The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin-echo envelope modulation and electron nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed 15N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by ca. two orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His6 site of human calprotectin.

Molybdenum nitrogenase catalyzes the reduction of dinitrogen into ammonia, which requires the coordinated transfer of eight electrons to the active site cofactor (FeMoco) through the intermediacy of an [8Fe-7S] cluster (P-cluster), both housed in the molybdenum–iron protein (MoFeP). Previous studies on MoFeP from two different organisms, Azotobacter vinelandii (Av) and Gluconacetobacter diazotrophicus (Gd), have established that the P-cluster is conformationally flexible and can undergo substantial structural changes upon two-electron oxidation to the POX state, whereby a backbone amidate and an oxygenic residue (Ser or Tyr) ligate to two of the cluster’s Fe centers. This redox-dependent change in coordination has been implicated in the conformationally gated electron transfer in nitrogenase. Here, we have investigated the role of the oxygenic ligand in Av MoFeP, which natively contains a Ser ligand (βSer188) to the P-cluster. Three variants were generated in which (1) the oxygenic ligand was eliminated (βSer188Ala), (2) the P-cluster environment was converted to the one in Gd MoFeP (βPhe99Tyr/βSer188Ala), and (3) two oxygenic ligands were simultaneously included (βPhe99Tyr). Our studies have revealed that the P-cluster can become compositionally labile upon oxidation and reversibly lose one or two Fe centers in the absence of the oxygenic ligand, while still retaining wild-type-like dinitrogen reduction activity. Our findings also suggest that Av and Gd MoFePs evolved with specific preferences for Ser and Tyr ligands, respectively, and that the structural control of these ligands must extend beyond the primary and secondary coordination spheres of the P-cluster. The P-cluster adds to the increasing number of examples of inherently labile Fe–S clusters whose compositional instability may be an obligatory feature to enable redox-linked conformational changes to facilitate multielectron redox reactions.

Human calprotectin (CP, S100A8/S100A9 oligomer) is a metal-sequestering host-defense protein that prevents bacterial acquisition of Mn(II). In this work, we investigate Mn(II) competition between CP and two solute-binding proteins that Staphylococcus aureus and Streptococcus pneumoniae, Gram-positive bacterial pathogens of significant clinical concern, use to obtain Mn(II) when infecting a host. Biochemical and electron paramagnetic resonance (EPR) spectroscopic analyses demonstrate that CP outcompetes staphylococcal MntC and streptococcal PsaA for Mn(II). This behavior requires the presence of excess Ca(II) ions, which enhance the Mn(II) affinity of CP. This report presents new spectroscopic evaluation of two Mn(II) proteins important for bacterial pathogenesis, direct observation of Mn(II) sequestration from bacterial Mn(II) acquisition proteins by CP, and molecular insight into the extracellular battle for metal nutrients that occurs during infection.

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 His 6 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 His 6 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)-His 6 site characterized by the zero-field splitting parameters D = 525 MHz and E/D = 0.3. Further electron-nuclear double resonance studies with globally 15 N-labeled mCP provided hyperfine couplings from the coordinating ε-nitrogen atoms of the His ligands (a iso = 4.3 MHz) as well as the distal δ-nitrogen atoms (a iso = 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.

To mimic a hypothetical pathway for protein evolution, we previously tailored a monomeric protein (cyt cb 562) for metal-mediated self-assembly, followed by redesign of the resulting oligomers for enhanced stability and metal-based functions. We show that a single hydrophobic mutation on the cyt cb 562 surface drastically alters the outcome of metal-directed oligomerization to yield a new trimeric architecture, (TriCyt1) 3. This nascent trimer was redesigned into second and third-generation variants (TriCyt2) 3 and (TriCyt3) 3 with increased structural stability and preorganization for metal coordination. The three TriCyt variants combined furnish a unique platform to 1) provide tunable coupling between protein quaternary structure and metal coordination, 2) enable the construction of metal/pH-switchable protein oligomerization motifs, and 3) generate a robust metal coordination site that can coordinate all mid-to-late first-row transition-metal ions with high affinity.