For present purposes, a protein-bound metal site consists of one or more metal ions and all protein side chain and exogenous bridging and terminal ligands that define the first coordination sphere of each metal ion. Such sites can be classified into five basic types with the indicated functions: (1) structural -- configuration (in part) of protein tertiary and/or quaternary structure; (2) storage -- uptake, binding, and release of metals in soluble form: (3) electron transfer -- uptake, release, and storage of electrons; (4) dioxygen binding -- metal-O{sub 2} coordination and decoordination; and (5) catalytic -- substrate binding, activation, and turnover. The authors present here a classification and structure/function analysis of native metal sites based on these functions, where 5 is an extensive class subdivided by the type of reaction catalyzed. Within this purview, coverage of the various site types is extensive, but not exhaustive. The purpose of this exposition is to present examples of all types of sites and to relate, insofar as is currently feasible, the structure and function of selected types. The authors largely confine their considerations to the sites themselves, with due recognition that these site features are coupled to protein structure at all levels. In themore » next section, the coordination chemistry of metalloprotein sites and the unique properties of a protein as a ligand are briefly summarized. Structure/function relationships are systematically explored and tabulations of structurally defined sites presented. Finally, future directions in bioinorganic research in the context of metal site chemistry are considered. 620 refs.« less

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Structural and Functional Aspects of Metal Sites in Biology

Zinc enzymology is, compared to some other current areas of metallobiochemistry, a maturing field, but in addition to further developments of structure-function relationships it has also provided a number of surprising new results and ideas in the last few years. In fact, the number of studies makes it impossible to provide a comprehensive review of the recent literature on zinc enzymology here, and the authors therefore focus on those zinc enzymes for which structure-function relationships are possible on the basis of structural and biochemical data. This means that, with a few exceptions, only zinc enzymes for which NMR or crystal structures are available are included here. Another seemingly simple, yet experimentally sometimes complex issue concerns the choice of which metalloenzyme is a zinc enzyme. Since there is in principle no difference in chemical catalysis by low-affinity compared to high-affinity metal sites, some of these enzymes are also included in this article, especially if they are or have been discussed as zinc enzymes, or are active with zinc. 552 refs.

Recent Advances in Zinc Enzymology

Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynaptic neurotoxins acting on ion channels are not dealt with here. These neurotoxins can be grouped in three large families: 1) the clostridial neurotoxins that act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins; 2) the snake presynaptic neurotoxins with phospholipase A(2) activity, whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions; and 3) the excitatory latrotoxin-like neurotoxins that induce a massive release of neurotransmitter at peripheral and central synapses. Their modes of binding, sites of action, and biochemical activities are discussed in relation to the symptoms of the diseases they cause. The use of these toxins in cell biology and neuroscience is considered as well as the therapeutic utilization of the botulinum neurotoxins in human diseases characterized by hyperfunction of cholinergic terminals.

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.2.717

Neurotoxins Affecting Neuroexocytosis

To resolve cellular heterogeneity, we developed a combinatorial indexing strategy to profile the transcriptomes of single cells or nuclei, termed sci-RNA-seq (single-cell combinatorial indexing RNA sequencing). We applied sci-RNA-seq to profile nearly 50,000 cells from the nematode Caenorhabditis elegans at the L2 larval stage, which provided >50-fold “shotgun” cellular coverage of its somatic cell composition. From these data, we defined consensus expression profiles for 27 cell types and recovered rare neuronal cell types corresponding to as few as one or two cells in the L2 worm. We integrated these profiles with whole-animal chromatin immunoprecipitation sequencing data to deconvolve the cell type–specific effects of transcription factors. The data generated by sci-RNA-seq constitute a powerful resource for nematode biology and foreshadow similar atlases for other organisms.

/pdf/comprehensive-single-cell-transcriptional-profiling-of-a-4eahlwn5oc.pdf

Comprehensive single-cell transcriptional profiling of a multicellular organism

Zinc is required for the activity of > 300 enzymes, covering all six classes of enzymes. Zinc binding sites in proteins are often distorted tetrahedral or trigonal bipyramidal geometry, made up of the sulfur of cysteine, the nitrogen of histidine or the oxygen of aspartate and glutamate, or a combination. Zinc in proteins can either participate directly in chemical catalysis or be important for maintaining protein structure and stability. In all catalytic sites, the zinc ion functions as a Lewis acid. Researchers in our laboratory are dissecting the determinants of molecular recognition and catalysis in the zinc-binding site of carbonic anhydrase. These studies demonstrate that the chemical nature of the direct ligands and the structure of the surrounding hydrogen bond network are crucial for both the activity of carbonic anhydrase and the metal ion affinity of the zinc-binding site. An understanding of naturally occurring zinc-binding sites will aid in creating de novo zinc-binding proteins and in designing new metal sites in existing proteins for novel purposes such as to serve as metal ion biosensors.

Function and mechanism of zinc metalloenzymes.

The structure of the zinc peptidase astacin in complex with a phosphinic peptide suggests that a special role is played by the side chain of a zinc-bound tyrosine, which is shifted to form a hydrogen bond to the phosphinyl group—a mimic of the carboxyanion of the transition state.

Structure of astacin with a transition-state analogue inhibitor

Refined 1.8 A X-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L. Structure determination, refinement, molecular structure and comparison with thermolysin.

For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.

Biosynthesis of Astacus protease, a digestive enzyme from crayfish.

Astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., represents a structurally distinct group of metalloproteinases termed the 'astacin family'. This protein family includes oligomeric membrane-bound proteins with zinc proteinase domains found in rodent kidneys (meprins A and B) and human small intestine (N-benzoyl-L-tyrosyl-4-aminobenzoate hydrolase). Another branch of this family comprises morphogenetically active proteins, which induce bone formation (human bone morphogenetic protein 1), or which play specific roles during the embryonic development of amphibians, fishes, echinoderms, and insects. The X-ray crystal structure of astacin has recently been solved to a resolution of 0.18 nm [Bode et al. (1992) Nature 358, 164-167]. This structure is different from hitherto known metalloendopeptidase structures and has been used in the present study to analyze the structures of the other members of the astacin protein family. Computer-assisted modelling of the proteolytic domain of the alpha-subunit of meprin A based on the astacin structure is possible if five single and one double residue deletions and three single residue insertions are implied. The proteinase domains of the other astacins can be included in the model-based sequence alignment by introducing additionally three insertions and one deletion. All of these insertions and deletions are observed in loop segments connecting regular secondary structure elements and should leave the overall structure unaltered. The topology of residues forming the zinc-binding active site of astacin corresponds to almost identical arrangements in all other astacins, suggesting that these are likewise metalloproteinases. Based on this similarity, it is proposed that the active-site metal ion of the astacins is penta-coordinated by three histidine residues, a tyrosine residue and a water molecule in a trigonal bipyramidal geometry. Other remarkable common features are a hydrophobic cluster in the N-terminal domain and a conserved, solvent-filled cavity buried in the C-terminal domain. Most interestingly, the amino-termini of all astacins can be modelled to start in a corresponding internal water cavity as seen in the astacin template, where the terminal alanine residue forms a water-linked salt bridge to Glu103, directly adjacent to His102, the third zinc ligand. Therefore, an activation mechanism for the astacins reminiscent of that of the trypsin-like proteinases had been suggested, which now seems to be probable also for the other astacins. Besides these common traits, there are some minor differences which may have important consequences on the function of the astacins.(ABSTRACT TRUNCATED AT 400 WORDS)

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb17915.x

Implications of the three‐dimensional structure of astacin for the structure and function of the astacin family of zinc‐endopeptidases

In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1033.2003.03891.x

Robert Zwilling

Papers

Structure of astacin with a transition-state analogue inhibitor

Refined 1.8 A X-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L. Structure determination, refinement, molecular structure and comparison with thermolysin.

Biosynthesis of Astacus protease, a digestive enzyme from crayfish.

Implications of the three‐dimensional structure of astacin for the structure and function of the astacin family of zinc‐endopeptidases

The astacin protein family in Caenorhabditis elegans