Date of Degree


Document Type


Degree Name





Rupal Gupta

Committee Members

Sharon Loverde

Sebastien Poget

Richard Magliozzo

Brian Gibney

Subject Categories

Biochemistry | Structural Biology


nutritional immunity, protein oligomerization


S100A12 protein belongs to the S100 family of calcium-binding proteins and participates in the innate immune system. Antimicrobial proteins from the S100 family of the proteins (S100A12, S100A8/A9, etc.) are secreted and expressed by neutrophils during microbial infection and perform their antimicrobial activity through metal sequestration. While most S100 proteins function intracellularly, S100A12 is highly expressed and secreted into the extracellular space by neutrophils during infection. Sequestration of Zn2+ by S100A12 is aided by the nanomolar zinc binding affinity of the protein at neutral pH conditions, which is further enhanced upon calcium-binding. The Zn2+ binding scaffold in S100A12 is composed of a His3Asp motif. Upon Zn2+ binding, human S100A12 self-assembles into multimeric assemblies, the order of which depends on the protein concentration. While apo-S100A12 retains its strong metal affinity in the pH range of 7.0 – 10.0, its Co2+/Zn2+ affinity progressively diminishes as the pH is reduced from 7.0 to 5.3. In the presence of calcium, the viable pH range of metal-binding increases, and S100A12 retains its strong Co2+/Zn2+ binding up to pH 5.7 in the Ca2+ bound state. The calcium induces enhanced Co2+/Zn2+ binding assisting efficient metal sequestration during neutrophil activation, occurring at sub-neutral pH conditions.

We have employed various biophysical and molecular biology methods and tools such as nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR), and single point mutations to understand the structural and functional nature of binding both zinc and calcium to the S100A12. Our data shows the different roles of the calcium-binding motifs in the function of the S100A12. Also, we determined that the modulation of viable pH range for Co2+/Zn2+ binding by calcium is employed by restraining pH-dependent conformational changes to the EF-hand 1 loop that contains the Co2+/Zn2+ binding Asp25. We also performed a structural characterization of the metal-dependent self-assembly of S100A12. The structural characterization demonstrated that the cooperative binding of Zn2+ to its metal-binding motifs and Ca2+ binding to protein’s EF-hand motifs induces reversible protein self-assembly. Both magic angle spinning and solution NMR spectroscopy on apo-, Zn2+-, Ca2+-, and Zn2+, Ca2+- S100A12 demonstrated the significant chemical shift perturbations (CSPs) induced by metal binding, which indicate conformational changes throughout the polypeptide chain. We demonstrated that observed perturbations are not from changes in the secondary structure of S100A12, and the secondary structure of the protein remains primarily preserved. Notably, the reported metal-induced CSPs were observed for the functionally relevant hinge region, which participates in target recognition and chemotaxis.