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

Abstract

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 αIV 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.

Antimicrobial peptides: Defending the mucosal epithelial barrier

“…Extracellular calprotectin can also undergo oxidation [ 169 ], thereby increasing its vulnerability to proteolysis and fragmentation into smaller peptides of yet unknown biological activity [ 170 ]. In the extracellular space, calprotectin is available to bind receptors ( Table 1 , Figure 2) , assemble heterodimers and other oligomeric forms [ 189 ], and sequester metal ions [ 190 ]. Soluble calprotectin may be recognized as a damage-associated molecular pattern (DAMP) or alarmin to activate neutrophils or function as an antimicrobial protein to suppress microbial growth [ 191 ].…”

“…Each S100 subunit contains two Ca 2+ binding sites [ 195 , 196 ]. In the absence of Ca 2+ ions, S100A8/A9 exists as a heterodimer, and upon Ca 2+ binding, the two heterodimers self-associate to form a heterotetramer [ 189 , 196 ]. Hetero-multimerization can also be promoted by oxidative cross-linking during inflammation [ 197 ].…”

“…Each heterodimer contains two distinct transition-metal-binding sites. When compared with the heterodimer, calcium-induced tetramers show enhanced binding affinity for Mn, Fe, Ni, Cu, and Zn and increased protease stability [ 189 , 196 ]. By sequestering transition metals, calprotectin heterotetramers starve microbes that infect the surrounding inflammatory tissue.…”

“…In the presence of calprotectin, the lower gingival inflammatory cell infiltrate could increase the alkaline pH environment, albeit with lower tissue Ca ++ which is released from neutrophils. Inflammation is also generally accompanied by interstitial acidosis, which serves as another danger signal [ 224 ], and release of cellular calcium into the tissues [ 189 ]. In human periodontitis [ 164 , 165 ], the neutrophil-rich inflammatory environment would also be expected to contain increased extracellular levels of calprotectin [ 191 ].…”

Hard–Soft Cooperativity of an Allosteric Tweezer

“…As illustrated in Scheme , this allosteric tweezer operates by (i) chelation of a metal, (ii) anion binding to the coordinated metal, (iii) structural folding to provide a sandwich-like cavity, and (iv) uptake of another metal into the cavity. To the best of our knowledge, this work represents the first example of an artificial receptor having multiple allosteric sites working in a cascade fashion …”