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Structure of the S100A4/myosin-IIA complex

  • Udupi A Ramagopal1, 3,
  • Natalya G Dulyaninova1,
  • Kristen M Varney2,
  • Paul T Wilder2,
  • Sridevi Nallamsetty2,
  • Michael Brenowitz1,
  • David J Weber2,
  • Steven C Almo1 and
  • Anne R Bresnick1Email author
BMC Structural Biology201313:31

https://doi.org/10.1186/1472-6807-13-31

Received: 3 July 2013

Accepted: 13 November 2013

Published: 20 November 2013

Abstract

Background

S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood.

Results

We determined the X-ray crystal structure of the S100A4Δ8C/MIIA1908-1923 peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA1908-1923 peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site.

Conclusion

The structure of the S100A4/MIIA1908-1923 peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908–1923 of the myosin-IIA heavy chain represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.

Keywords

X-ray crystallographyNMRS100A4Myosin-IICytoskeletonCoiled-coil

Background

S100A4 is a member of the S100 family of small, dimeric, EF-hand proteins. The S100 proteins, of which there are 21 family members in humans, primarily function as Ca2+-sensors to modulate a multitude of biological processes. The majority of S100 proteins are symmetric dimers characterized by the presence of two EF-hand Ca2+-binding loops per subunit; an N-terminal pseudo EF-hand (EF1) comprising 14 residues and a C-terminal canonical EF-hand (EF2) comprising 12 residues [1, 2]. Calcium-binding to the C-terminal EF-hand induces a structural rearrangement involving the reorientation of helix 3 relative to helix 4, resulting in the exposure of a hydrophobic cleft that forms the binding surface for target proteins [3]. As a consequence, target binding for most S100 proteins is strictly Ca2+-dependent.

The reported equilibrium dissociation constants (KD) for the S100A4/Ca2+ interaction are relatively weak in the absence of target (CaKD = 54 μM, EF1; CaKD = 3.3 μM, EF2) and increase approximately 10-fold in the presence of target (CaKD = 3.6 μM, EF1; CaKD = 0.26 μM, EF2) [4]. Studies with other S100 proteins suggest that the increased Ca2+-binding affinity observed in the presence of target is due to a ligand-induced reduction in S100 backbone and side chain dynamics [5, 6]. Localized target-mediated enhancement of the S100A4/Ca2+ interaction would permit high intracellular S100A4 expression levels (e.g. 3 – 5 μM [7]) without the depletion of free intracellular Ca2+ levels and the disruption of Ca2+ oscillations. Proteomic and localization studies have shown that S100A4 is enriched in the pseudopodia of migrating cells [810]. Moreover a S100A4 biosensor, which reports on Ca2+-bound S100A4, has shown that activated S100A4 localizes to the leading edge of polarized, migrating cells [11, 12]. The enrichment of S100A4 in protrusive structures is consistent with cell-based functional studies demonstrating that S100A4 expression modulates the migratory capacities of a broad range of cell types [7, 9, 13, 14]. Consistent with a role in regulating cell motility, S100A4 has a number of reported cytoskeletal and scaffold protein targets including non-muscle tropomyosin 2, liprin-β1 and non-muscle myosin-IIA [1517].

Non-muscle cells can express three myosin-II heavy chain isoforms (NMHC-IIA – MYH9, NMHC-IIB – MYH10, NMHC-IIC – MYH14), which exhibit 64-80% amino acid identity [18]. Each heavy chain is comprised of an N-terminal motor domain that contains the ATP- and actin-binding sites, an intermediate rod domain that dimerizes to form an α-helical coiled-coil and a C-terminal tailpiece. Despite the fairly high sequence conservation between the non-muscle myosin-II isoforms, studies with assembly-competent myosin-II rod constructs, which lack the myosin-II motor domain, demonstrate that S100A4 preferentially recognizes and disassembles myosin-IIA rods [19, 20]. Although recent reports indicate that S100A4 binds with high affinity to isolated myosin-IIC peptides, S100A4-mediated regulation of myosin-IIC assembly has not yet been examined [21]. For myosin-IIA, studies in mammary adenocarcinoma cells have shown that the S100A4/myosin-IIA interaction regulates the placement of pseudopodial protrusions during chemotactic migration [9]. In addition, macrophages with a genetic deletion for S100A4 exhibit a myosin-IIA overassembly defect that results in impaired cellular protrusions and defective chemotaxis [7]. Together these data support a role for the S100A4/myosin-IIA interaction in modulating pseudopodial structures, and as a consequence, chemotaxis. Consistent with its function in modulating cellular motility, S100A4 overexpression enhances the invasive capabilities and metastatic dissemination of tumor cells [2225].

We report the structure of the Ca2+-S100A4/myosin-IIA complex, which demonstrates an unusual mode of S100 protein target recognition, and is in accordance with the recently described X-ray and NMR structures of S100A4 bound to longer myosin-IIA peptides [21, 26]. In addition, biophysical studies of the Ca2+-S100A4/myosin-IIA complex suggest that instability of the myosin-IIA coiled-coil is a contributing factor to S100A4-mediated myosin-IIA depolymerization.

Results

Interaction of S100A4 with myosin-IIA

Our previous studies showed that S100A4 binds the MIIA1904-1927 peptide with a stoichiometry of one peptide per S100A4 dimer [27]. To examine the interaction of S100A4 with a longer, more physiologically-relevant myosin-IIA construct, we used MIIA1851-1960, which contains approximately 75 residues from the myosin-IIA coiled-coil and the entire C-terminal tailpiece, and encompasses the myosin-II extended assembly competence domain [28, 29]. MIIA1851-1960 inhibits binding of the MIIA1904-1927 peptide to S100A4 with an IC50 of 21.2 ± 2.0 nM (Figure 1A). Circular dichroism spectroscopy of MIIA1851-1960 demonstrated that the peptide is primarily α-helical as evidenced by characteristic minima at 222 and 208 nm (Figure 1B). An examination of the thermal stability of MIIA1851-1960 revealed a relatively modest transition midpoint (Tm) of 27°C for unfolding (Figure 1C), which was reversible (data not shown). The dimeric state of MIIA1851-1960 was confirmed by sedimentation equilibrium studies performed at 22°C (Table 1, Additional file 1: Figure S1). While our thermal denaturation studies suggest some MIIA1851-1960 monomer may be present, we did not detect any monomer in sedimentation equilibrium studies which were performed at higher protein concentrations than the thermal stability assays.
Figure 1
Figure 1

Properties of MIIA 1851-1960 . (A) Competition fluorescence anisotropy measurements of S100A4 binding to MIIA1851-1960, IC50 = 21.2 ± 2.0 nM. Values represent the mean ± standard deviation from two independent experiments. (B) Far-UV spectra of 40 μM MIIA1851-1960 monomer. (C) The thermal stability of MIIA1851-1960 (40 μM monomer concentration) was assayed by monitoring the ellipticity at 222 nm. Data represent the average of two independent experiments.

Table 1

Summary of sedimentation equilibrium data

MIIA 1851-1960: S100A4 a

Predicted MW (Da) b

Experimental MW (Da)

MIIA1851-1960

24,795

25,651 ± 308

0.5 MIIA:1.0 S100A4

35,592

35,271 ± 425

1.0 MIIA:1.0 S100A4

32,893

32,918 ± 490

adimer: dimer ratio.

bPredicted molecular weights are based on sequence, which were confirmed by mass spectrometry; S100A4 dimer – 23,194.6 g/mol; MIIA1851-1960 monomer – 12,397.5 g/mol.

Sedimentation equilibrium studies of the S100A4/MIIA1851-1960 complex using a 0.5:1 molar ratio of MIIA1851-1960 dimer to S100A4 dimer revealed a species with a weight-average molecular mass of 35,271 ± 425 Da, which is consistent with the predicted mass of a S100A4 dimer with a single bound MIIA1851-1960 polypeptide chain (35,592 Da) (Table 1). At a 1:1 molar ratio of MIIA1851-1960 dimer to 1 S100A4 dimer we observed a weight-average molecular mass of 32,918 ± 490 Da, which likely corresponds to a mixture of S100A4 dimers with a single bound MIIA1851-1960 polypeptide chain and MIIA1851-1960 dimers (Table 1).

Given the similarity in the molecular weights of the S100A4 and MIIA1851-1960 dimers, the S100A4/MIIA1851-1960 complex was further evaluated in sedimentation velocity experiments where MIIA1851-1960 was added to S100A4 at increasing molar ratios. The time derivative plots for MIIA1851-1960 alone (Figure 2A) and S100A4 alone (Additional file 1: Figure S2) showed that each protein sedimented as a single homogenous species with S values of 1.91 S and 1.9 S, respectively (Additional file 1: Table S1). At a molar ratio of 0.5:1 MIIA1851-1960 dimer to S100A4 dimer there was a shift in the S value to 2.87 S consistent with the formation of an S100A4/MIIA1851-1960 complex (Figure 2B). At increasing molar ratios of MIIA1851-1960 dimer to S100A4 dimer (1:1, 1.5:1), the best fit for the time derivative plot was to a two-component model. This analysis indicated a peak at 2.89 S that corresponded to the S100A4/MIIA1851-1960 complex and a second peak at 1.9 S, which was consistent with the presence of unperturbed MIIA1851-1960 dimer (Figure 2C and 2D, Additional file 1: Table S1). The 2.89 S species had an estimated mass of 31.5 ± 0.2 kDa. Given the thermal instability of MIIA1851-1960 and our observation that S100A4 forms a thermostable dimer at submicromolar concentrations [4, 27, 30], the 2.89 S species likely corresponds to a S100A4 dimer bound to a single MIIA1851-1960 polypeptide (calculated mass of 35,592 Da), which is consistent with our previous chemical cross-linking studies [27].
Figure 2
Figure 2

Sedimentation velocity of the S100A4/MIIA 1851-1960 complex. Plots of sedimentation coefficient distribution g*(s) versus S20,w for MIIA1851-1960 alone (A) and MIIA1851-1960/S100A4 mixtures at molar ratios of 0.5:1 (B), 1:1 (C), and 1.5:1 (D) of MIIA1851-1960 dimer:S100A4 dimer, which are represented by the open circles. The green line is the best fit to MIIA1851-1960, the blue line is the best fit to the S100A4 dimer/MIIA1851-1960 monomer complex and the red line is the best fit for all species in the S100A4/MIIA1851-1960 mixtures.

Characterization of S100A4Δ8C

Since we were unable to crystallize the wild-type full-length S100A4 with myosin-IIA peptides and our previous structural studies demonstrated that at high protein concentrations residues Glu91-Gln97 mediate interactions between symmetry-related S100A4 dimers in the crystalline state [4], we created a series of S100A4 C-terminal truncations (S100A4Δ3C, S100A4Δ4C, S100A4Δ6C, S100A4Δ7C, S100A4Δ8C, S100A4Δ9C and S100A4Δ13C; where “Δ-number” represents the number of residues deleted from the S100A4 C-terminus) that were expected to reduce S100A4 self-association. The S100A4Δ8C construct was used since it exhibited minimal self-association in the myosin-IIA peptide-bound state as assessed by dynamic light scattering and NMR. The ability of S100A4Δ8C to bind myosin-IIA was assessed in an anisotropy assay using FITC-labeled MIIA1904-1927, which binds wild-type S100A4 with a KD of 0.26 ± 0.03 μM [27]. The measured dissociation constant for S100A4Δ8C (0.51 ± 0.05 μM) was comparable to that observed for the wild-type S100A4 (Figure 3A). In addition, binding was Ca2+-dependent as no binding was observed in the presence of EGTA (data not shown). Using assembly competent myosin-IIA rods (MIIA1339−1960), we monitored the ability of S100A4Δ8C to disassemble preformed myosin-IIA filaments. At a molar stoichiometry of one S100A4Δ8C dimer per myosin-IIA rod, S100A4Δ8C disassembled approximately 85% of the myosin-IIA filaments, which was similar to the disassembly observed in the presence of wild-type S100A4 (Figure 3B and 3C).
Figure 3
Figure 3

S100A4Δ8 exhibits comparable myosin-IIA binding activity to wild-type S100A4. (A) Fluorescence anisotropy measurements of S100A4Δ8 binding to FITC-MIIA1904-1927. Values represent the mean ± standard deviation from two independent experiments. A KD of 0.51 ± 0.05 μM was determined from the fit to a single site saturation binding curve. (B) Representative gel of myosin-IIA disassembly assays performed at a ratio of 1:1 S100A4 dimer:myosin-IIA rod. 1 – myosin-IIA in the absence of S100A4; 2 – myosin-IIA in the presence of S100A4Δ8; 3 – myosin-IIA in the presence of wild-type S100A4 (m = reaction mixture, s = supernatant). (C) Quantification of disassembly assays. Values represent the mean ± standard deviation from two independent experiments.

X-ray Structures of S100A4Δ8C and the S100A4Δ8C/MIIA1908-1923 peptide complex

Both the S100A4Δ8C and S100A4Δ8C/MIIA1908-1923 peptide structures were determined by molecular replacement using the native Ca2+-S100A4 structure (2Q91) truncated at Glu91 as the search model [4] (Table 2). For the apo-S100A4Δ8C structure there were four molecules in the asymmetric unit (two S100A4Δ8C dimers), with continuous density from residues Cys3-Gly92 for all molecules. Residues in the favored, allowed and disallowed regions in the Ramachandran plot account for 97.4%, 2.0% and 0.6% of the total residues, respectively. The S100A4Δ8C/MIIA1908-1923 structure contained a single S100A4 dimer in the asymmetric unit with the MIIA1908-1923 peptide bound asymmetrically across the dimer interface. The electron density was continuous from residues Ala2-Gly92 for both molecules in the asymmetric unit, except for residues Gly47-Arg49 (loop region between helix 2 and 3) of subunit A, which were not modeled. Continuous difference density was observed in difference Fourier syntheses (using Fo-Fc coefficients; contoured at 3σ) following the first round of refinement, indicating the presence of a highly ordered peptide, asymmetrically bound to the S100A4 dimer (Figure 4A and 4B). Residues in the favored, allowed and disallowed regions in the Ramachandran plot account for 96.8%, 2.2% and 1.2% of the total residues, respectively. The two residues in the disallowed region, which deviate slightly from expected values, are Arg49 (subunit B) from the disordered region and the C-terminal Gly92.
Figure 4
Figure 4

Structure of the S100A4Δ8C/MIIA 1908-1923 complex. (A) Ribbon diagram of the S100A4Δ8C/MIIA1908-1923 complex showing the S100A4 subunits in green (subunit A) and brown (subunit B) and the MIIA1908-1923 peptide in blue. (B) Refined model of the S100A4Δ8C/MIIA1908-1923 peptide structure. The myosin-IIA peptide (residues Asp1908-Leu1917) is shown in stick representation superimposed onto the Fo-Fc electron density map, contoured at 3.0σ.

Table 2

Crystallographic data and refinement statistics

 

S100A4Δ8C

S100A4Δ8C/MIIA 1908-1923

PDB ID

4HSZ

4ETO

Source

X29, BNL

X4A, BNL

Wavelength (Å)

1.075

0.979

Resolution limits (Å)

46.9 – 2.25

50 – 1.54

Space group

P1

P21

Unit cell (Å) a, b, c and α, β, γ (°)

a = 28.80, b = 34.36, c = 95.31 and α = 95.48, β = 95.30, γ = 114.82

a = 30.28, b = 91.99, c = 32.86 and β = 112.6

Number of observations

31076

87461

Number of unique reflections

14935

24461

aCompleteness (%)

96.4 (94.1)

99.6 (100)

Mean I/σI

19.6 (2.4)

27.8 (2.76)

bRmerge on I

6.3 (42.2)

6.3 (49.6)

Bwilson2)

30.023

18.4

Refinement statistics

  

Resolution limits (Å)

46.9 – 2.25

46.0 – 1.54

Number of reflections (work/free)

14159/750

23168/1247

Cutoff criteria I/σI

0

0

Protein/water atoms

2904/9

1530/86

Rwork/Rfree (5% of data)

0.228/0.277

0.209/0.251

cBonds (Å)/angles (°)

0.007/0.984

0.020/1.823

Mean B (Å2)

56.2

20.14

aParentheses indicate statistics for the high–resolution data bin for x–ray and refinement data.

b R merge = ∑hkli|I(hkl)i – < I(hkl) > |/∑hkli < I(hkl)i >.

cValues indicate root–mean–square deviations in bond lengths, bond angles, and B-factors of bonded atoms.

The 1.54 Å structure of the S100A4/MIIA1908-1923 complex revealed a single MIIA1908-1923 peptide binding across the S100A4 dimer interface (Figure 4A). Electron density was observed only for residues Asp1908-Leu1921 of the MIIA1908-1923 peptide. As shown in Figure 5A, there is an extensive H-bond network between myosin-IIA residues Asn1911, Ser1915 and Lys1918, and Ser64 and Gln73 on subunit B. In addition peptide residues Glu1913 and Lys1920 form H-bonds with Gln73 and Lys57 of subunit A. Met1910, Val1914, Leu1917 and Leu1921, which correspond to the a and d positions of the myosin-IIA coiled-coil (a – Val1914, Leu1921; d – Met1910, Leu1917), also partcipate in S100A4 binding. Met1910 and Val1914 intercalate between helices 4 and 4′ at the S100A4 dimer interface, while Leu1917 and Leu1921 insert into the hydrophobic cleft of subunit A (Figure 5B).
Figure 5
Figure 5

Interactions within the S100A4Δ8C/MIIA 1908-1923 complex. Binding of the MIIA1908-1923 peptide (blue) to the S100A4Δ8C dimer involves direct electrostatic interactions (A) and water mediated electrostatic interactions (not shown) as well as hydrophobic interactions (B). Hydrogen bonds are shown as red dotted lines.

The total buried surface area upon complex formation is appproximately 1348 Å2, where 684 Å2 and 664 Å2 are contributed by the MIIA1908-1923 peptide and S100A4, respectively. Overall, nearly 380 contacts with a separation of less than 5.0 Å and 8 hydrogen bonds are observed at the myosin-IIA peptide and S100A4 interface. The calculated rms deviations for Cα atoms for residues 3–86 from the Ca2+-bound S100A4 (2Q91) with the Ca2+-bound S100A4Δ8C (4HSZ), the S100A4Δ8C/MIIA1908-1923 peptide complex (4ETO) and the previously reported S100A4/MIIA1893-1935 complex (3ZWH) were 1.2 Å, 1.3 Å and 1.1 Å, respectively; indicating that neither myosin-IIA binding, nor the C-terminal truncation, alters the overall conformation of the S100A4 dimer. The most significant differences were observed in the loop connecting helices 2 and 3 (residues 45–52).

To examine myosin-IIA peptide binding to S100A4Δ8C in solution, the MIIA1908-1923 peptide used in crystallization studies was titrated into a 15N-labeled sample of Ca2+-bound S100A4Δ8C. A comparison of these data to a similar titration performed with wild-type S100A4 [4] revealed that of the 44 correlations shown previously to shift upon MIIA1908-1923 binding to wild-type S100A4, 34 of these chemical shift perturbations were observed in titrations with S100A4Δ8C. These included residues in helix 1 (Cys3 Met12, Val13, Phe16, and Lys18), the pseudo-EF-hand (Gly21 Asn30, Lys31), the hinge (Arg40, Glu41, Leu42, Arg49, Thr50, and Asp51), helix 3 (Glu52, Phe55, Asn56, Leu58, Met59, Ser60 and Leu62), the typical EF-hand (Asp63, Ser64, Asn68, Gln73, and Glu74), helix 4 (Cys76, Val77, Met85, and Cys86), and the C-terminal tail (Asn87, Gln97). Following the addition of the MIIA1908-1923 peptide, differences in chemical shift were also observed in the 1H-15N correlations for Ser14, His17, Ser20, Phe27, Gly47, Leu79, Ala83, Glu88, Phe89, Gly92 and for Phe93, but these changes did not occur or were not the same as perturbations found in titrations with wild-type S100A4. In addition, there were fewer overlapping 1H-15N correlations for the S100A4Δ8C/peptide complex since correlations arising from residues 94–101 were absent in this S100A4 construct. Overall, these NMR data confirm that S100A4Δ8C binds the MIIA1908-1923 peptide in a very similar structure/orientation in solution as observed for wild-type S100A4.

Crystal packing interactions

An examination of the crystal packing interactions for S100A4Δ8C shows that the C-terminal tail (residues Glu88-Glu91) of subunit A interacts with the hydrophobic cleft formed between helices 3 and 4 of the crystallographically related subunit B (Figure 6A) and vice versa. These interactions result in the formation of rods of S100A4 dimers running parallel to the crystallographic b-axis for the AB subunits and parallel to the ab diagonal for the CD subunits (Additional file 1: Figure S3). The inter-dimer contacts between the C-terminal tail of one S100A4 dimer and the target binding cleft of the symmetry related molecule are primarily mediated by Phe89 and Phe90 of the C-terminal tail, and residues Phe45, Leu46, Phe55, and Leu58 in the hinge and helix 3 of the symmetry related molecule (Figure 6B). A comparison of these S100A4 inter-dimer interactions with the S100A4/MIIA1893-1935 structure (3ZWH, Figure 6C) revealed that residues Pro1927-Val1930 of the myosin-IIA peptide adopt a similar type I β-turn as S100A4 residues Glu88-Glu91, with Phe1928 and Val1929 residing deep within the hydrophobic cavity formed by the hinge and helix 3 (Figure 6D). Although the orientation of the S100A4 C-terminal helix (Figure 4D, red) as it approaches the hydrophobic cleft is almost opposite to that of the myosin-IIA peptide (Figure 4D, green), the Cα atoms for Glu88-Glu91 of S100A4 and Pro1927-Val1930 of myosin-IIA have an overall rms deviation of 0.921 Å; demonstrating that when bound in the hydrophobic cleft, the C-terminal tail of S100A4 and myosin-IIA present similar geometric and chemical features.
Figure 6
Figure 6

Self-association of S100A4Δ8 molecules in the crystal lattice. (A) The Ca2+-S100A4Δ8 dimer is shown with one red and one gray subunit. The S100A4Δ8C dimers in the crystal are positioned such that the C-terminal tail of subunit B (red) inserts into the hydrophobic cleft (subunit A-gray) of the adjacent S100A4 dimer. Similarly, the C-terminal tail of subunit A (gray) from the same adjacent dimer inserts into the hydrophobic cleft on subunit B (red). (B) High magnification view of the boxed area in (A). Phe89 and Phe90 of the C-terminal tail are buried inside the hydrophobic cleft of the symmetry-related molecule. (C) Superimposition of the S100A4/MIIA1893-1935 structure (3ZWH; peptide – green, S100A4 dimer – orange and pistachio) onto the S100A4Δ8C/MIIA1908-1923 structure (peptide – blue, S100A4Δ8C dimer – gray and red). (D) Myosin-IIA residues Pro1927-Val1930 (green) from the S100A4/MIIA1893-1935 structure (3ZWH) occupy the same region of the hydrophobic cleft as the C-terminal tail of S100A4Δ8C. S100A4Δ8C residues Glu88-Glu91 (magenta) and myosin-IIA residues Pro1927-Val1930 (green) adopt a similar conformation. Note that the S100A4 (red) and myosin-IIA (green) helices approach the hydrophobic cleft from opposite orientations.

Solution studies of a larger S100A4/MIIA1893-1923 peptide complex

Since MIIA1893-1923 bound S100A4 with a higher affinity than MIIA1908-1923 [4], and exhibited better spectral properties than MIIA1908-1923, we examined this longer peptide in NMR studies to further investigate the solution properties of the asymmetric binding mode observed for myosin-IIA in the S100A4Δ8C/MIIA1908-1923 complex. As expected for a complex with a dissociation constant in the nanomolar range, MIIA1893-1923-bound S100A4 exhibited resonances with slow exchange on the chemical shift timescale. This property resulted in spectra exhibiting narrow linewidths, which is consistent with little, if any, broadening contributions from intermediate exchange. The 1H-15N HSQC of the Ca2+-S100A4/MIIA1893-1923 complex showed that peak doubling occurred for approximately two-thirds of the observable 1H-15N correlations throughout all regions of the protein (Figure 7). The resonance doubling complicated the assignment of backbone and side chain resonances, but it was still possible to sequence-specifically assign all of the observed correlations using standard NMR techniques. The observed peak doubling is consistent with the binding mode of the shorter MIIA1908-1923 peptide in our X-ray structure, in which peptide binding breaks the symmetry of the S100A4 dimer interface that is observed in the Ca2+-bound S100A4, resulting in two distinct electronic shielding environments for several residues.
Figure 7
Figure 7

NMR assignments for MIIA 1893-1923 -bound Ca 2+ -S100A4. (A) A 1H-15N heteronuclear single quantum coherence spectrum (HSQC) of MIIA1893-1923-bound S100A4 shown together with the resonance assignments. (B) Insert of the HSQC spectrum illustrating the assignments for the most crowded region of the HSQC spectrum. The two weak contours labeled with an asterisk (*) were not assigned since they did not have observable correlations to any other residues.

A comparison of the 1H-15N HSQC spectra of the S100A4/MIIA1893-1923 complex (peak doubling) to that of the Ca2+-S100A4 (no peak doubling) revealed that it was not possible to analyze the chemical shift perturbations due to the ambiguities that peak doubling introduced. To address this problem, a Proxyl spin-label was covalently attached to the C-terminus of the MIIA1893-1923 peptide and the distance-dependent effects (1/r6) of paramagnetic relaxation were mapped onto the Ca2+-S100A4 structure (Figure 8) [31]. At the lowest concentration of MIIA1893-1923-Cys-Proxyl peptide (2.6 μM), large reductions in cross-peak intensities were observed for residues in loop 1 (Asp25), the beginning of helix 3 (Asp51, Glu52) and in the C-terminal loop (Lys100). These relaxation effects, plus those observed at the next titration point (5.3 μM), were consistent with the C-terminus of the MIIA1893-1923 peptide being located near residues in helix 3 and the hinge (Leu46, Gly47, Asp51, Glu52; Figure 9A and 9B). Although the cross-peak intensities for residues Arg99 and Lys100 were also significantly reduced by low MIIA1893-1923-Cys-Proxyl peptide concentrations, their exact positions in the Ca2+-S100A4 structure were uncertain since the C-terminal loop is mobile in solution. In addition, these residues were not observed in the Ca2+-S100A4 X-ray structure [4], which is consistent with high structural plasticity and dynamic behavior. Other residues in Ca2+-S100A4 were affected to a lesser degree, and only at higher concentrations of the spin-labeled peptide (i.e. ≥ 21 μM), including residues in helix 1 (Leu5, Glu6, Val13, Ser14, and His17), loop 1 (Lys26), the hinge (Lys48), and helix 3 (Thr50). However, data at higher spin-labeled peptide concentrations were interpreted cautiously because of the potential for outer sphere effects from free spin-labeled peptide [3133]. Altogether, these solution data support the asymmetric binding mode for the myosin-IIA peptide and indicate that the peptide can bind in either of two orientations across the S100A4 dimer interface.
Figure 8
Figure 8

1 H- 15 N heteronuclear single quantum coherence spectrum (HSQC) of full-length Ca 2+ -S100A4 at 600 MHz. Black: S100A4 bound to the MIIA1893-1923 peptide (0.25 mM S100A4 subunit and 0.75 mM MIIA1893-1923). Green: Following the addition of 42 μM MIIA1893-1923-Cys-Proxyl peptide. Residues exhibiting a significant reduction in intensity are labeled.

Figure 9
Figure 9

Paramagnetic relaxation effects observed for 1 H- 15 N HSQC correlations of Ca 2+ -S100A4 in a titration with a MIIA 1893-1923 peptide spin-labeled at its C-terminus. (A) 1H-15N HSQC correlations for Glu23, Asp51, and Lys100 of Ca2+-bound S100A4 in the presence of increasing amounts of spin-labeled MIIA1893-1923 peptide (0, 2.6, 5.3, 11, 21, 42, and 95 μM). (B) Ribbon diagram of Ca2+-bound S100A4 (PDB 2Q91) with residues affected by the spin label highlighted in blue. Residues exhibiting the strongest effects are shown as spheres. Arg99 and Lys100 are not shown on the ribbon diagram since these residues are not observed in the X-ray structure due to the mobility of the C-terminal loop [4].

Discussion

To date, structural studies of S100-target complexes have revealed a variety of binding modes with respect to target recognition, which are typically characterized by the formation of symmetric complexes with a 1:1 S100 subunit:target stoichiometry (Figure 10) [3439]. For S100B and S100A1-target complexes, target binding is primarily mediated by interactions with the hydrophobic cleft formed by helices 3 and 4 of the S100 protein, although the orientation of the target peptide within the hydrophobic cleft can vary significantly [3436]. Other target binding modes include interactions with helix 4 of one S100 subunit and helix 1′ of the second S100 subunit as observed for S100A10 and S100A11-target complexes [38, 39], and in the case of the S100A6/SIP complex, target binding involves bidentate interactions with the hydrophobic cleft of one S100 subunit and helix 1′ of the second S100 subunit [37]. The structure of the S100A4Δ8C/MIIA1908-1923 peptide complex demonstrates a fourth binding mode, in which the myosin-IIA peptide binds asymmetrically across helices 4 and 4′ at the S100A4 dimer interface. Asymmetric target binding is emerging as a common feature amongst the S100 protein family as this binding mode is also observed for AHNAK bound to the S100A10/annexin A2 complex and SMARCA3 bound to the S100A10/annexin A2 complex [4042].
Figure 10
Figure 10

Comparison of six S100-target complexes. (A) S100B-p53 (1DT7); (B) S100B-NDR kinase (1PSB); (C) S100A1-TRTK12 (2KBM); (D) S100A10-annexin2 (1BT6); (E) S100A6-SIP (2JTT) and (F) S100A10-annexin2-AHNAK (4DRW). The S100 subunits are shown in green and tan, and the peptide ligand in blue. For the S100A10-annexin2-AHNAK ternary complex, the annexin2 peptide is shown in orange.

The recent structures of S100A4 bound to longer myosin-IIA peptides have demonstrated that the N- and C-termini of the peptide insert into the hydrophobic clefts formed by helices 3 and 4 of each S100A4 subunit to mediate high affinity binding [21, 26]; however, our structural studies indicate that a significantly shorter myosin-IIA peptide is sufficient to promote an asymmetric binding mode. A contributing factor to the asymmetric disposition of the myosin-IIA peptide is that it makes contacts with dimer-related residues on helices 4 and 4′ of each S100 subunit. In particular, Val77, Cys81 and Met84 of each S100A4 subunit form interactions with myosin-IIA that position the peptide nearly orthogonally across the dimer interface. Our previous biochemical studies support the contribution of these residues to myosin-IIA binding as substitution of Cys81 with Ala or Ser reduces S100A4 binding to myosin-IIA by 20- and 55-fold, respectively, and disrupts S100A4-mediated myosin-IIA disassembly [43].

In addition to crystallographic and biochemical data, NMR data supports a model in which residues 1908 to 1923 of the myosin-IIA heavy chain represent the core sequence for S100A4 recognition. In this regard, we examined whether this small peptide was sufficient to induce what is termed the “final folding” event that occurs upon S100 protein-target complex formation [6]. We examined whether conformational exchange in S100A4, which is observed in the absence of bound target, could be stabilized upon binding the MIIA1908-1923 peptide. As found for other wild-type and mutant S100 proteins [6, 44], significant exchange broadening was observed in the Ca2+-bound S100A4Δ8C. Notably, this exchange broadening completely disappeared upon S100A4Δ8C/MIIA1908-1923 complex formation (data not shown). As with other S100 proteins, this behavior likely accounts for the higher Ca2+-binding affinity observed for S100A4 when bound to MIIA1908-1923 [4]. This may be considered analogous to a “mini-folding” event in which target binding shifts the S100A4 equilibrium population from a highly dynamic, but weak Ca2+-binding state to a high affinity Ca2+ binding state that is composed of an S100A4 population with a narrower distribution of dynamic features. While the regulation of S100A4 protein dynamics upon target binding will be the subject of future studies, it is important to note that the MIIA1908-1923 peptide represents the core of the S100A4/myosin-IIA interaction since it is adequately sized and positioned to eliminate conformational exchange upon binding either S100A4Δ8C or wild-type protein [4]. Importantly, as most target-free S100 proteins have a relatively low affinity for Ca2+ (i.e., versus bound to target) this allows for numerous S100 proteins to exist at high cellular concentrations (> 1 μM) without significantly depleting intracellular [Ca2+]free levels and short-circuiting Ca2+ oscillations. Thus, reasonably high S100A4 levels (e.g., 3–5 μM [7]) can be poised inside the cell as myosin-IIA becomes available for complex formation and downstream cytoskeletal modulation.

An interesting feature of the S100A4/myosin-IIA complex is that myosin-IIA residues contributing to the coiled-coil interface also mediate S100A4 binding. These observations suggest a mechanism in which S100A4 binding induces partial unwinding of the myosin-IIA coiled-coil, and thus promotes myosin-IIA filament disassembly. The relatively low Tm (45°C) of the myosin-IIA rod (residues 1102–1960) [45], and the relative instability of the coiled-coil region that encompasses the S100A4 binding site suggests that this region of the myosin-IIA coiled-coil likely undergoes significant thermally-driven structural fluctuations (Figure 1C) [21, 45]. Modest fluctuations in the structure of the coiled-coil would allow initial docking of S100A4 onto a single myosin-IIA polypeptide chain followed by partial wrapping of the myosin-IIA heavy chain across the S100A4 dimer and further unzipping of the coiled-coil. The free myosin-IIA heavy chain would then be available for subsequent capture by a second S100A4 dimer. Although turbidity studies with short myosin-IIA rod constructs (residues 1712–1960 and 1761–1960) support a binding stoichiometry of one S100A4 dimer per myosin-IIA polypeptide chain [21, 46]; sedimentation assays with longer myosin-IIA rod constructs (residues 1339–1960) have shown that a stoichiometry of one S100A4 dimer to two myosin-IIA polypeptide chains (one myosin-IIA heavy chain dimer) is sufficient to promote maximal filament disassembly [19, 43]. While S100A4 binds with low nanomolar affinities to isolated myosin-IIA peptides and short, relatively unstable myosin-IIA rod constructs, binding to longer myosin-IIA rods or full-length myosin-IIA appears to be significantly weaker [19, 47]. These observations suggest that the loss of energy due to myosin-IIA heavy chain unzipping can modulate S100A4 binding affinity, as a significant fraction of the binding energy must be used to drive the dissociation of the two polypeptide chains in the myosin-IIA rod. Future studies directed at examining the requirement for the binding of one versus two S100A4 dimers per myosin-IIA heavy chain dimer for coiled-coil unzipping, as well as the reduction in free energy that occurs during unzipping will require the use of longer and more stable myosin-IIA coiled-coil constructs.

This proposed model of S100A4-mediated myosin-II heavy chain unzipping is analogous to the binding of single-stranded DNA binding protein and ribosomal protein S1 to single-stranded DNA and RNA segments, respectively, that transiently form during thermal breathing of the nucleic acid base pairs [48, 49]. While the detailed mechanisms of polymer unzipping associated with these nucleic acid binding proteins will be different due to their multi-domain nature and/or oligomeric state, these biological systems provide a conceptual framework for considering the mechanism of S100A4 coiled-coil unzipping.

Conclusions

Direct visualization of the S100A4/myosin-IIA complex has defined the chemical determinants required for myosin-IIA binding and the overall organization of the complex. These studies have identified residues 1908–1923 of the myosin-IIA heavy chain as a core sequence for formation of the S100A4/myosin-IIA complex. Moreover, these studies highlight the potential role of structural dynamics within the myosin-IIA coiled-coil and S100A4 in mediating the Ca2+-dependent S100A4/myosin-IIA interaction.

Methods

S100A4 purification

For the production of the untagged, full-length protein, the codon-optimized human S100A4 sequence was subcloned into the Nde1/BamH1 sites of pET11b (Novagen). The S100A4Δ8C deletion mutant (deletion of the C-terminal eight amino acids) was subcloned into the Nde1/BamH1 sites of pET11b. BL21(DE3)* cells were transformed with the codon-optimized human wild-type or Δ8C S100A4 and cultures were grown in Luria broth at 37°C to an OD600 of 0.8-1.0. Protein expression was induced with 0.7 mM IPTG and the cultures were grown for an additional 18–22 hrs at 27°C. The cells were harvested at 8000 rpm for 10 min, and the cell pellets were resuspended in lysis buffer (50 mM Tris pH 7.5, 10% glycerol, 300 mM KCl, 5 mM DTT, 1 mM EDTA, 1 mM PMSF and 5 μg/ml each of chymostatin, leupeptin, and pepstatin). The cell lysates were frozen at −80°C, thawed on ice, and sonicated. Following centrifugation of the lysate at 30000 g for 30 min, ammonium sulfate was added to the supernatant to 43% saturation, the sample was stirred for 20 min on ice and centrifuged at 30000 g for 10 min. CaCl2 was added to the supernatant to a final concentration of 2 mM, and the sample was applied to a Phenyl-Sepharose column (GE Healthcare) equilibrated in buffer P (20 mM Tris pH 7.5, 2 mM CaCl2, 300 mM KCl, 5 mM DTT, 1 mM EDTA, and 0.02% NaN3) containing 25 g ammonium sulfate/100 ml. The column was washed with 3 column volumes of buffer P containing 25 g ammonium sulfate/100 ml and then with 3 column volumes of buffer P without ammonium sulfate, and S100A4 was eluted with 20 mM Tris pH 7.5, 5 mM EGTA, 2 mM DTT, 2 mM TCEP, 1 mM EDTA and 0.02% NaN3. Fractions containing S100A4 were pooled and dialyzed against 2.0 L of buffer Q (20 mM Tris pH 7.5, 2 mM DTT, 2 mM TCEP and 0.02% NaN3). The dialyzed pool was applied to a Hi Prep 16/10 QXL column (GE Healthcare) and the column washed with 3 column volumes of buffer Q followed by a 200 ml gradient of 0–0.5 M NaCl in buffer Q. The purified proteins were stored at −80°C. For crystallization, S100A4 was gel-filtered on a HiLoad Superdex 75 26/60 column (GE Healthcare) and concentrated using a Vivaspin 15R ultrafiltration spin column (Sartorius Stedium, Life Technology).

For NMR studies, 15N-labeled wild-type S100A4 was purified from bacterial cells grown in M9 media containing 15N NH4Cl as described previously [4].

Myosin-IIA peptides

The FITC-Ahx-TETADAMNREVSSLKNKLRRGDLP-CONH2 (FITC-MIIA1904-1927), Ace-TETADAMNREVSSLKNKLRRGDLP-CONH2 (MIIA1904-1927) and Ace-DAMNREVSSLKNKLRR-CONH2 (MIIA1908-1923) peptides were synthesized by Bio-Synthesis Inc. to a purity of >95% as determined by HPLC and mass spectrometry. A 5 mM stock solution of FITC-MIIA1904-1927 was prepared in 20 mM Tris pH 7.5, 150 mM KCl, 1 mM DTT and 0.02% NaN3, aliquoted and stored at −80°C. The concentration of the FITC-MIIA1904-1927 peptide was determined using the extinction coefficient for FITC. The concentrations of the MIIA1908-1923 and MIIA1904-1927 peptides were determined by quantitative amino acid analysis (Keck Biotechnology Resource Laboratory at Yale University, New Haven, CT). A 25 mM stock solution of MIIA1908-1923 was prepared in 10 mM Tris pH 7.5, 10 mM NaCl, and 0.02% NaN3, aliquoted and stored at −80°C. For NMR studies, two peptides Ac-RRKLQRELEDATETADAMNREVSSLKNKLRR-CONH2 (MIIA1893-1923) and Ac-RRKLQRELEDATETADAMNREVSSLKNKLRRC-CONH2 (MIIA1893-1923-Cys), which contained an additional Cys on the C-terminus were made by solid-state peptide synthesis to a purity of >95% as determined by HPLC and mass spectrometry. Stock peptide solutions were prepared in metal free double distilled H2O and concentrations were determined by quantitative amino acid analysis (BioSynthesis Inc.).

Myosin-IIA proteins

A codon-optimized C-terminal fragment of the human myosin-IIA heavy chain that corresponds to residues 1851–1960 (MIIA1851-1960) was cloned into the NdeI/EcoR1 sites of pET26b and expressed in BL21(DE3)* cells using TB autoinducing media. MIIA1851-1960 was purified by sequential chromatography on High Q Sepharose anion exchange (GE Healthcare) and Bio-Scale Ceramic Hydroxyapatite Type I (Bio-Rad) columns as described previously [4] and then gel-filtered on a Sephacryl S200 26/60 column (GE Healthcare). The MIIA1851-1960 protein concentration was determined using the Bradford protein assay (Bio-Rad) and a MIIA1851-1960 standard of known concentration. The concentration of the MIIA1851-1960 standard was determined by amino acid analysis (Keck Biotechnology Resource Laboratory at Yale University, New Haven, CT). The mass of MIIA1851-1960 was confirmed by mass spectrometry.

Peptide spin labeling

For the MIIA1893-1923-Cys peptide, 25 μl of a 29.77 mM stock peptide solution was added to 92 μl of metal free (Chelex-100 treated) double distilled H2O, 20 μl 500 mM TES pH 7.2, 2 μl 500 mM EDTA and incubated with pre-washed immobilized TCEP disulfide reducing gel (Pierce) for >1 hour at room temperature. The beads containing the immobilized TCEP and other particulates were removed using a 0.45 μm Nanosep MF GHP centrifugal device (Pall Life Sciences) before the addition of 61 μl freshly prepared 50 mg/ml 3-maleimido-Proxyl (Sigma-Aldrich) in DMSO. Purification of the Proxyl-labeled peptide (MIIA1893-1923-Cys-Proxyl) was performed by separation of the unlabeled and labeled peptides on a C18 column using a 0-90% acetonitrile gradient and monitoring at 214 nm and 280 nm. The unlabeled peptide does not absorb at 280 nm, whereas the Proxyl-labeled peptide has an extinction coefficient of 500 M-1 cm-1 at 280 nm. The reaction was complete in 4 hours at room temperature and further incubation resulted in the production of second labeled form of the peptide, likely from the slower modification of methionine by the 3-maleimido- Proxyl [50]. The purified MIIA1893-1923-Cys-Proxyl peptide (>99%) was resuspended in D6-DMSO (Cambridge Isotopes Labs) and the peptide concentration was determined using the extinction coefficient of the Proxyl label.

S100A4 crystallization

S100A4Δ8C was freshly dialyzed against 20 mM Tris pH 7.5, 150 mM KCl, 1 mM DTT, 0.02% NaN3, and 0.5 mM CaCl2 and clarified by ultracentrifugation at 80000 rpm for 10 minutes at 22°C in TLA 120.2 rotor. S100A4 protein concentrations were determined using the Bradford protein assay (Bio-Rad) and a S100A4 standard of known concentration. The concentration of the S100A4 standard was determined by quantitative amino acid analysis (Keck Biotechnology Resource Laboratory at Yale University, New Haven, CT).

S100A4Δ8C was mixed with the MIIA1908-1923 peptide in 1:1 ratio of S100A4 subunit per peptide and incubated for 1 hour at room temperature. Diffraction quality crystals of the complex were obtained by sitting drop vapor diffusion at 20°C by mixing 1 μl of the protein complex (15.5 mg/mL) with 1 μl of reservoir solution containing 0.1 M HEPES pH 7.0 and 30% (v/v) Jeffamine M-600. Rod-shaped crystals appeared after 3–4 days, which were flash cooled in liquid nitrogen without any additional cryo-protectant. Ca2+-bound S100A4Δ8C crystals were grown using a similar protocol except the reservoir solution contained 0.1 M HEPES pH 7.0, 0.1 M magnesium chloride, and 15% PEG 4000. Thin-plate shaped crystals appeared in one week and the crystals were harvested with mother liquor supplemented with 15% glycerol as a cryoprotectant for flash-cooling. Data were collected at beamlines X4A and X29A (Brookhaven National laboratory) for the S100A4Δ8C/MIIA1908-1923 peptide and Ca2+-bound S100A4Δ8C crystals, respectively. Data were integrated and scaled with HKL2000 [51]. The S100A4Δ8C/MIIA1908-1923 peptide complex crystals exhibited diffraction consistent with the space group P21 (a = 30.28 Å, b = 91.99 Å, c = 32.86 Å, β = 112.6°) and apo-S100A4Δ8C crystals were triclinic with space group P1 (a = 28.80 Å, b = 34.36 Å, c = 95.31 Å, α = 95.48°, β = 95.30°, γ = 114.82°).

Structure determination

The S100A4Δ8C and S100A4Δ8C/MIIA1908-1923 peptide structures were determined by molecular replacement to 2.5 Å and 1.54 Å, respectively using the program MOLREP [52] and the Ca2+-S100A4 structure (PDB 2Q91) as the search model. Iterative cycles of model building and refinement were performed using COOT [53] and REFMAC5 [54]. For the S100A4Δ8C/MIIA1908-1923 peptide structure, continuous difference density was observed in the Fo-Fc map contoured at the 3σ level following the first round of refinement, indicating the presence of a highly ordered peptide, asymmetrically bound to the S100A4 dimer. The final S100A4Δ8C/MIIA1908-1923 peptide model contained 2 S100A4 subunit chains (Ala2-Gly92) (named A and B), 1 myosin-IIA peptide, 4 calcium ions and 86 water molecules, resulting in a Rwork and Rfree of 20.9% and 25.1%, respectively (PDB 4ETO). The final Ca2+-S100A4Δ8C model contained 4 subunit chains (Cys3-Gly92), 8 calcium ions and 10 water molecules with a Rwork and Rfree of 22.8% and 27.7%, respectively (PDB 4HSZ).

Anisotropy assays

Fluorescence anisotropy measurements were performed at 22°C using a Fluoromax-3-spectrofluorometer (Jobin Yvon). Individual reactions (200 μl) contained 50 nM FITC-MIIA1904-1927 and 0–30 μM S100A4Δ8C dimer in 20 mM Tris pH 7.5, 150 mM KCl, 1 mM DTT, 0.02% NaN3, 0.5 mM CaCl2 and 0.5 mg/ml BSA. For competition anisotropy measurements individual reactions contained 50 nM FITC-MIIA1904-1927, 0.3 μM S100A4 dimer and 0–10 μM MIIA1851-1960 dimer as described above. Controls included experiments representing maximum anisotropy (15 μM wild-type S100A4 and S100A4Δ8C, 50 nM FITC-MIIA1904-1927 and calcium) and minimum anisotropy (in the absence of S100A4 and in the presence of S100A4 and 5 mM EGTA). Anisotropy was measured using excitation and emission wavelengths of 490 nm and 520 nm, respectively. Measurements were acquired at the magic angle of 55° between the vectors of polarization of the excitation and emission light using a G factor of 0.634 as determined previously for FITC on this instrument. For measurements of S100A4Δ8C binding, data from two independent experiments were plotted using Graphpad Prism v5, and the dissociation constant was calculated by fitting to a single site saturation binding equation allowing for a floating Ymin value [11]. For competition anisotropy assays data from two independent experiments were plotted using Graphpad Prism v5, and fit to a sigmoidal dose–response equation with a variable slope to obtain the IC50 value.

Promotion of disassembly assays

Assays comparing the ability of wild-type and the Δ8C S100A4 to depolymerize assembled myosin-IIA rods were performed as described in Li et al. [19], using 1.5 μM S100A4 dimer and 1.5 μM myosin-IIA rod dimer in 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 2 mM MgCl2, 0.5 mM CaCl2, and 0.02% NaN3. Reaction mixtures (100 μl) were incubated for 2 hr at 22°C and then centrifuged at 80,000 rpm (175000 g) for 10 min at 25°C in a TL-100 ultracentrifuge (Beckman). Samples of the reaction mixtures and supernatants were separated on a 12% Tris-Tricine SDS-polyacrylamide gel. Coomassie-stained gels were scanned and the extent of myosin-IIA polymerization was quantified by densitometry and analyzed with the ImageQuant version 5.2.

NMR spectroscopy

The Ca2+-loaded S100A4Δ8C/MIIA1908-1923 and S100A4/MIIA1893-1923 samples were prepared as described previously [4] and contained 0.25 mM S100A4 subunit and 0.75 mM MIIA peptide in 10 mM CaCl2, 0.35 mM NaN3, 10 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 10% D2O, and 10 mM Tris, and the pH was brought to 6.5 with HCl. The MIIA1893-1923-Cys-Proxyl peptide was titrated into the sample, and spectral changes were monitored after each addition via the 15N- HSQC signal. HSQC NMR data were collected at 37°C with a 600 MHz (600.13 MHz for protons) Bruker DMX NMR spectrometer equipped with pulsed-field gradients, four frequency channels, and a triple resonance, z-axis gradient cryogenic probe, Data were processed with NMRPipe [55], and proton chemical shifts were reported with respect to the H2O or HDO signal taken as 4.658 ppm relative to external trimethylsilylpropionic acid (0.0 ppm). The 15 N chemical shifts were indirectly referenced as described previously using the following ratio of the zero-point frequency: 0.10132905 for 15 N to 1H [56].

Circular dichroism spectroscopy

MIIA1851-1960 was dialyzed into 20 mM Tris pH 7.5, 150 mM KCl, 0.5 mM CaCl2, 0.02% NaN3 and clarified by ultracentrifugation at 80,000 rpm for 10 minutes at 4°C in TLA 120.2 rotor. MIIA1851-1960 (40 μM monomer concentration) was transferred to a 1 mm path-length quartz cuvette and scanned in Jasco J-815 Spectrometer equipped with a PFD-425 temperature controller. Spectra from 190 to 260 nm were obtained at 22°C using a scan speed of 100 nm/minute, a 1 nm bandwidth, and a sensitivity of 100 mdeg. Thermal melt curves were obtained by monitoring ellipticity at 222 nm while the sample temperature was increased from 2 – 85°C in 2°C intervals at a rate of 60°C/h, using a 1 nm bandwidth. The data were fit to a sigmoidal dose–response with a variable slope using Graphpad Prism v5.

Analytical ultracentrifugation

Sedimentation equilibrium and velocity experiments were conducted at 22°C in a Beckman XL-I analytical ultracentrifuge using a Ti60 rotor and Raleigh interference optics. Wild-type S100A4 and MIIA1851-1960 were dialyzed into 20 mM Tris pH 7.5, 150 mM KCl, 0.5 mM CaCl2, 1 mM TCEP and 0.02% NaN3 and clarified by ultracentrifugation at 80,000 rpm for 10 minutes at 4°C in TLA 120.2 rotor. Sedimentation equilibrium analysis of MIIA1851-1960 and S100A4 was conducted in six channel centerpieces by sequentially equilibrating the protein at three concentrations for 24 hr at 12,000, 20,000 and 28,000 rpm and globally analyzing the data using HeteroAnalysis v1.1.44 (James L. Cole and Jeffrey W. Lary). The sedimentation velocity experiments were conducted at 53,000 rpm using double sector centerpieces and analyzed using DCDT + v2.4.0 [57, 58]. The best-fit parameters and their 95% joint confidence intervals are reported. The calculated masses of the wild-type S100A4 and MIIA1851-1960 monomers are 11,597.3 and 12,397.5 Da, respectively. Values of ν-bar = 0.715 for S100A4/MIIA851-1960 and 0.735 for S100A4, ρ = 1.0059, μ = 1.00586 cp were calculated using Sednterp v1.06 (Hayes, B., T. Laue & J. Philo, Sedimentation Interpretation Program. 2003, University of New Hampshire) from the amino acid and buffer compositions, respectively.

Abbreviations

MIIA: 

Non-muscle myosin-IIA

EF: 

EF-hand

FITC: 

Fluorescein isothiocyanate

DTT: 

Dithiothreitol

TCEP: 

Tris (2-carboxyethyl) phosphine.

Declarations

Acknowledgements

This work was supported with National Institutes of Health grants CA129598 (ARB), GM094662 (SCA), GM094665 (SCA), GM58888 (DJW), CA107331 (DJW) and P30CA13330 to the Albert Einstein College of Medicine Cancer Center.

Authors’ Affiliations

(1)
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, USA
(2)
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA
(3)
Division of Biological Sciences, Bangalore, India

References

  1. Zimmer DB, Cornwall EH, Landar A, Song W: The S100 protein family: history, function, and expression. Brain Res Bull 1995, 37(4):417–429. 10.1016/0361-9230(95)00040-2View ArticlePubMedGoogle Scholar
  2. Marenholz I, Heizmann CW, Fritz G: S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochem Biophys Res Commun 2004, 322(4):1111–1122. 10.1016/j.bbrc.2004.07.096View ArticlePubMedGoogle Scholar
  3. Zimmer DB, Wright Sadosky P, Weber DJ: Molecular mechanisms of S100-target protein interactions. Microsc Res Tech 2003, 60(6):552–559. 10.1002/jemt.10297View ArticlePubMedGoogle Scholar
  4. Malashkevich VN, Varney KM, Garrett SC, Wilder PT, Knight D, Charpentier TH, Ramagopal UA, Almo SC, Weber DJ, Bresnick AR: Structure of Ca2 + −bound S100A4 and its interaction with peptides derived from nonmuscle myosin-IIA. Biochemistry 2008, 47(18):5111–5126. 10.1021/bi702537sPubMed CentralView ArticlePubMedGoogle Scholar
  5. Wright NT, Inman KG, Levine JA, Cannon BR, Varney KM, Weber DJ: Refinement of the solution structure and dynamic properties of Ca(2+)-bound rat S100B. J Biomol NMR 2008, 42(4):279–286. 10.1007/s10858-008-9282-yPubMed CentralView ArticlePubMedGoogle Scholar
  6. Liriano MA, Varney KM, Wright NT, Hoffman CL, Toth EA, Ishima R, Weber DJ: Target binding to S100B reduces dynamic properties and increases Ca(2+)-binding affinity for wild type and EF-hand mutant proteins. J Mol Biol 2012, 423(3):365–385. 10.1016/j.jmb.2012.07.011PubMed CentralView ArticlePubMedGoogle Scholar
  7. Li ZH, Dulyaninova NG, House RP, Almo SC, Bresnick AR: S100A4 regulates macrophage chemotaxis. Mol Biol Cell 2010, 21(15):2598–2610. 10.1091/mbc.E09-07-0609PubMed CentralView ArticlePubMedGoogle Scholar
  8. Kim EJ, Helfman DM: Characterization of the metastasis-associated protein, S100A4. Roles of calcium binding and dimerization in cellular localization and interaction with myosin. J Biol Chem 2003, 278(32):30063–30073. 10.1074/jbc.M304909200View ArticlePubMedGoogle Scholar
  9. Li ZH, Bresnick AR: The S100A4 metastasis factor regulates cellular motility via a direct interaction with myosin-IIA. Cancer Res 2006, 66(10):5173–5180. 10.1158/0008-5472.CAN-05-3087View ArticlePubMedGoogle Scholar
  10. Wang Y, Ding SJ, Wang W, Jacobs JM, Qian WJ, Moore RJ, Yang F, Camp DG 2nd, Smith RD, Klemke RL: Profiling signaling polarity in chemotactic cells. Proc Natl Acad Sci U S A 2007, 104(20):8328–8333. 10.1073/pnas.0701103104PubMed CentralView ArticlePubMedGoogle Scholar
  11. Garrett SC, Hodgson L, Rybin A, Toutchkine A, Hahn KM, Lawrence DS, Bresnick AR: A biosensor of S100A4 metastasis factor activation: inhibitor screening and cellular activation dynamics. Biochemistry 2008, 47(3):986–996. 10.1021/bi7021624PubMed CentralView ArticlePubMedGoogle Scholar
  12. House RP, Garrett SC, Bresnick AR: Moving Aggressively: S100A4 and tumor invasion. In Signaling Pathways and Molecular Mediators in Metastasis, Volume part 1. Edited by: Fatatis A. Netherlands: Springer; 2012:91–113.Google Scholar
  13. Takenaga K, Nakamura Y, Endo H, Sakiyama S: Involvement of S100-related calcium-binding protein pEL98 (or mts1) in cell motility and tumor cell invasion. Jpn J Cancer Res 1994, 85(8):831–839. 10.1111/j.1349-7006.1994.tb02955.xView ArticlePubMedGoogle Scholar
  14. Jenkinson SR, Barraclough R, West CR, Rudland PS: S100A4 regulates cell motility and invasion in an in vitro model for breast cancer metastasis. Br J Cancer 2004, 90(1):253–262. 10.1038/sj.bjc.6601483PubMed CentralView ArticlePubMedGoogle Scholar
  15. Takenaga K, Nakamura Y, Sakiyama S, Hasegawa Y, Sato K, Endo H: Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin. J Cell Biol 1994, 124(5):757–768. 10.1083/jcb.124.5.757View ArticlePubMedGoogle Scholar
  16. Kriajevska M, Fischer-Larsen M, Moertz E, Vorm O, Tulchinsky E, Grigorian M, Ambartsumian N, Lukanidin E: Liprin beta 1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, is a new target for the metastasis-associated protein S100A4 (Mts1). J Biol Chem 2002, 277(7):5229–5235. 10.1074/jbc.M110976200View ArticlePubMedGoogle Scholar
  17. Kriajevska MV, Cardenas MN, Grigorian MS, Ambartsumian NS, Georgiev GP, Lukanidin EM: Non-muscle myosin heavy chain as a possible target for protein encoded by metastasis-related mts-1 gene. J Biol Chem 1994, 269(31):19679–19682.PubMedGoogle Scholar
  18. Golomb E, Ma X, Jana SS, Preston YA, Kawamoto S, Shoham NG, Goldin E, Conti MA, Sellers JR, Adelstein RS: Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family. J Biol Chem 2004, 279(4):2800–2808.View ArticlePubMedGoogle Scholar
  19. Li Z-H, Spektor A, Varlamova O, Bresnick AR: Mts1 regulates the assembly of nonmuscle myosin-IIA. Biochemistry 2003, 42(48):14258–14266. 10.1021/bi0354379View ArticlePubMedGoogle Scholar
  20. Mitsuhashi M, Sakata H, Kinjo M, Yazawa M, Takahashi M: Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. J Biochem 2011, 149(3):253–263. 10.1093/jb/mvq134View ArticlePubMedGoogle Scholar
  21. Kiss B, Duelli A, Radnai L, Kekesi KA, Katona G, Nyitray L: Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism. Proc Natl Acad Sci U S A 2012, 109(16):6048–6053. 10.1073/pnas.1114732109PubMed CentralView ArticlePubMedGoogle Scholar
  22. Davies BR, Davies MP, Gibbs FE, Barraclough R, Rudland PS: Induction of the metastatic phenotype by transfection of a benign rat mammary epithelial cell line with the gene for p9Ka, a rat calcium-binding protein, but not with the oncogene EJ-ras-1. Oncogene 1993, 8(4):999–1008.PubMedGoogle Scholar
  23. Davies MP, Rudland PS, Robertson L, Parry EW, Jolicoeur P, Barraclough R: Expression of the calcium-binding protein S100A4 (p9Ka) in MMTV-neu transgenic mice induces metastasis of mammary tumours. Oncogene 1996, 13(8):1631–1637.PubMedGoogle Scholar
  24. Ambartsumian NS, Grigorian MS, Larsen IF, Karlstrom O, Sidenius N, Rygaard J, Georgiev G, Lukanidin E: Metastasis of mammary carcinomas in GRS/A hybrid mice transgenic for the mts1 gene. Oncogene 1996, 13(8):1621–1630.PubMedGoogle Scholar
  25. Xue C, Plieth D, Venkov C, Xu C, Neilson EG: The gatekeeper effect of epithelial-mesenchymal transition regulates the frequency of breast cancer metastasis. Cancer Res 2003, 63(12):3386–3394.PubMedGoogle Scholar
  26. Elliott PR, Irvine AF, Jung HS, Tozawa K, Pastok MW, Picone R, Badyal SK, Basran J, Rudland PS, Barraclough R, et al.: Asymmetric mode of Ca(2)(+)-S100A4 interaction with nonmuscle myosin IIA generates nanomolar affinity required for filament remodeling. Structure 2012, 20(4):654–666. 10.1016/j.str.2012.02.002PubMed CentralView ArticlePubMedGoogle Scholar
  27. House RP, Pozzuto M, Patel P, Dulyaninova NG, Li ZH, Zencheck WD, Vitolo MI, Weber DJ, Bresnick AR: Two functional S100A4 monomers are necessary for regulating nonmuscle myosin-IIA and HCT116 cell invasion. Biochemistry 2011, 50(32):6920–6932. 10.1021/bi200498qPubMed CentralView ArticlePubMedGoogle Scholar
  28. Sohn RL, Vikstrom KL, Strauss M, Cohen C, Szent-Gyorgyi AG, Leinwand LA: A 29 residue region of the sarcomeric myosin rod is necessary for filament formation. J Mol Biol 1997, 266(2):317–330. 10.1006/jmbi.1996.0790View ArticlePubMedGoogle Scholar
  29. Cohen C, Parry DA: A conserved C-terminal assembly region in paramyosin and myosin rods. J Struct Biol 1998, 122(1–2):180–187.View ArticlePubMedGoogle Scholar
  30. Vallely KM, Rustandi RR, Ellis KC, Varlamova O, Bresnick AR, Weber DJ: Solution structure of human mts1 (S100A4) as determined by NMR spectroscopy. Biochemistry 2002, 41(42):12670–12680. 10.1021/bi020365rView ArticlePubMedGoogle Scholar
  31. Mahoney NM, Rastogi VK, Cahill SM, Girvin ME, Almo SC: Binding orientation of proline-rich peptides in solution: polarity of the profilin − ligand interaction. J Am Chem Soc 2000, 122(32):7851–7852. 10.1021/ja001240hView ArticleGoogle Scholar
  32. Weber DJ, Mullen GP, Mildvan AS: Conformation of an enzyme-bound substrate of staphylococcal nuclease as determined by NMR. Biochemistry 1991, 30(30):7425–7437. 10.1021/bi00244a009View ArticlePubMedGoogle Scholar
  33. Weber DJ, Abeygunawardana C, Bessman MJ, Mildvan AS: Secondary structure of the MutT enzyme as determined by NMR. Biochemistry 1993, 32(48):13081–13088. 10.1021/bi00211a018View ArticlePubMedGoogle Scholar
  34. Rustandi RR, Baldisseri DM, Weber DJ: Structure of the negative regulatory domain of p53 bound to S100B(betabeta). Nat Struct Biol 2000, 7(7):570–574. 10.1038/76797View ArticlePubMedGoogle Scholar
  35. Wright NT, Cannon BR, Wilder PT, Morgan MT, Varney KM, Zimmer DB, Weber DJ: Solution structure of S100A1 bound to the CapZ peptide (TRTK12). J Mol Biol 2009, 386(5):1265–1277. 10.1016/j.jmb.2009.01.022PubMed CentralView ArticlePubMedGoogle Scholar
  36. Bhattacharya S, Large E, Heizmann CW, Hemmings B, Chazin WJ: Structure of the Ca2+/S100B/NDR kinase peptide complex: insights into S100 target specificity and activation of the kinase. Biochemistry 2003, 42(49):14416–14426. 10.1021/bi035089aView ArticlePubMedGoogle Scholar
  37. Lee YT, Dimitrova YN, Schneider G, Ridenour WB, Bhattacharya S, Soss SE, Caprioli RM, Filipek A, Chazin WJ: Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: a novel mode for S100 protein target recognition. Biochemistry 2008, 47(41):10921–10932. 10.1021/bi801233zPubMed CentralView ArticlePubMedGoogle Scholar
  38. Rety S, Sopkova J, Renouard M, Osterloh D, Gerke V, Tabaries S, Russo-Marie F, Lewit-Bentley A: The crystal structure of a complex of p11 with the annexin II N-terminal peptide. Nat Struct Biol 1999, 6(1):89–95. 10.1038/4965View ArticlePubMedGoogle Scholar
  39. Rety S, Osterloh D, Arie JP, Tabaries S, Seeman J, Russo-Marie F, Gerke V, Lewit-Bentley A: Structural basis of the Ca(2+)-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I. Structure Fold Des 2000, 8(2):175–184. 10.1016/S0969-2126(00)00093-9View ArticlePubMedGoogle Scholar
  40. Dempsey BR, Rezvanpour A, Lee TW, Barber KR, Junop MS, Shaw GS: Structure of an asymmetric ternary protein complex provides insight for membrane interaction. Structure 2012, 20(10):1737–1745. 10.1016/j.str.2012.08.004View ArticlePubMedGoogle Scholar
  41. Ozorowski G, Milton S, Luecke H: Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2. Acta Crystallogr D Biol Crystallogr 2013, 69(Pt 1):92–104.PubMed CentralView ArticlePubMedGoogle Scholar
  42. Oh YS, Gao P, Lee KW, Ceglia I, Seo JS, Zhang X, Ahn JH, Chait BT, Patel DJ, Kim Y, et al.: SMARCA3, a chromatin-remodeling factor, is required for p11-dependent antidepressant action. Cell 2013, 152(4):831–843. 10.1016/j.cell.2013.01.014PubMed CentralView ArticlePubMedGoogle Scholar
  43. Dulyaninova NG, Hite KM, Zencheck WD, Scudiero DA, Almo SC, Shoemaker RH, Bresnick AR: Cysteine 81 is critical for the interaction of S100A4 and myosin-IIA. Biochemistry 2011, 50(33):7218–7227. 10.1021/bi200853yPubMed CentralView ArticlePubMedGoogle Scholar
  44. Markowitz J, Rustandi RR, Varney KM, Wilder PT, Udan R, Wu SL, Horrocks WD, Weber DJ: Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53. Biochemistry 2005, 44(19):7305–7314. 10.1021/bi050321tView ArticlePubMedGoogle Scholar
  45. Franke JD, Dong F, Rickoll WL, Kelley MJ, Kiehart DP: Rod mutations associated with MYH9-related disorders disrupt nonmuscle myosin-IIA assembly. Blood 2005, 105(1):161–169. 10.1182/blood-2004-06-2067View ArticlePubMedGoogle Scholar
  46. Badyal SK, Basran J, Bhanji N, Kim JH, Chavda AP, Jung HS, Craig R, Elliott PR, Irvine AF, Barsukov IL, et al.: Mechanism of the Ca(2) + −dependent interaction between S100A4 and tail fragments of nonmuscle myosin heavy chain IIA. J Mol Biol 2011, 405(4):1004–1026. 10.1016/j.jmb.2010.11.036PubMed CentralView ArticlePubMedGoogle Scholar
  47. Ford HL, Silver DL, Kachar B, Sellers JR, Zain SB: Effect of Mts1 on the structure and activity of nonmuscle myosin II. Biochemistry 1997, 36(51):16321–16327. 10.1021/bi971182lView ArticlePubMedGoogle Scholar
  48. Eggington JM, Kozlov AG, Cox MM, Lohman TM: Polar destabilization of DNA duplexes with single-stranded overhangs by the Deinococcus radiodurans SSB protein. Biochemistry 2006, 45(48):14490–14502. 10.1021/bi061178mView ArticlePubMedGoogle Scholar
  49. Qu X, Lancaster L, Noller HF, Bustamante C, Tinoco I Jr: Ribosomal protein S1 unwinds double-stranded RNA in multiple steps. Proc Natl Acad Sci U S A 2012, 109(36):14458–14463. 10.1073/pnas.1208950109PubMed CentralView ArticlePubMedGoogle Scholar
  50. Kosen PA: Spin labeling of proteins. Methods Enzymol 1989, 177: 86–121.View ArticlePubMedGoogle Scholar
  51. Otwinowski W, Minor F: Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 1997, 276: 307–326.View ArticleGoogle Scholar
  52. Vagin A, Teplyakov A: MOLREP: an automated program for molecular replacement. J Appl Cryst 1997, 30: 1022–1025. 10.1107/S0021889897006766View ArticleGoogle Scholar
  53. Emsley P, Cowtan K: Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004, 60(Pt 12 Pt 1):2126–2132.View ArticlePubMedGoogle Scholar
  54. Murshudov GN, Vagin AA, Dodson EJ: Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 1997, 53(Pt 3):240–255.View ArticlePubMedGoogle Scholar
  55. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A: NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 1995, 6(3):277–293.View ArticlePubMedGoogle Scholar
  56. Edison AS, Abildgaard F, Westler WM, Mooberry ES, Markley JL: Practical introduction to theory and implementation of multinuclear, multidimensional nuclear magnetic resonance experiments. Methods Enzymol 1994, 239: 3–79.View ArticlePubMedGoogle Scholar
  57. Stafford WF 3rd: Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal Biochem 1992, 203(2):295–301. 10.1016/0003-2697(92)90316-YView ArticlePubMedGoogle Scholar
  58. Philo JS: Improved methods for fitting sedimentation coefficient distributions derived by time-derivative techniques. Anal Biochem 2006, 354(2):238–246. 10.1016/j.ab.2006.04.053View ArticlePubMedGoogle Scholar

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