In this article, we reexamine aspects of Ca2+
and NM-MHC IIA tail fragment binding to S100A4 and extend the study through new kinetic measurements. Ca2+
binds to the S100A4 dimer with an affinity of around 2–6 μM. ITC measurements indicate a stoichiometry of one Ca2+
per S100A4 monomer and a Kd
of 1.7 μM, with a second site with Kd
= 6 μM revealed at temperatures of ≥ 30 °C. Regarding the assignment of the sites to EF1 or EF2, the E33Q mutant appears more similar to wild-type S100A4 than the D63N mutant and has an intact EF2 site, as determined by tyrosine and Quin-2 fluorescence (a; Fig. S1b
). The weaker binding site of native S100A4 seen by ITC (a) would then correspond to EF1, as assumed by Malashkevich et al.17
The resolved Quin-2 signal therefore reflects Ca2+
that was bound to EF2, with Ca2+
release from EF1 being too fast to measure. In the presence of target peptides, the amplitude of the Quin-2 signals increases by no more than 20%, suggesting that the release of any Ca2+
bound to EF1 remains too fast to measure. The small increase in amplitude of the resolved phase could reflect the increase in the initial degree of binding to EF2 at a fixed free [Ca2+
], in line with the decrease in Kd
. Previously, we reported a stoichiometry of binding of 1.7 ± 0.18 Ca2+
bound per S100A4 monomer, based on the amplitude of the tyrosine fluorescence change on addition of Ca2+
These signals were much noisier than those recorded using the Quin-2 indicator reported here. Furthermore, the calculated stoichiometry is critically dependent on the accuracy of the S100A4 concentration determination. S100A4 lacks tryptophan, and the absorbance from the two tyrosine residues is weak, so the conclusions are dependent on the accurate correction for turbidity and the lack of significant UV-absorbing contaminants. However, the unchanged amplitude of the Quin-2 signal on Ca2+
dissociation from the wild type compared with the E33Q mutant (a) argues strongly that a single site (EF2) per monomer is involved. Nevertheless, there is communication between the EF1 domain and the EF2 domain on dissociation of Ca2+
from EF2, as indicated by tyrosine fluorescence (Fig. S1c
), which could account for the NMR findings of Dutta et al.
, who concluded that EF1 had a higher affinity.15
We confirm that a 16-amino-acid myosin peptide derivative, F-M16N
, binds to S100A4 in the presence of Ca2+
with an affinity of around 1 μM,17
but we consider the fluorescein moiety to make a large contribution to the binding energy because the corresponding unlabelled M16N
peptide binds 2 orders of magnitude more weakly. The data of Malashkevich et al.
imply that the unlabelled M16N
peptide binds almost as tightly as longer fragments, although no raw data are shown for this peptide.17
In our analysis (b), M32 and F-M16N
were assumed to bind to each monomer independently. However, the stoichiometry of M32 binding (Fig. S3a
) suggests that one M32 peptide may span two monomers of S100A4, so it could compete with two molecules of F-M16N
. Fitting to such a model yields a Kd
of 1.5 μM for the M32 peptide (i.e., half that of the simple model). From our measurements, a 32-mer peptide (corresponding to A1907-G1938 of the myosin sequence) represents a minimal binding region. Longer fragments (M111 and M200) bind with even higher affinity, indicating that additional amino acids of the myosin heavy chain contribute to the binding interaction and/or the capacity of the longer fragments to form coiled-coil structures enables higher-order structures to form with increased avidity. We are currently exploring longer peptides to address this question and consider that both factors are involved.
Based on the S100A4 concentration determined from the absorbance at 280 nm, one mol of M32 peptide binds to two S100A4 monomers. In support of this conclusion, the ITC titration curve of an R1893-R1923 myosin fragment of Malashkevich et al.
(their Fig. 1117
) shows a stoichiometry close to 0.5 mol of peptide per mole of S100A4 (dimer), in which concentrations of the components were determined from quantitative amino acid analysis. The site between helix III and helix IV of S100A4, exposed in the presence of Ca2+
, could accommodate an α-helical target of about 16 residues. The longer M32 minimal peptide identified above suggests that the myosin binding site may extend beyond a single S100A4 monomer, as was reported for the novel interactions of a 31-mer SIP (S
rotein) fragment with the S100A6 dimer, as determined by NMR.35
The conclusion regarding the S100A4: peptide target stoichiometry reported here requires confirmation by comparable structural methods.
A corresponding stoichiometry is also seen in the interaction of S100A4 with M200, where 2 mol of S100A4 monomer was required to solubilise 1 mol of M200 aggregate (when expressed as M200 polypeptide chain concentration) (b). Murakami et al.31
found that a S100A4/M200 ratio of 5:1 was required for solubilisation in their assays, whereas Li et al.11
found that 1 mol of S100A4 dimer was required to disassemble 1 mol of NM-MHC IIA rods. These stoichiometries complicate the calculation of the equilibrium parameters for the models described in and . If the binding sites remained independent, then Kd
values would be altered by just a statistical factor but would more likely extend the potential for cooperativity. However, until accurate stoichiometries are determined by direct structural methods, and provide a useful start point for the discussion of thermodynamic coupling. As a consequence of this coupling in , the very weak binding of target proteins to S100A4 in the absence of Ca2+
requires the target-bound form of S100A4 to bind Ca2+
with a very high affinity (). In the case of bound F-M16N
, the estimated Kd
limit for Ca2+
binding is < 20 nM. It is important to stress that this would not be the observed Kd
binding to a mixture of F-M16N
and S100A4, unless the peptide concentration was much greater than millimolar (i.e., exceeded K0
in ), because the observed constant would reflect both the binding of peptide to S100A4 and the binding of Ca2+
to this complex. A similar argument applies to M200, but here the thermodynamic coupling factor appears even greater (possibly because of avidity due to the two potential binding sites from each chain of the coiled coil of M200). The physiological consequence of this is that the myosin tail and S100A4 could bind at resting cytoplasmic Ca2+
levels and cause filament disassembly, provided at least one of the protein components is present at a concentration exceeding several micromolars (cf. Fig. S6c
). Unfortunately, there are no reliable estimates of the effective concentrations of S100A4 in cells, although total concentrations of micromolar might be expected.22
Cell excitation that leads to elevated [Ca2+
] would promote further myosin filament disassembly. However, the modelling shown in indicates that a sustained rise in [Ca2+
] lasting tens of seconds is required in order to achieve a significant effect.
In the absence of added Ca2+
(i.e., where the free “contaminant” [Ca2+
] in buffers would be of the order of 1 μM), mixing S100A4 with excess Quin-2 produced a small-amplitude signal (corresponding to 0.1 mol of Ca2+
per mole of S100A4 monomer) with a rate constant of around 1 s− 1
. This is similar to the rate constants obtained for target-bound S100A4 and suggests that this process might correspond to the dissociation of Ca2+
from a small fraction of the S100A4 tetramers. The crystal structure of the Ca2+
-bound form of the S100A4 tetramer shows that the C termini of two of the monomers are positioned in the target binding sites of two opposing subunits.18
Indeed, in this article, we drew attention to the C-terminal sequence ExFPxxxP, which is similar to the DLPFVVP sequence identified within the minimal myosin target peptide (). An S100A4 construct lacking the last 13 C-terminal residues fails to from higher oligomers beyond the dimer.17
The tetramer and higher oligomers of the wild type are therefore unlikely to bind to the myosin target site with significant affinity because of the competition with the C-terminus. Based on thermodynamic coupling, the tetramer would also be expected to have a higher affinity for Ca2+
than for the dimer. Indeed, ultracentrifuge data17
suggest that the affinity would be about 20-fold higher. A Ca2+
value of around 0.1 μM for the tetramer would suggest that overexpression of S100A4 could lead to oligomerisation of S100A4 at resting cytoplasmic [Ca2+
]. In addition, phenothiazine drugs have been reported to induce S100A4 oligomers and to inhibit interaction with NM-MHC IIA.36
It is possible that there are natural effectors that operate by this mechanism.
S100A4 solubilises M200 filament aggregates in the presence of Ca2+
; in terms of kinetics, this construct serves as a model system for studying the mechanism of depolymerisation of intact NM-MHC IIA filaments. S100A4 binds to M200 filament aggregates and actively depolymerises them, rather than just binding to the monomer and perturbing the myosin filament equilibrium. However, the (M200)n.
filament complex is only a transient species that leads to a solubilised M200.S100A4.Ca2+
complex. This has implications for in vivo
studies, since the degree of colocalisation of S100A4 with myosin filaments may be rather limited under steady-state conditions, and any solubilisation would lead to a more general cytoplasmic distribution of both proteins. Zhang et al.
found that an eCFP-S100A4 construct did interact with a NM-MHC IIA–eYFP rod construct in the cytoplasm of HeLa cells, using fluorescence lifetime imaging.37
As these experiments required 5 min of acquisition time, the time-averaged Ca2+
concentration was likely to be close to resting levels and thus corroborates the potential for these proteins to interact at Ca2+
concentrations significantly lower than the Kd
for S100A4 in the absence of target. However, the in vivo
case is complicated by further regulatory mechanisms such as myosin phosphorylation38
and cross-reactivity between other S100 proteins and their targets.