Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin
- Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, China
- Hong Kong University of Science and Technology, China
- Pennsylvania State University College of Medicine, United States
- Chinese Academy of Sciences, China
Figures
Figure 1 with 2 supplements
Biochemical characterizations of the Myo3/Espin1 interaction.
(A) Domain organizations of Espin1, Myo3a and Myo3b. (B) Sequence alignment of THDI of Myo3a and Myo3b showing that there are a pair of repeating sequences within THDI, which we term as ARB1 and …
Figure 1—figure supplement 1
ITC results of Myo3a-ARBs binding to Espin1-AR.
(A–C) ITC results showing that Myo3a-ARB12 (A) as well as each individual ARB (B for ARB1 and C for ARB2) can bind to Espin1-AR with strong affinities.
Figure 1—figure supplement 2
Analytical gel filtration chromatography analysis of the Espin1-AR and Myo3b-ARB12 interaction.
(A–C) The profiles of mixtures of Trx-Espin1-AR and Myo3b-ARB12 with different molar-ratios. The black and red curves in each panel represent the elution profiles of Trx-Espin1-AR alone and …
Figure 2 with 2 supplements
The overall structure of the Myo3-ARB/Espin1-AR complex.
(A) An omit map showing the binding of Myo3b-ARB1 to Espin1-AR. The Fo-Fc density map was generated by deleting the Myo3b-ARB1 part from the final model and contoured at 3.0σ. The Myo3b-ARB1 fitting …
Figure 2—figure supplement 1
Structural-based sequence alignments of AR of Espin1 from different vertebrate species and Espin-like proteins from mammals.
Residues that are identical and highly similar are shown in red and yellow boxes, respectively. Residues that are directly involved in ARBs binding are highlighted with solid magenta stars. …
Figure 2—figure supplement 2
Superhelical model of Espin1-AR.
The superhelical model of Espin1-AR viewed from the top (left) and side (right). To illustrate the curvature nature of the ANK repeats, we artificially connected several Espin1-AR units in a …
Figure 3 with 2 supplements
The detailed Myo3/Espin1-AR interaction.
(A) The Myo3b-ARB1/Espin1-AR interface is divided into three regions corresponding to the ‘YY’ motif (A1), the ‘KxL’ motif (A2) and the N-terminal positively charged residues (A3) of Myo3b-ARB1. The …
Figure 3—figure supplement 1
The ITC titration curves for calculating the dissociation constants shown in Figure 3C.
Note that Panels A & G are the same as Figure 1C2 and C3.
Figure 3—figure supplement 2
Comparison of Myo3b-ARB2/Espin1-AR and Myo3b-ARB1/Espin1-AR structures.
(A) An omit map showing the binding of Myo3b-ARB2 to Espin1-AR. The Fo-Fc density map was generated by deleting the Myo3b-ARB2 part from the final model and contoured at 2.5σ. The Myo3b-ARB2 fitting …
Figure 4 with 1 supplement
Biochemical characterization of the Espin1 auto-inhibition.
(A) Domain organization of Espin1 showing that the Espin1-AI in the middle may bind to Espin1-AR at the N-terminus. (B) Sequence alignment of Espin1-AI from different vertebrate species, and …
Figure 4—figure supplement 1
FPLC-MALS results of Espin1-1-529 and Espin1-FL.
The FPLC-SLS results showing that Espin1-1-529 (left) and Espin1-FL (right) each exists as a monomer in solution.
Figure 5 with 1 supplement
Myo3-ARBs/Espin1 interaction is critical for the filopodia tip localizations of Espin1 and Myo3.
(A) Representative fluorescence images of COS7 cells co-expressing RFP-Espin1 and various GFP-Myo3a experimental constructs. A1, Myo3aΔKΔABM WT; A2, Myo3aΔKΔABM mARB; A3, Myo3aΔKΔABM dARB. Scale …
Figure 5—figure supplement 1
ARBs are required for both Myo3b and Espin1 filopodia tip localization.
Representative COS7 cells images of RFP-Espin1 co-transfected with different constructs of GFP-Myo3b. Row 1, Myo3bΔKΔABM WT; Row 2, Myo3bΔKΔABM mARB; Row 3, Myo3bΔKΔABM dARB. Scale bar: 5 µm.
Figure 6 with 1 supplement
Myo3 binding promotes Espin1’s higher order actin bundling activity.
(A) Representative images of actin bundles induced by Espin1 with and without the presence of various forms of Myo3-ARBs under fluorescent microscopy (left) or transmission electron microscopy …
Figure 6—figure supplement 1
Representative TEM images of actin bundles.
(A–D) Representative images of actin bundles induced by Espin1 with and without the presence of various forms of Myo3-ARBs under transmission electron microscopy. A: F-actin+Espin1 with a 4:1 molar …
Author response image 1
ITC results showing that Myo3a-ARB2 binds to Espin1-1-494 with similar affinity compared to Espin1-AR.
https://doi.org/10.7554/eLife.12856.019Tables
Table 1
Statistics of X-ray Crystallographic Data Collection and Model refinement Numbers in parentheses represent the value for the highest resolution shell. a. Rmerge=Σ Ii- <I> / ΣIi, where Ii is the …
Data sets | Espin1-AR/Myo3b-ARB1 5ET1 | Espin1-AR/Myo3b-ARB2 5ET0 |
|---|---|---|
Space group | P21 | P2 |
Wavelength (Å) | 0.9791 | 0.9795 |
Unit Cell Parameters (Å) | a=72.74, b=71.14, c=76.88 α=γ=90°, β=96.88° | a=39.74, b=68.78, c=173.45 α=γ=90°, β=90.04° |
Resolution range (Å) | 50-1.65 (1.68–1.65) | 50-2.30 (2.42–2.30) |
No. of unique reflections | 93433 (4625) | 39636 (5866) |
Redundancy | 3.7 (3.7) | 3.7 (3.8) |
I/σ | 18.5 (1.7) | 7.7 (1.9) |
Completeness (%) | 99.8 (99.9) | 94.9 (96.6) |
Rmergea (%) | 8.9 (91.6) | 10.3 (79.9) |
CC* for the highest resolution shell e | 0.866 | 0.878 |
CCi/2 for the highest resolution shell e | 0.599 | 0.627 |
Structure refinement | ||
Resolution (Å) | 50-1.65 (1.71–1.65) | 10-2.3 (2.38–2.30) |
Rcryst b/Rfree c (%) | 16.94/19.11 (25.77/28.64) | 22.32/25.34 (26.74/30.90) |
rmsd bonds (Å) / angles (°) | 0.006 / 0.795 | 0.010 / 1.113 |
Average B factor (Å2) d | 23.2 | 60.5 |
No. of atoms | ||
Protein atoms | 5374 | 4985 |
Water | 378 | 23 |
Ligands | 30 | 0 |
No. of reflections | ||
Working set | 89061 | 37660 |
Test set | 4345 | 1925 |
Ramachandran plot regions d | ||
Favored (%) | 98.9 | 98.4 |
Allowed (%) | 1.1 | 1.6 |
Outliers (%) | 0 | 0 |
