The C-terminal random coil region tunes the Ca²⁺-binding affinity of S100A4 through conformational activation.
S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1-88, Δ13) changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15-0.25 Å-1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.
Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden.
Annette
A
Duelli
Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary.
Bence
B
Kiss
Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden.
Ida
I
Lundholm
Institute of Chemistry, Laboratory of Structural Chemistry and Biology, Eötvös Loránd University, Budapest, Hungary.
Andrea
A
Bodor
European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Hamburg, Germany.
Maxim V
MV
Petoukhov
European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Hamburg, Germany.
Dmitri I
DI
Svergun
Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary.
László
L
Nyitray
Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden.
Gergely
G
Katona
D000465:Algorithms; D002118:Calcium / Q000737:chemistry*; D002151:Calorimetry; D018360:Crystallography, X-Ray; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009362:Neoplasm Metastasis; D024507:Nonmuscle Myosin Type IIA / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D013816:Thermodynamics
D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C077591:S100A4 protein, human; D024507:Nonmuscle Myosin Type IIA; D002118:Calcium