Metal ions in biological catalysis: from enzyme databases to general principles

329

588

The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn 2+ and Zn 2+ with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn 2+ sequestration. The asymmetry of the CP heterodimer creates a single Mn 2+ -binding site from six histidine residues, which distinguishes CP from all other Mn 2+ -binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn 2+ and Zn 2+ being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn 2+ sequestration. These data establish the importance of Mn 2+ sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.bacterial pathogenesis | Staphylococcus aureus | antibiotic resistance | protein crystal structure | isothermal titration calorimetry B acterial pathogens are a significant threat to global public health. This threat is compounded by the fact that these organisms are rapidly becoming resistant to all relevant antimicrobials. Of particular note is the recent emergence of antibiotic-resistant strains of Staphylococcus aureus as a leading cause of bacterial infection in the United States (1) and arguably the most important threat to the public health of the developed world. Consequently, the identification of therapeutics to treat bacterial pathogens is paramount to our continued ability to limit this infectious threat.One promising area of potential therapeutic development involves targeting bacterial access to essential transition metals. This strategy is based on the fact that all bacterial pathogens require these nutrient metals to colonize their hosts (2-5). In vertebrates, the bacterial need for nutrient transition metals is counteracted by the sequestration of these metals by the host. This limitation of essential nutrients, termed "nutritional immunity," is a potent defense against infection (6). Although a variety of metals is required for microbial growth, studies of nutritional immunity have been primarily restricted to the struggle for iron (Fe) between host and pathogen (7-9).An innate immune factor, calprotectin (CP), is abundant in neutrophils and plays a key role in nutritional immunity. CP can be found at sites of infection in excess of 1 mg/mL and is required for the control of a number of medically relevant bacteria and fungi including S. aureus, Candida albicans, and Aspergillus fumigates (10-14). The antimicrobial activity of CP is due to the chelation of the essential nutrients Zn 2+ (Zn) and Mn 2+ (Mn), which results in bacterial metal starvation and is reversed by the addition of these metals in excess (11, 13). Moreover, CP-deficient mice have increased microbial burdens following systemic challenge, und...

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confidence: 99%

Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens

Damo

Kehl-Fie

Sugitani

et al. 2013

329

588

“…Many biological pathways contain at least one metalloenzyme (2) and consequently require Mg, K, Ca, Fe, Mn, and Zn to sustain life (3). Other elements, like Cu, Mo, Ni, Se, and Co, are required by many-though not all-organismal lineages, and the utilization of trace elements varies greatly between species (3).…”

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confidence: 99%

History of biological metal utilization inferred through phylogenomic analysis of protein structures

Dupont

Butcher

Valas³

et al. 2010

275

245

The fundamental chemistry of trace elements dictates the molecular speciation and reactivity both within cells and the environment at large. Using protein structure and comparative genomics, we elucidate several major influences this chemistry has had upon biology. All of life exhibits the same proteome size-dependent scaling for the number of metal-binding proteins within a proteome. This fundamental evolutionary constant shows that the selection of one element occurs at the exclusion of another, with the eschewal of Fe for Zn and Ca being a defining feature of eukaryotic proteomes. Early life lacked both the structures required to control intracellular metal concentrations and the metal-binding proteins that catalyze electron transport and redox transformations. The development of protein structures for metal homeostasis coincided with the emergence of metal-specific structures, which predominantly bound metals abundant in the Archean ocean. Potentially, this promoted the diversification of emerging lineages of Archaea and Bacteria through the establishment of biogeochemical cycles. In contrast, structures binding Cu and Zn evolved much later, providing further evidence that environmental availability influenced the selection of the elements. The late evolving Zn-binding proteins are fundamental to eukaryotic cellular biology, and Zn bioavailability may have been a limiting factor in eukaryotic evolution. The results presented here provide an evolutionary timeline based on genomic characteristics, and key hypotheses can be tested by alternative geochemical methods.Archean-Proterozoic | biogeochemistry | bioinorganic chemistry | evolution | metal homeostasis M etalloproteins contain one or more ions of an inorganic element in their 3D structure and are said to comprise 30% of all proteins (1). Many biological pathways contain at least one metalloenzyme (2) and consequently require Mg, K, Ca, Fe, Mn, and Zn to sustain life (3). Other elements, like Cu, Mo, Ni, Se, and Co, are required by many-though not all-organismal lineages, and the utilization of trace elements varies greatly between species (3). The different elements have a range of affinities for most coordinating environments in the order Mg +2 /Ca +2 < Mn +2 < Fe +2 < Co +2 < Ni +2 < Cu +2 ∼Zn +2 , an equilibrium series known as the Irving-Williams Series (4). This array of outer-sphere chemistry provides significant catalytic diversity, yet has consequences for both biological and environmental chemistry. Within the cell, metals compete for protein binding sites; hence, extensive protein networks involving transporters and metalsensing regulatory proteins are required to maintain the proper subcellular concentrations of each element, and in some cases directly shuttle each metal to its requisite metalloprotein in the proper cellular compartment (1, 5, 6). It has been hypothesized that the establishment of a metal homeostasis system is required for distinct phylotypes of cells to develop (7).Environmentally, for similar fundamental chemical reasons, the...

“…ferric uptake regulator | graded transcription regulation | regulatory metal V arious transition metal ions are essentially required for cell growth and survival because they stabilize the folded conformations or mediate chemical reactions of metalloproteins, which constitute about one-third of all proteins (1,2). Zinc is an abundant transition metal that serves as a cofactor for diverse enzymes and regulatory proteins.…”

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confidence: 99%

Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur

Shin

Jung

et al. 2011

108

189

Zinc is one of the essential transition metals in cells. Excess or lack of zinc is detrimental, and cells exploit highly sensitive zinc-binding regulators to achieve homeostasis. In this article, we present a crystal structure of active Zur from Streptomyces coelicolor with three zinc-binding sites (C-, M-, and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C- and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mutations did not affect sporulation and derepressed only a sensitive gene. Biochemical and spectroscopic analyses of representative metal site mutants revealed that the C-site serves a structural role, whereas the M- and D-sites regulate DNA-binding activity as an on-off switch and a fine-tuner, respectively. Consistent with differential effect of mutations on target genes, zinc chelation by TPEN derepressed some genes ( znuA, rpmF2 ) more sensitively than others ( rpmG2 , SCO7682) in vivo. Similar pattern of TPEN-sensitivity was observed for Zur-DNA complexes formed on different promoters in vitro. The sensitive promoters bound Zur with lower affinity than the less sensitive ones. EDTA-treated apo-Zur gained its DNA binding activity at different concentrations of added zinc for the two promoter groups, corresponding to free zinc concentrations of 4.5 × 10 −16 M and 7.9 × 10 −16 M for the less sensitive and sensitive promoters, respectively. The graded expression of target genes is a clever outcome of subtly modulating Zur-DNA binding affinities in response to zinc availability. It enables bacteria to detect metal depletion with improved sensitivity and optimize gene-expression pattern.