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Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

ABSTRACT: Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96-114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn(II) coordination sphere in solution, affording an unprecedented biological His6 site. These solution studies are in agreement with a Mn(II)-CP crystal structure reported recently (Damo, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3841). Remarkably high-affinity Mn(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103-105 to 104-106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against Escherichia coli and Staphylococcus aureus. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn(II) binding; the S100A7, S100A9(C3S), S100A12, and S100B homodimers do not exhibit such Mn(II)-binding capacity.

SUBMITTER: Brophy MB

PROVIDER: S-EPMC3892207 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

Brophy Megan Brunjes MB Nakashige Toshiki G TG Gaillard Aleth A Nolan Elizabeth M EM

Journal of the American Chemical Society 20131118 47

Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96-114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-ex ...[more]

PMID: 24245608

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Project description:Manganese is an essential metal ion that bacterial pathogens need to acquire from the vertebrate host during infection. In the mammalian nutritional immunity strategy to combat bacterial infection, the host restricts bacterial access to Mn(II) by sequestering this metal nutrient using the protein calprotectin (CP). The role of murine calprotectin (mCP) in Mn(II) sequestration has been demonstrated in vivo, but the molecular basis of this function has not been evaluated. Herein, biochemical assays and electron paramagnetic resonance (EPR) spectroscopy are employed to characterize the Mn(II) binding properties of mCP. We report that mCP has one high-affinity Mn(II) binding site. This site is a His6 site composed of His17 and His27 of mS100A8 and His92, His97, His105, and His107 of mS100A9. Similar to the human ortholog (hCP), Ca(II) binding to the EF-hand domains of mCP enhances the Mn(II) affinity of the protein; however, this effect requires ≈10-fold more Ca(II) than was previously observed for hCP. Mn(II) coordination to the His6 site also promotes self-association of two mCP heterodimers to form a heterotetramer. Low-temperature X-band EPR spectroscopy revealed a nearly octahedral Mn(II) coordination sphere for the Mn(II)-His6 site characterized by the zero-field splitting parameters D = 525 MHz and E/D = 0.3. Further electron-nuclear double resonance studies with globally 15N-labeled mCP provided hyperfine couplings from the coordinating ε-nitrogen atoms of the His ligands (aiso = 4.3 MHz) as well as the distal δ-nitrogen atoms (aiso = 0.25 MHz). Mn(II) competition assays between mCP and two bacterial Mn(II) solute-binding proteins, staphylococcal MntC and streptococcal PsaA, showed that mCP outcompetes both proteins for Mn(II) under conditions of excess Ca(II). In total, this work provides the first coordination chemistry study of mCP and reveals striking similarities in the Mn(II) coordination sphere as well as notable differences in the Ca(II) sensitivity and oligomerization behavior between hCP and mCP.

Project description: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.

Project description:Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts, including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to those grown in control cultures but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum, indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here, we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to posttranslationally inhibit zinc metalloprotease activity and thereby affect the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. IMPORTANCE The factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases, suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.

Dataset Information

Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

Publications

Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

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