Molecular determinants of S100B oligomer formation

Abstract

Background: S100B is a dimeric protein that can form tetramers, hexamers and higher order oligomers. These forms have been suggested to play a role in RAGE activation.

Methodology/principal findings: Oligomerization was found to require a low molecular weight trigger/cofactor and could not be detected for highly pure dimer, irrespective of handling. Imidazol was identified as a substance that can serve this role. Oligomerization is dependent on both the imidazol concentration and pH, with optima around 90 mM imidazol and pH 7, respectively. No oligomerization was observed above pH 8, thus the protonated form of imidazol is the active species in promoting assembly of dimers to higher species. However, disulfide bonds are not involved and the process is independent of redox potential. The process was also found to be independent of whether Ca(2+) is bound to the protein or not. Tetramers that are purified from dimers and imidazol by gel filtration are kinetically stable, but dissociate into dimers upon heating. Dimers do not revert to tetramer and higher oligomer unless imidazol is again added. Both tetramers and hexamers bind the target peptide from p53 with retained stoichiometry of one peptide per S100B monomer, and with high affinity (lgK = 7.3±0.2 and 7.2±0.2, respectively in 10 mM BisTris, 5 mM CaCl(2), pH 7.0), which is less than one order of magnitude reduced compared to dimer under the same buffer conditions.

Conclusion/significance: S100B oligomerization requires protonated imidazol as a trigger/cofactor. Oligomers are kinetically stable after imidazol is removed but revert back to dimer if heated. The results underscore the importance of kinetic versus thermodynamic control of S100B protein aggregation.

Figure 1. Identification of the trigger.

Highly pure S100B dimer was mixed with potential triggers and lyophilized, dissolved, incubated for 24 h at 4 °C, and analyzed by analytical gel filtration on a Superdex 200 column at room temperature. The chromatograms shown are for samples with 20 mg/ml S100B, 1 mM EDTA, 1 mM DTT, 0.25 M NaCl, pH 7.0, and no (top), 20 mM (middle) or 90 mM imidazol .

Figure 2. NMR spectra reveal no imidazol in S100B tetramer or dimer isolated by gel filtration.

¹H NMR spectra in D₂O for imidazol and the dimer and tetramer fractions as purified by gel filtration after triggering of 20 mg/ml S100B with 90 mM imidazol in1 mM EDTA, 1 mM DTT and 0.25 M NaCl.

Figure 3. Tetramer formation - dependence on imidazol concentration and pH.

Analytical gel filtration on a Superdex 200 column was used to estimate the fraction of S100B in tetrameric state after triggering experiments starting with 20 mg/ml highly pure S100B dimer in 1 mM EDTA, 1 mM DTT and 0.25 M NaCl with different concentrations of imidazol at pH 7.0 (A) or with 90 mM imidazol at different pH values (B).

Figure 4. Kinetic stability.

An imidazol-triggered sample of S100B was separated into hexamer, tetramer and dimer by gel filtration and the fractions reinjected on the Superdex 200 analytical gel filtration column before (top two chromatograms) and after (bottom two chromatograms) heating to 90 °C for 15 minutes.

Figure 5. Dynamic light scattering.

An imidazol-triggered sample of S100B was separated into hexamer (dotted line), tetramer (dashed line) and dimer (solid line) by gel filtration and the fractions analyzed by dynamic light scattering.

Figure 6. p53 binding.

Titration of 0.9 µM p53 peptide with S100B dimer (O), tetramer (•), or hexamer (▴), together with fitted lines. The fluorescence intensity recorded during each titration has been normalized.