Multilevel Changes in Protein Dynamics upon Complex Formation of the Calcium‐Loaded S100A4 with a Nonmuscle Myosin IIA Tail Fragment

Abstract

Dysregulation of Ca -binding S100 proteins plays important role in various diseases. The asymmetric complex of Ca -bound S100A4 with nonmuscle myosin IIA has high stability and highly increased Ca affinity. Here we investigated the possible causes of this allosteric effect by NMR spectroscopy. Chemical shift-based secondary-structure analysis did not show substantial changes for the complex. Backbone dynamics revealed slow-timescale local motions in the H1 helices of homodimeric S100A4; these were less pronounced in the complex form and might be accompanied by an increase in dimer stability. Different mobilities in the Ca -coordinating EF-hand sites indicate that they communicate by an allosteric mechanism operating through changes in protein dynamics; this must be responsible for the elevated Ca affinity. These multilevel changes in protein dynamics as conformational adaptation allow S100A4 fine-tuning of its protein-protein interactions inside the cell during Ca signaling.

Ezrin interacts with S100A4 via both its N- and C-terminal domains

“…Interestingly, significant chemical shift changes occur in the H1 helix, which is located far from the canonical ligand binding site. Similar tendencies were previously observed in the S100A4-NMIIA complex [32, 39], however, the Δ δ values were more significant for the myosin peptide.…”

“…For these measurements an S100A4 variant C-terminally truncated by 9 residues (S100A4-Δ9) was used due to the aggregation tendencies of the wild-type protein [32]. The truncated version shows similar binding affinities to the N-ERMAD and the C-ERMAD (as shown in S2H and S2I Fig, respectively).…”

“…Elution was done by using EDTA-containing buffer; then the eluted fractions were concentrated and stored at −70°C. 15 N-labeled S100A4-Δ9 was expressed and purified as described in [32]. …”

“…All chemical shifts were referenced to the internal DSS resonance, while 15 N chemical shift values were referenced indirectly using the corresponding gyromagnetic ratios according to IUPAC convention. Sequence specific assignment of H N , N and side chain proton resonances was done using a 15 N-labeled protein at 300 K and on the basis of standard 3D HSQC-TOCSY (mixing time 70 ms) and 3D HSQC-NOESY (mixing time: 150 ms) measurements and also relying on earlier results from the assigned S100A4-Δ13 protein (BMRB entry code: 25136) [32]. All spectra were processed with TOPSPIN and analyzed using CARA (ETH Zürich) and SPARKY softwares [36, 37].…”

“…Δ δ chemical shift mapping values were calculated as described earlier [32]. In the NMR titration experiment, unlabeled C-ERMAD (7.5 mM) was added gradually to the NMR tube containing 15 N-labeled S100A4-Δ9 dimer (250 μM).…”