The structure and regulation of human muscle α-actinin

Abstract

The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.

Graphical abstract

Figure 1

Complete Structure of α-Actinin-2 in Closed Conformation (A) Domain composition of the α-actinin dimer. Color code, as in all the following figures: ABD, red; neck, yellow; SR1–SR4, green; EF1-2, violet; EF3-4, blue. (B) The dimeric structure of α-actinin-2 assembled from two halves of the α-actinin-2 protomer (ABD-SR1-SR2/SR3-SR4-CaM) through a crystallographic 2-fold axis (dashed line; ellipse in C). Overall dimensions are indicated. (C) Same as in (B), rotated 90° around the horizontal axis. See also Table S1.

Figure 2

Close-Up of the Functional Domain Interactions (A) PIP2 binding site on α-actinin-2 and the EF3-4-neck interaction. ABD and EF3-4 are presented with their solvent-accessible surface areas. The R residues responsible for PIP2 binding are highlighted in blue on the ABD surface. (B) Detail of EF1-2 interactions with SR4. (C) Detail of EF3-4 interactions with the neck region and SR1. (D) Comparison of α-actinin-2 CAMD with TnC bound to TnI, aligned on EF3-4 and the C-terminal lobe of TnC. Left: cartoon representation of α-actinin EF1-2 and EF3-4 with the interacting portion of the neck from the juxtaposed subunit (yellow) and a part of SR4 from the same subunit. Right: cartoon representation of TnC bound to TnI. N-terminal lobe, violet, as in EF1-2, and the C-terminal lobe, blue, as in EF3-4. Bound N-terminal TnI fragment, yellow; C-terminal TnI helix, green. Calcium ions are shown as black spheres (on TnC). The C-terminal lobe of TnC is aligned to EF3-4. To show the direction of bound helices, residues defining the neck domain of actinin and TnI fragments are indicated. (E) Sequence alignment of EF1-2 and EF3-4 and the C-terminal lobe of TnC. The corresponding calcium-binding positions in Ca²⁺/CaM are indicated by black dots. Charged residues involved in calcium binding are boxed. Fully conserved residues are highlighted in red. (F) Comparison of interactions between Zr-7 and the neck α helices with EF3-4. Electrostatic surface representation of the EF hands and cartoon representation for Zr-7 (cyan) and the α-actinin-2 neck (yellow). Side chains of key hydrophobic residues are shown as sticks; sequence numbers are boxed. Structural sequence alignment between titin Zr-7 and the neck. Residues involved in the interface with EF3-4 are highlighted in yellow. Residues involved in H bonding are boxed. Asterisks denote the mutations in the NEECK mutant. Black dots denote the CaM-binding motif 1-4-5-8. Underneath is shown the sequence of the NEEK mutant. (G) α-actinin-2 with docked PIP2 (the overall top-scoring pose is shown as a yellow stick model) together with the EF3-4-neck interaction. The top-scoring pose with two PIP2 tails in contact with the neck region is presented in Figure S2F. ABD and the neck region are presented with their solvent-accessible surface areas colored by electrostatic potential and the rest by cartoon representation and color coded as in Figure 1. The three R residues responsible for PIP2 binding are indicated. See also Table S2.

Figure S1

Conformational Variability and Substructures of α-Actinin-2, Related to Figure 2 (A) Loop connecting α helices 1 and 2 in SR4 undergoes a conformational change upon EF1-2 binding. Ribbon representation of superimposed SR4 from the full-length structure of α-actinin-2 (green) and previously determined rod domain structure (light blue) (PDB entry 1HCI) (Ylänne et al., 2001). (B) Cartoon representation of EF-hand lobes in different conformations. On top from left to right: the open conformation of the N-terminal lobe from calmodulin (PDB code 1CDM) (Meador et al., 1993), the semi-open conformation from the C-terminal lobe of the myosin essential light chain (PDB code 1SCM) (Xie et al., 1994) and the closed conformation from the calcium free C-terminal lobe of calmodulin (PDB code 1DMO) (Zhang et al., 1995). On bottom the EF1-2 and EF3-4 from the α-actinin-2 CAMD. All structures are aligned on the N-terminal α-helix. The N-terminal and C-terminal amino acids as also the middle helices h2 and h3 are indicated on the structures. The h2 and h3 α helices on both EF1-2 and EF3-4 are tilted compared to the positions on the closed lobe conformation, similarly to the semi-open conformation.

Figure S2

Molecular Structures of L-α-Phosphatidylinositol 4,5-Diphosphate Used Mapping of PIP2 Binding Sites and Docking Experiments, Related to Figures 2 and 3 (A) Top: Native PIP2 containing two long-chain fatty acid C20:4 and C18:0 (PubChem database compound ID: 53480324 - National Center for Biotechnology Information, http://pubchem.ncbi.nlm.nih.gov). The maximum length of long-chain fatty acid C18:0 accounted from phosphate at the sn-1 position is indicated (Å). Middle, bottom: PIP2 analogs labeled on sn-1 with the 26-carbon Bodipy®TMR label attached via a 6-carbon linker. Either palmitic acid C16:0 or caproic C6:0 was esterified on the sn-2 position for the bodipy®TMR-PIP2-C16 (PIP2-C16^∗, middle) and bodipy®TMR-PIP2-C6 (PIP2-C6^∗, bottom), respectively. Chemical structures of PIP2 were edited using Accelrys Draw 4.1 (Accelrys Software Inc). (B) PIP2 binding to α-actinin-2 variants measured by fluorescence anisotropy. Unlabelled α-actinin-2 was titrated into a fixed concentration of fluorescent PIP2-C6^∗ (4 μM). Increases in fluorescence anisotropy signal indicate α-actinin-2 is binding to PIP2-C6^∗. NEECK mutant showed similar PIP2 binding, while single mutant R163E, double mutant R163E/R169E, and triple R163E/R169E/R192E mutants showed decreased PIP2 binding (insert: relative PIP2 binding analysis). PIP2-C6^∗ analog was used for the screening of the phosphoinositide head-group binding to arginine residues in order to minimize the interference of a longer chain fatty acid, such as PIP2-C16^∗, in the binding assay. Based on this assay, triple PIP2 mutant, which displayed about 50% reduction of the anisotropy signal, was used as reference to investigate the relative effect of PIP2 binding inhibition on Zr-7 binding regulation compared to WT (Figures 3D–3F). Average and error bars (SDs) of three experimental replicas are plotted. (C) Definition of the search region for flexible docking of PIP2 using Gold (D) Fraction of docked solutions belonging to each of the 4 types as defined in Experimental Procedures. (E) The average number of contacts to PIP2 phosphates by different R residues in the Arginine platform among docked solutions. (F) α-actinin-2 structure with docked PIP2 (top scoring pose with two PIP2 tails in contact with the neck region are represented as yellow stick model) together with the EF3-4:neck interaction. ABD and the neck region are presented with their solvent accessible surface areas colored by electrostatic potential, the rest by cartoon representation and color coded as in Figure 1. The three R residues responsible for PIP2 binding are highlighted. The PIP2 molecule used in the flexible docking experiments containing palmitic acid (C16:0) esterified on the sn-1 and sn-2 (i.e., without bodipy-TMR fluorescence tag).

Figure 3

Structural Plasticity and Regulation of α-Actinin-2 Assessed by EPR and MST (A) Cluster of the ten cysteine residues (Cα in dark yellow spheres) of α-actinin-2 used for the computed DEER distance distribution shown in (B). The inset shows pairs with interspin distances <20 Å. (B) Experimental distance distribution of spin-labeled α-actinin WT (black) and simulation of the distance distribution (gray) based on spin-labeled cysteine residues from the crystal structure using the program MMM (see Extended Experimental Procedures and Table S3). (C) DEER traces (for better comparison, adjusted by the modulation depth) and distance distributions from Q band DEER experiments using a DEER dipolar evolution time of 1 μs at 50 K of α-actinin-2 WT (black), WT plus PIP2-C16^∗ (orange), WT plus PIP2-C16^∗ plus Zr-7 (magenta), WT plus Zr-7 (light blue), and the NEECK mutant (green). The arrow indicates the change in the time domain trace, which is reflected in the variation of the fraction of distances <20 Å (inset). (D) PIP2-C16^∗ binding to α-actinin-2 measured by MST. (E) CAMD, α-actinin-2 WT, and NEECK variant binding to Zr-7 measured by MST. The affinity determined for α-actinin-2 (+PIP2-C16^∗) binding to Zr-7 is in a similar range of affinity for α-actinin-2 CAMD and is implicated in Zr-7 interaction, as well as for the NEECK variant. (F) α-actinin-2 variant binding to Zr-7 with and without PIP2-C16^∗ measured by MST. Unlabeled Zr-7 was titrated into a fixed concentration of fluorescently labeled α-actinin-2 (50 nM). Average and error bars (SDs) of three MST experimental replicas are plotted. Mean and SD of K_d values were calculated from these plots. See also Tables S3 and S4.

Figure S3

Interspin Distance Measurements by Continuous-Wave and Pulsed EPR, Related to Figure 3 (A) Spin-normalized cw EPR spectra (in H₂O) detected at 160 K of α-actinin-2 WT spin labeled with 1:10-fold cysteine:MTSL stoichiometric ratios (black) and with 1:1:6 ratio of cysteine:MTSL(paramagnetic):MTSL(diamagnetic) (red). The increase in the spectral intensity in the spin diluted sample is indicative of decreased dipolar broadening arising from interspin distances < 2 nm. (B) Spin-normalized cw EPR spectra (in D₂O) at 160 K of α-actinin-2 WT (black) and NEECK (green). The increased spectral intensity in the NEECK mutation indicates decreased dipolar broadening (disappearance of short interspin distances). (C) Q-band DEER primary traces (V(t)/V(0)), form factors (F(t)/F(0)) and distance distributions (P(r)). A dipolar evolution time of 1 μs was used to obtain a good S/N ratio focusing on the distance features < 1.8 nm (borderline region of the DEER sensitivity). (D) Longer DEER traces with a dipolar evolution time of 2.8 μs (V(t)/V(0)), form factors (F(t)/F(0)) and distance distributions (P(r)). Samples and color codes are the same as in (C). (E) Low temperature CW EPR spectra (spin normalized) of the corresponding sample used for DEER (full line), compared with the WT and NEECK mutant (dashed lines). The comparison corroborates the DEER results. Color codes are the same as in (C).

Figure 4

Solution Structure of α-Actinin-2 and the NEECK Mutant Derived from SAXS (A) Experimental SAXS data of WT (black) and the NEECK mutant (green) of α-actinin-2. SAXS curves are computed from a rigid-body (RB) model for WT (gray) and NEECK (black). The logarithm of scattering intensity (I) is plotted as a function of the momentum transfers (s, Å⁻¹). Successive curves are displaced by one logarithmic unit for better visualization. Distance distribution functions (inset) P(r) for WT and NEECK assume slightly different shapes. RB modeling fits the experimental WT data with χ 1.25 (gray line) and experimental NEECK data with χ 1.14 (dashed black line). The fit discrepancy for NEECK increased to 1.32, assuming a helical neck (solid black line). (B) Characterization of hydrodynamic properties of α-actinin WT and the NEECK mutant by SEC-MALLS. The lines across the protein elution volume show the molecular masses (MWs) of proteins. SEC-MALLS shows that NEECK has the same molecular weight as WT α-actinin-2 but a higher Stokes radius Rs (inset; data are represented as mean ± SD of three experiments), corroborating the open conformation for NEECK suggested by SAXS (C). AU, arbitrary units. (C) RB model of NEECK in solvent-accessible surface representation. The neck region was modeled as a flexible linker between the rigid bodies ABD and rod, with no contact restraint. Only one RB model is shown for clarity out of three independent BUNCH runs (Figure S5A). (D) The best RB model of WT α-actinin-2 in solvent-accessible surface representation superimposed on the crystal structure. For WT RB modeling, only ABD was allowed a variable position, whereas EF hands 3-4 were fixed in contact with the neck. In all models, N-terminal residues missing from the crystal structure were modeled as dummy atoms. Arrows highlight the movement of ABD and EF hands 3-4 relative to the superimposed crystal structure. See also Table S5.

Figure S4

Neck Peptide of Wild-Type α-Actinin-2 Is Unstructured when Not Bound to the CaM-like Domain, Related to Figure 4 Fingerprint area of the 2D NOESY spectrum of the WT neck-peptide in 90:10 H₂O:D₂O with neck-peptide in 25mM phosphate pH 6.5, 25 mM NaCl, 3 mM DTT and 0.5 mM EDTA recorded at 298K. Hα_i-HN_i and Hα_i-HN_i+1 cross peaks are shown in red and the sequential walk of the peptide is indicated with horizontal and vertical lines. No additional NOE cross peaks were found that indicated an existence of any kind of stable α-helical structural in the fingerprint area.

Figure S5

Structural Plasticity of NEECK as Assessed by SAXS and Room Temperature Continuous-Wave EPR, Related to Figure 4 (A) BUNCH runs starting from random initial structures provided models of NEECK mutant with varying positions of ABD and EF3-4 assuming nonhelical neck. Three representative BUNCH models (distinguished by degree of transparency) are superimposed, showing that ABDs deviate from the linear alignment of the rod domains and that EF3-4 are in open conformations. (B–E) The flexibility of the ABD domains and EF3-4 is also supported by EOM analysis of NEECK. The central rod part (green) was fixed as in the crystal structure whereas ABD (red) and EF3-4 (blue) domains position were optimized together with the N-termini residues. (B) The low discrepancy between experimental data (green plot) and those computed from EOM models (dashed gray plot) of NEECK mutant is defined by χ value of around 1.0. Size and shape descriptor distributions: R_g (C) and D_max (D). (D) The distributions correspond to the pool of 1,000 structures (solid lines), and the optimized ensemble (dashed lines) calculated using chromosomes with N = 20 structures; dotted lines are references for R_g and D_max values calculated from X-ray structure of WT α-actinin-2. The overall R_g and D_max analysis of the select optimized ensemble show bimodal distribution, which indicates distinct population of conformers (compacted and extended). The peak of compacted models is slightly shifted to smaller distances, due to the larger deviation of ABD domains from the linear alignment of the rod domains, compared to the peak of extended models. (E) Selected ensemble of models were superimposed and distinguished by degree of transparency. In the lower panel models are rotated 45° around the long axis of the dimer. (F) Cw EPR spectra (normalized by peak intensity) of spin labeled α-actinin-2 WT (black) and α-actinin-2 NEECK mutant (green), detected at room temperature. ΔH_pp indicates the peak-to-peak line-width of the central peak. The narrower lines in the room temperature EPR spectrum of the NEECK mutant spectrum (green) with ΔH_pp of 0.26 mT indicates an increase in mobility of at least one part of the spin-labeled structure compared to the WT (ΔH_pp = 0.46 mT).

Figure 5

Mutations Affecting Regulation of α-Actinin-2 with PIP2 Do Not Influence F-Actin Binding but Impact α-Actinin-2 Z-Disk Dynamics (A) Binding of α-actinin-2 variants to F-actin and titin Zr-7. α-actinin-2 WT, NEECK, and PIP2 mutants (PIP2 mut) were cosedimented with actin, and equal amounts of supernatant (s) and pellet (p) fractions were subjected to SDS-PAGE and visualized by Coomassie blue. (B and C) FRAP measurements of α-actinin-2 dynamics in live NRCs expressing GFP-labeled α-actinin-2 variants (WT, PIP2 mutants, and NEECK). (B) Snapshots at prebleach and two time points postbleach; the bleached region of interest (ROI) is highlighted by a dotted box. Note that NEECK fluorescence does not recover within the 144 s time course shown here, whereas rapid recovery is observed for WT α-actinin. Insets: ROIs enlarged 2-fold. (C) Quantification of fluorescence intensity recovery. Note that the slowed fluorescence recovery of the PIP2 mutant is mirrored by treatment with 500 μM neomycin (Neo). Bold lines, exponential fits; shaded lines, average values. Error bars indicate SD.

Figure S6

PIP2 and Vinculin in Relation to the Z-Disk and Temporal Increase of the Titin T12 Epitope in α-Actinin WT- and NEECK-Transfected Cardiomyocytes, Related to Figures 5 and 6 (A) PIP2 is localized to the cardiac Z-disk. Immunofluorescence in untransfected NRC using PIP2 antibody shows a striated pattern that coincides with the Z-disk titin epitope T12 (arrows). Note the noncardiomyocytes above the striated cell, in which PIP2 is localized in a vesicular pattern. (B) The α-actinin-2 NEECK mutant does not colocalize in abnormal Z-disks. GFP-labeled NEECK α-actinin was transiently expressed in NRC for 18-48 hr. The NEECK mutant leads to widening of the Z-disk (upper row) and ultimately formation of rod-like structures (lower row). Note that vinculin shows no appreciable colocalization with α-actinin-2 but remains predominantly restricted to focal adhesions. Red: monoclonal anti-vinculin stain, green: α-actinin-2 NEECK-GFP, Blue: actin (Alexa643-phalloidine). (C) The optically resolvable T12 distance was measured in confocal immunofluorescence images 2 and 3 days after transfection of the GFP-labeled constructs. Note the expansion to an average width of over 600 nm at day 2 and over 900 nm at day 3, while the optical T12 distance in WT α-actinin transfected cells does not exceed 300 nm. Data from 9 independent cells each. Dashed lines: means.

Figure 6

Constitutively Activated α-Actinin-2 Disrupts Z-Disks and Leads to Myofibril Disassembly GFP-labeled WT and NEECK α-actinin-2 were transiently expressed in NRCs for 18–48 hr. (A) WT α-actinin shows normal Z-disk localization, and the titin T12 epitope is resolved as a single line in standard confocal microscopy. (B) In contrast, NEECK leads to widening of the Z-disk and splitting of the T12 epitope after ∼18 hr (asterisk); doublet T12 lines are highlighted by arrows. (C) After 48 hr, Z-disks are completely disrupted and Z-disk titin, actin, and mutant α-actinin are localized in rod-like structures. (D and E) Superresolution microscopy reveals that epitopes of N-terminal Z1Z2 of titin and their ligand telethonin are unresolvable in WT-transfected cells; NEECK causes widening of Z-disks. Doublet Z1Z2/telethonin lines are highlighted by arrows and the central α-actinin region is indicated by arrowheads. Insets show 2-fold enlargement. Z-disk titin (T12 epitope) or telethonin, red; mutant α-actinin-GFP, green; actin (Alexa 688-phalloidin) or titin Z1Z2, blue.

Figure 7

Molecular Mechanism of α-Actinin-2 Phosphoinositide-Based Activation and Model for F-Actin/α-Actinin Crosslinking in the Z-Disk (A) Reaction mechanism depicting α-actinin activation by PIP2. α-actinin in the absence of PIP2 is in equilibrium between highly populated closed [A_C] and low populated open states [A_O]. Addition of PIP2 generates an activated state [A^∗:PIP2], with lower activation energy for opening. Binding of Zr-7 recruits α-actinin to the open conformation, leading to an increase of [A_O:Zr-7] due to higher α-actinin affinity for Zr-7 compared to the neck. (B) Actin binding sites 1–3 (orange) mapped onto the molecular surface of ABD in α-actinin-2 in closed conformation. Color coding of domains is as in Figure 1. (C) α-actinin-2 crystal structure superimposed on F-actin decorated by the CH1 domain of α-actinin-2 (PDB ID code 3LUE). (D) Model of α-actinin-2 crosslinking antiparallel actin filaments. α-actinin-2 in open conformation (NEECK) was modeled assuming structural plasticity in the flexible neck, which allows for suitable orientation of ABDs. Titin Zr-7 is in cartoon presentation (cyan). See also Movie S1.

Figure S7

Amino Acid Sequence Conservation and Genetic Variants Mapping on Solvent-Accessible Areas of α-Actinin-2, Related to “Impact of Pathogenic Mutations” in Discussion (A) Representative vertebrate sequences of all 4 α-actinin isoforms were aligned using Clustal Omega (Sievers et al., 2011), and sequence conservation (variable red, average white, conserved blue) mapped on the solvent accessible surface using Consruf server (Celniker et al., 2013). Species included are Homo sapiens, Bos taurus, Mus musculus, Macaca mulatta, Canis familiaris, Orcinus orca, Danio rerio, Gallus gallus, Falco peregrinus, Oryctolagus cuniculus, Lepisosteus oculatus, Equus caballus. (B) Genetic variants reported in literature (red; see Discussion) were mapped on the solvent accessible surface of α-actinin-2 (white).