Zinc‐Mediated Oligomerization of S100A12

Members of the S100 class of the EF‐hand family of calcium binding proteins are important in diverse biological functions. In addition to binding calcium, several S100 proteins also bind zinc; a property associated with the extracellular function of S100 proteins. In general, S100 protein affinity for zinc is high (with K d ~subnanomolar) and calcium dependent. For example, S100A12 sequesters the transition metal zinc as part of the host response to starve bacterial pathogens, a process termed nutritional immunity. Additionally, S100A12 activates the inflammation receptor RAGE (receptor for advanced glycation end products), an interaction which may be modulated by zinc. While metal binding to S100 proteins has been demonstrated to affect protein oligomerization, several questions remain regarding the nature of these interactions. S100A12 is a dimeric protein comprised of two subunits arranged in an antiparallel fashion. There are four calcium‐binding EF‐hand motifs in each dimer, and there are two zinc binding sites located at the dimer interface. Here, we report biophysical experiments that characterize the oligomerization of S100A12 in response to metal binding. Dynamic light scattering experiments indicate calcium does not induce oligomerization of S100A12. The hydrodynamic radius of apo S100A12 was measured to be 2.5 nm, a size consistent with its dimeric structure. Measurements of the hydrodynamic radius were invariant after titration with calcium up to a molar ratio of 10:1. We determined the molecular mass of calcium‐S100A12 to be ~20 kDa using composition‐gradient multi‐angle light scattering experiments. This contrasts with calprotectin (CP), a homologous S100 protein, that undergoes a transition from dimer to tetramer upon calcium binding. We measured the hydrodynamic radii of CP to be 3.5 nm and 4.5 nm for the apo (dimer) and calcium bound form (tetramer), respectively. In contrast, zinc addition to calcium‐S100A12 resulted in oligomerization. Multi‐angle light scattering experiments revealed a concentration dependent oligomerization of up to hexamers of S100A12. To understand the molecular level details of zinc induced oligomerization of S100A12, we determined the crystal structure of S100A12 in complex with calcium and zinc to 3.0 angstroms resolution. Crystals were obtained in the orthorhombic space group P2221 with a total of 8 subunits in the asymmetric unit arranged as two identical tetramers. Together, our results demonstrate that zinc induces oligomerization of S100A12. These data will further our understanding of the role of S100A12 in nutritional immunity and in inflammation. Support or Funding Information This material is based upon work supported by the National Science Foundation under Grant nos. HRD‐1547757 and HRD‐1400969. Any opinions, findings, interpretations, conclusions or recommendations expressed in this material are those of its authors and do not represent the views of the National Science Foundation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .