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 αß heterodimeric apo protein into a Ca(II)-bound (αß)2 heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αß 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 αß heterodimer. These studies provide a structural model in which the apo αß 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 αß heterodimer causes the active state to predominate, resulting in self-association and formation of the (αß)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.

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry.

Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.

Exosome swarms eliminate airway pathogens and provide passive epithelial immunoprotection through nitric oxide.

BACKGROUND: Nasal mucosa-derived exosomes (NMDEs) harbor immunodefensive proteins and are capable of rapid interepithelial protein transfer. OBJECTIVES: We sought to determine whether mucosal exposure to inhaled pathogens stimulates a defensive swarm of microbiocidal exosomes, which also donate their antimicrobial cargo to adjacent epithelial cells. METHODS: We performed an institutional review board-approved study of healthy NMDE secretion after Toll-like receptor (TLR) 4 stimulation by LPS (12.5 µg/mL) in the presence of TLR4 inhibitors. Interepithelial transfer of exosomal nitric oxide (NO) synthase and nitric oxide was measured by using ELISAs and NO activity assays. Exosomal antimicrobial assays were performed with Pseudomonas aeruginosa. Proteomic analyses were performed by using SOMAscan. RESULTS: In vivo and in vitro LPS exposure induced a 2-fold increase in NMDE secretion along with a 2-fold increase in exosomal inducible nitric oxide synthase expression and function through TLR4 and inhibitor of nuclear factor κB kinase activation. LPS stimulation increased exosomal microbiocidal activity against P aeruginosa by almost 2 orders of magnitude. LPS-stimulated exosomes induced a 4-fold increase in NO production within autologous epithelial cells with protein transfer within 5 minutes of contact. Pathway analysis of the NMDE proteome revealed 44 additional proteins associated with NO signaling and innate immune function. CONCLUSIONS: We provide direct in vivo evidence for a novel exosome-mediated innate immunosurveillance and defense mechanism of the human upper airway. These findings have implications for lower airway innate immunity, delivery of airway therapeutics, and host microbiome regulation.

Oxidative Post-translational Modifications Accelerate Proteolytic Degradation of Calprotectin.

Oxidative post-translational modifications affect the structure and function of many biomolecules. Herein we examine the biophysical and functional consequences of oxidative post-translational modifications to human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer, calgranulins A/B oligomer). This abundant metal-sequestering protein contributes to innate immunity by starving invading microbial pathogens of transition metal nutrients in the extracellular space. It also participates in the inflammatory response. Despite many decades of study, little is known about the fate of CP at sites of infection and inflammation. We present compelling evidence for methionine oxidation of CP in vivo, supported by using 15N-labeled CP-Ser (S100A8(C42S)/S100A9(C3S)) to monitor for adventitious oxidation following human sample collection. To elucidate the biochemical and functional consequences of oxidative post-translational modifications, we examine recombinant CP-Ser with methionine sulfoxide modifications generated by exposing the protein to hydrogen peroxide. These oxidized species coordinate transition metal ions and exert antibacterial activity. Nevertheless, oxidation of M81 in the S100A9 subunit disrupts Ca(II)-induced tetramerization and, in the absence of a transition metal ion bound at the His6 site, accelerates proteolytic degradation of CP. We demonstrate that native CP, which contains one Cys residue in each full-length subunit, forms disulfide bonds within and between S100A8/S100A9 heterodimers when exposed to hydrogen peroxide. Remarkably, disulfide bond formation accelerates proteolytic degradation of CP. We propose a new extension to the working model for extracellular CP where post-translational oxidation by reactive oxygen species generated during the neutrophil oxidative burst modulates its lifetime in the extracellular space.

The Hexahistidine Motif of Host-Defense Protein Human Calprotectin Contributes to Zinc Withholding and Its Functional Versatility.

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP coordinates a variety of divalent first-row transition metal ions, which is implicated in its antimicrobial function, and its ability to sequester nutrient Zn(II) ions from microbial pathogens has been recognized for over two decades. CP has two distinct transition-metal-binding sites formed at the S100A8/S100A9 dimer interface, including a histidine-rich site composed of S100A8 residues His17 and His27 and S100A9 residues His91 and His95. In this study, we report that CP binds Zn(II) at this site using a hexahistidine motif, completed by His103 and His105 of the S100A9 C-terminal tail and previously identified as the high-affinity Mn(II) and Fe(II) coordination site. Zn(II) binding at this unique site shields the S100A9 C-terminal tail from proteolytic degradation by proteinase K. X-ray absorption spectroscopy and Zn(II) competition titrations support the formation of a Zn(II)-His6 motif. Microbial growth studies indicate that the hexahistidine motif is important for preventing microbial Zn(II) acquisition from CP by the probiotic Lactobacillus plantarum and the opportunistic human pathogen Candida albicans. The Zn(II)-His6 site of CP expands the known biological coordination chemistry of Zn(II) and provides new insight into how the human innate immune system starves microbes of essential metal nutrients.

Calcium-induced Tetramerization and Zinc Chelation Shield Human Calprotectin from Degradation by Host and Bacterial Extracellular Proteases.

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/14 oligomer, calgranulins A and B) is a protein component of the innate immune system that contributes to the metal-withholding response by sequestering bioavailable transition metal ions at sites of infection. Human CP employs Ca(II) ions to modulate its quaternary structure, transition metal binding properties, and antimicrobial activity. In this work, we report the discovery that Ca(II)-induced self-association of human CP to afford heterotetramers protects the protein scaffold from degradation by host serine proteases. We present the design and characterization of two new human CP-Ser variants, S100A8(C42S)(I60E)/S100A9(C3S) and S100A8(C42S)(I60K)/S100A9(C3S), that exhibit defective tetramerization properties. Analytical size exclusion chromatography and analytical ultracentrifugation reveal that both variants, hereafter I60E and I60K, persist as heterodimers in the presence of Ca(II) only, and form heterotetramers in the presence of Mn(II) only and both Ca(II) and Mn(II). Coordination to Fe(II) also causes I60E and I60K to form heterotetramers in both the absence and presence of Ca(II). The Ca(II)-bound I60E and I60K heterodimers are readily degraded by trypsin, chymotrypsin and human neutrophil elastase, whereas the Ca(II)-bound CP-Ser heterotetramers and the Ca(II)- and Mn(II)-bound I60E and I60K heterotetramers are resistant to degradation by these host proteases. The staphylococcal extracellular protease GluC cuts the S100A8 subunit of CP-Ser at the C-terminal end of residue 89 to afford a ΔSKHE variant. The GluC cleavage site is in close proximity to the His3Asp metal-binding site, which coordinates Zn(II) with high affinity, and Zn(II) chelation protects the S100A8 subunit from GluC cleavage. Taken together, these results provide new insight into how Ca(II) ions and transition metals modulate the chemistry and biology of CP, and indicate that coordination to divalent cations transforms human CP into a protease-resistant form and enables innate immune function in the hostile conditions of an infection site.

Rapid formal hydrolysis of peptide-αthioesters.

In the presence of 2-mercaptoethanol peptide-(α)thioesters undergo smooth conversion to their corresponding peptide-(α)carboxylates. This general and operationally simple reaction extends the utility of a promising new strategy for cleaving resin-bound Boc/benzyl-protected peptides without the use of hydrogen fluoride.