monoclonal) | BD Bioscience | Cat. # 610620 RRID: AB_397952 | IF (1:500) |
| Antibody | Anti-Rsu1 (rabbit polyclonal) | Thermo Fisher Scientific | Cat. # A305-422A RRID: AB_2631813 | WB (1:4000) |
| Antibody | Anti-PINCH (mouse monoclonal) | BD Bioscience | Cat. # 612711 | WB (1:1000) |
| Antibody | Anti-Parvin (mouse) | Millipore | Cat. # MABT157 | WB (1:1000) |
| Antibody | Anti-ILK (mouse polyclonal) | BD Bioscience | Cat. # 611803 RRID: AB_399283 | WB (1:1000) |
| Antibody | Anti-GAPDH (mouse monoclonal) | TRANSGEN | Cat. # HC301-02 RRID: AB_2629434 | WB (1:3000) |
| Antibody | Anti-GFP (mouse monoclonal) | TRANSGEN | Cat. # HT801 | WB (1:3000) |
| Antibody | Anti-Mouse IgG (H+L), Alexa Fluor 594 (donkey) | Thermo Fisher Scientific | Cat. # A21203; RRID: AB_141633 | IF (1:1000) |
| Antibody | Anti-mouse IgG, HRP-linked Antibody (horse) | Cell Signaling | Cat. # 7076 RRID: AB_330924 | WB (1:10000) |
| Antibody | Anti-rabbit IgG, HRP-linked Antibody (goat) | Cell Signaling | Cat. # 7074 RRID: AB_2099233 | WB (1:10000) |
| Commercial assay or kit | Western ECL substrate | BIO-RAD | Cat. # 170–5061 | |
| Software, algorithm | ASTRA6 | WYATT Technology | PRID:SCR_016255 | |
| Software, algorithm | MicroCal PEAQ-ITC integrated Software package | Malvern Panalytical | ||
| Software, algorithm | OriginPro | OriginLab | Learning Edition | |
| Software, algorithm | GraphPad Prism | GraphPad Software | RRID: SCR_002798 | |
| Software, algorithm | Image pro plus | MEDIA CYBERNETICS | RRID: SCR_016879 | |
| Software, algorithm | Image J | National Institutes of Health | RRID: SCR_003070 |
