As the internal region of ALP is involved in localization of the protein and interaction with α-actinin 27, it was of great interest to study the structure and function of this region in more detail. According to the SMART server 34, the PDZ domain of human ALP ends at amino acid residue 83 while the LIM domain begins at residue 294. In our previous studies 27, we used the fragment containing residues 112–284, but found that during purification this fragment often degraded. For this reason, we tried to find optimal protein fragments of the ALP internal region and we tested several different constructs around the area that had sequence features characteristic for folded domains when predicted with servers like Foldindex 33 (Fig 2) and DisEMBL 35. We found that a 167 residue construct (ALP107-273) was optimally stable, and could be purified in high amounts and concentrated up to 30 mg/ml. SDS-PAGE analysis of this fragment revealed a single protein band of the expected molecular weight of 18265 Da (Fig 1B). The molecular weight of ALP107-273 was also verified by MALDI-TOF mass spectrometry. In gel filtration chromatography, the fragment eluted as a single peak immediately prior to the globular protein standard of 44 000 Da (Fig 1C), suggesting that it was either a very elongated monomer, dimer or trimer.

To test whether ALP107-273 was functional, we measured the interaction of the purified protein fragment with α-actinin by surface plasmon resonance (SPR) and tested the localization of a yellow fluorescent protein (YFP) fusion protein construct in living cells. The SPR measurements were made either by immobilizing ALP107-273 on the chip, using the α-actinin rod fragments as a soluble analyte, or the other way around. A clear interaction was observed in both experimental setups (Figs 3A and 3B) and the apparent dissociation equilibrium constants (K_d) were in the low micromolar range. When an α-actinin rod fragment consisting of four spectrin repeats (R1-R4) was immobilized and ALP107-273 used as the analyte, the K_d was 2.7 × 10^-7 M. With immobilized ALP107-273 and soluble R1-R4, the K_d was 4.5 × 10^-6 M. These values are in the same range as our previous measurements that used longer ALP fragments 27. ALP107-273 interacted with a shorter piece of the α-actinin rod region that contained two spectrin repeats (R2-R3) with an apparently higher affinity (Kd = 6.8 × 10^-9) than it did with R1-R4 (Fig 3C).

The YFP-fusion construct of ALP107-273 was expressed in U2OS cells together with the α-actinin cyan fluorescent protein (CFP). In these cells, α-actinin localizes to the leading lamellipodia, to focal adhesions and to actin stress fibres. In contractile stress fibres, α-actinin exhibits a punctate pattern (Fig 4A, D and red in 4C, F). ALP107-273 co-localized with α-actinin in the stress fibres, but no co-localization was seen in either the cell edge or the focal adhesions (Fig 4B, E and green in 4C, F).

Taken together, the SRP interaction measurements and live cell experiments using YFP fusion protein indicated that ALP107-273 interaction with α-actinin was similar to the full length ALP 27.

To study the structure of ALP107-273 we utilized circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The CD spectrum suggested that ALP107-273 is mostly unfolded (Fig 5A). No features characteristic of either α-helical or β-sheet structures were seen. In comparison, the CD spectrum of ALP1-273 showed a slight increase in β-strand content that might be attributed to the PDZ domain (Fig 5A).

The first ¹⁵N-HSQC spectrum measured in normal buffer conditions from uniformly ¹⁵N-labeled ALP107-273 was in line with the CD measurements as it showed the collapsed spectrum characteristic of an unfolded protein (Fig 5B, green). However, we were able to partially stabilize the structure by adding 40 mM Aspartic acid pH 4.0 (Fig. 5B, red) 3637. Under these conditions, iHNCA, HNCA, HNCACO, HNCO, HNCOCA, HNCACB and CBCA(CO)NH spectra were acquired and used for backbone assignment 38. The ALP107-273 fragment contained 167 residues, of which 16 were prolines. All together, 147 of the 151 non-proline residues could be assigned. When the ¹³C chemical shift values of all assigned Cα and Cβ atoms were compared to the average value of the corresponding residues in random coil conformation 39, no clear indications of either α helix or β sheet secondary structures were observed (Fig 6). Thus, NMR analysis indicated that ALP107-273 was mostly unstructured with no secondary structure.

The interaction with the α-actinin rod region could have induced some structural changes in ALP107-273. Assessing this by titration was complicated because the complex between ALP107-273 and the large α-actinin rod dimer of 114 kDa was undetectable by NMR. To be able to measure the spectrum of an unbound ALP107-273 that is in dynamic equilibrium between α-actinin bound and unbound states, we used a rather low concentration of ALP107-273 and observed increasing levels of ALP107-273 ¹⁵N-HSQC peaks upon addition of α-actinin (Fig 5C). Most of the peaks (110, or 75%) could be correlated with those assigned in the presence of 40 mM Aspartic acid. Only a minor fraction of the peaks were induced by ALP dilution alone (Fig 5D). The NMR titration studies suggested that structure of ALP107-273 was partially stabilized by the interaction with the α-actinin rod region.

As NMR studies suggested that ALP107-273 could exist in an elongated, unfolded conformation, even when interacting with α-actinin, we chose to map the interaction site using overlapping synthetic peptides (20–22 residues long). We focused our efforts on the minimum interaction area mapped by truncation mutagenesis, namely ALP residues 151–232, which included the ZM motif 27. The sequences of these peptides are shown in figure 7A. Only peptide 3 (PLEMELPGVKIVHAQFNTPMQL, corresponding to residues 175–196) bound to the immobilized α-actinin rod domain (composed of spectrin repeats R1-R4) in the SPR experiments. A clear interaction was seen with 50 μM and 80 μM peptide concentrations, whereas the other four peptides failed to interact even at 200 μM concentrations (Fig 7B). To verify the interaction of peptide 3 with the rod, peptide 3 was immobilized and the rod was used as the soluble analyte (Fig 7C). The rod interacted with the peptide with apparent K_d of 3.0 × 10^-6 M. However, in contrast to the ALP107-273 fragment, peptide 3 did not interact with R2-R3 (Fig 7C). A scrambled version of peptide 3 (MNKPEQALLVQIHPLVTEPFMG) did not interact with the rod domain, verifying the sequence specificity of the interaction with the rod (Fig 7D). Thus, an area of residues 175–196 containing the N-terminal half of the ZM motif of ALP appeared to interact directly with the α-actinin rod domain, but not with its two central spectrin repeats, R2-R3.

The peptide binding studies suggested that sequences before and at the ZM motif of ALP are sufficient for interaction with α-actinin. To further test if this region was really necessary for the interaction, we constructed five overlapping deletions in ALP107-273 between residues 162 and 244. The deletions were 18–37 amino acids long and each was designed to include a stretch between two proline residues (Fig 7A). Four deletions that overlapped with peptide 3 totally abrogated the ability of ALP107-273 to localize at stress fibres (Fig 8), whereas a deletion after the ZM motif did not affect the localization. As the ZM motif contains conserved tyrosine and serine residues that might be phosphorylated, we also made point mutations where these residues were changed to phenylalanine and alanine, respectively. This mutant also failed to localize to stress fibres (Fig 6). Taken together, the peptide binding studies and the mutant studies suggested that the ZM motif is sufficient and necessary for ALP interaction with α-actinin in the stress fibres.

Internal fragments of human ALP (

AF039018

ALP internal fragments and an α-actinin rod containing the spectrin repeats 1–4 (R1-R4) and a fragment containing spectrin repeats 2 and 3 (R2-R3) were expressed at +37°C for 3–4 h in Escherichia coli BL21(DE3) strain as previously described 27. Briefly, proteins were purified using nickel nitrilotriacetic acid-agarose (Qiagen) as a first step. The His-tag was removed by tobacco etch virus protease (Invitrogen). For further purification, size exclusion chromatography (Superdex 75 16/60 and 26/60 columns, Amersham Biosciences) for ALP, and ion exchange (ProteinPakQ 8HR columns, Waters) for α-actinin fragments were used. The molecular weight standards for the gel filtration were obtained from Bio-Rad Laboratories. For NMR analysis, ¹⁵N-labelled ALP107-273 and ¹⁵N¹³C-labelled ALP107-273 were produced in general M9-media with supplements 49. Expression was induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at +37°C for 8 hrs in E. coli BL21(DE3) strain. Low molecular weight markers used in SDS-polyacrylamide electrophoresis were obtained from Amersham Biosciences. Five ALP peptides were purchased from EZBiolab Inc. (Westfield, IN, USA).

A JASCO J-715 spectrometer (JASCO Corporation, Hachioji City, Tokyo, Japan) was used for circular dichroism measurements. Purified ALP proteins at a concentration of approximately 30 mg/ml were diluted to 0.15 mg/ml with 20 mM Tris, pH 8 buffer. The far-UV spectrum (195–250 nm) was measured at 20°C and the background effect of buffer was subtracted. The instrument settings were as follows: response 1 sec, scan speed 50 nm/min, cell length 0.1 mm, number of scans 16.

The pEYFP ALP107-273 and mutants were transfected into CHO cells as described previously 27. Following 24 hrs transfection, the cells were seeded for 4 hrs on coverslips coated with 10 μg/ml human plasma fibronectin (Sigma) and fixed with 4% paraformaldehyde in 140 mM NaCl, 10 mM Na-Phosphate pH 7.4. The coverslips were coded and the number of cells with YFP localization at stress fibres was counted by two observers who were blinded to the identity of the samples. At least 100 cells were scored from each coverslip, four coverslips were used per transfection, and the experiment was repeated at least three times.

Human osteosarcoma (U2OS) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine, penicillin and streptomycin (Sigma-Aldrich). Transfected cells were re-plated on fibronectin (10 μg/ml) coated glass bottom dishes (MatTek). General growth medium was used as imaging medium. The time lapse images were acquired with an IX-71 inverted microscope (Olympus) equipped with a Polychrome IV monochromator (TILL Photonics) with the appropriate filters, an Andor iXon (Andor) camera, a heated sample environment (+37°C) and CO₂-control. A PLAPON 60xO TIRFM 60x/1,45 (oil) objective (Olympus) was used. Software for image acquisition was TILL Vision version 4 (TILL Photonics).

After defrosting 100 μl of the purified ¹⁵N labelled ALP (1.6 mM) in buffer solution (20 mM TRIS, 150 mM NaCl, 1 mM EDTA and 1 mM DTT, pH 8), a NMR sample of pure ALP was prepared by adding 10% D₂O to the defrosted ALP and transferring the sample to a susceptibility matched Shigemi tube (Shigemi Inc., tube matched to water with 8 mm bottom). Initially, a ¹⁵N-HSQC spectrum of ALP107-273 was acquired as a reference spectrum. The titration series was initiated by preparing a sample containing 45 nmol ALP, 15 nmol rod domain of α-actinin (R1-R4, 277 μM and 236 μM in 20 mM TRIS and 50 mM NaCl, pH 8), 15 μl buffer solution of R1-R4 and 10% D₂O to a susceptibility matched Shigemi tube. A ¹⁵N-HSQC spectrum was measured and the titration was continued in a stepwise manner by adding 15 nmol of R1-R4 to the sample and acquiring a new ¹⁵N-HSQC spectrum at each titration point, until the ratio of 3:8 (ALP:R1-R4) was reached. As a control, titration of ALP was performed with R1-R4 buffer (20 mM TRIS and 50 mM NaCl, pH 8). In order to enable direct comparison of the ¹⁵N HSQC spectra at each data point, identical experimental and processing parameters were employed. Measurements were performed with a Bruker Avance DRX 500 MHz spectrometer (Bruker BioSciences, Billerica, Massachusetts, USA) and a 5 mm triple resonance inverse probehead at room temperature. Measurement of ¹⁵N/¹³C enriched ALP was performed with a Varian Unity INOVA 800 MHz spectrometer (Varian, Palo Alto, California).

A Biacore 3000 system (Biacore, Uppsala, Sweden) was used for surface plasmon resonance (SPR) analysis. Ligand immobilization was performed via amine coupling to gold sensor chips (CM5). The running buffer was 20 mM Tris, pH 7.4, 150 mM NaCl, 0.005% surfactant P20 (BR-1000-54, Biacore AB, Uppsala, Sweden).