Molecular Basis of Ca(II)-Induced Tetramerization and Transition-Metal Sequestration in Human Calprotectin

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αβ heterodimeric apo protein into a Ca(II)-bound (αβ)₂ heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αβ heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αβ heterodimer. These studies provide a structural model in which the apo αβ heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αβ heterodimer causes the active state to predominate, resulting in self-association and formation of the (αβ)₂ heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the His₃Asp and His₆ metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the His₃Asp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix α_IV of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His₆ site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.