Structure–Function Analysis of the ADAM Family of Disintegrin-Like and Metalloproteinase-Containing Proteins (Review)

The ADAMs belong to a disintegrin-like and metalloproteinase-containing protein family that are zinc-dependent metalloproteinases. These proteins share all or some of the following domain structure: a signal peptide, a propeptide, a metalloproteinase, a disintegrin, a cysteine-rich, and an epidermal growth factor (EGF)-like domains, a transmembrane region, and a cytoplasmic tail. ADAMs are widely distributed in many organs, tissues, and cells, such as brain, testis, epididymis, ovary, breast, placenta, liver, heart, lung, bone, and muscle. These proteins are capable of four potential functions: proteolysis, adhesion, fusion, and intracellular signaling. Because the number of ADAM genes has grown rapidly and the biological functions of most members are unclear, this review analyzes the protein structures and functions, their activation and processing, their known and potential activities, and their evolutionary relationships. A sequence alignment of human ADAMs is compiled and their homology and physical data are calculated. The conceivable functions of ADAMs in reproduction, development, and diseases are also discussed.     

Insight into the Structure of the “Unstructured” Tau Protein

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Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules: TEKTINS,Ca2+-BINDING PROTEINS,AND STABLE PROTOFILAMENTS*?

Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.     

Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabissativa

?1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ?1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75? resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.     

Exploration of Human Salivary Microbiomes—Insights into the Novel Characteristics of Microbial Community Structure in Caries and Caries-Free Subjects

Recently, high-throughput sequencing has improved the understanding of the microbiological etiology of caries, but the characteristics of the microbial community structure in the human oral cavity with and without caries are not completely clear. To better understand these characteristics, Illumina MiSeq high-throughput sequencing was utilized to analyze 20 salivary samples (10 caries-free and 10 caries) from subjects from the same town in Dongxiang, Gansu, China. A total of 5,113 OTUs (Operational Taxonomic Units, 97% cutoff) were characterized in all of the salivary samples obtained from the 20 subjects. A comparison of the two groups revealed that (i) the predominant phyla were constant between the two groups; (ii) the relative abundance of the genera Veillonella, Bifidobacterium, Selenomonas, Olsenella, Parascardovia, Scardovia, Chryseobacterium, Terrimonas, Burkholderia and Sporobacter was significantly higher in the group with caries (P < 0.05); and (iii) four genera with low relative abundance (< 0.01% on average), including two characteristic genera in caries (Chryseobacterium and Scardovia), significantly influenced the microbial community structure at the genus and OTU levels. Moreover, via co-occurrence and principal component analyses, the co-prevalence of the pathogenic genera was detected in the caries samples, but in the caries-free samples, the function of clustered genera was more random. This result suggests that a synergistic effect may be influencing the assembly of the caries microbial community, whereas competition may play a more dominant role in governing the microbial community in the caries-free group. Our findings regarding the characteristics of the microbial communities of the groups with and without caries might improve the understanding of the microbiological etiology of caries and might improve the prevention and cure of caries in the future.     

Structure of Cinaciguat (BAY 58–2667) Bound to Nostoc H-NOX Domain Reveals Insights into Heme-mimetic Activation of the Soluble Guanylyl Cyclase

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58–2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58–2667. The 2.3-Å resolution structure of BAY 58–2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58–2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58–2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58–2667 binding causes a rotation of the αF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His¹⁰⁵–heme covalent bond. The structure provides insights into how BAY 58–2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.     

Crystal Structure of the TNF-α-Inducing Protein (Tipα) from Helicobacter pylori: Insights into Its DNA-Binding Activity

Helicobacter pylori infection is one of the highest risk factors for gastroduodenal diseases including gastric cancer. Tumor necrosis factor-alpha (TNF-α) is one of the essential cytokines for tumor promotion, and thus, an H. pylori protein that induces TNF-α is believed to play a significant role in gastric cancer development in humans. The HP0596 gene product of H. pylori strain 26695 was identified as the TNF-α-inducing protein (Tipα). Tipα is secreted from H. pylori as dimers and enters the gastric cells. It was shown to have a DNA-binding activity. Here, we have determined the crystal structure of Tipα from H. pylori. Its monomer consists of two structural domains (“mixed domain” and “helical domain”). Tipα exists as a dimer in the crystal, and the dimeric structure represents a novel scaffold for DNA binding. A positively charged surface patch formed across the two monomers of the Tipα dimer by the loop between helices α1 and α2 may be important in DNA binding.     

Comparison of Macular Integrity Assessment (MAIA ?), MP-3, and the Humphrey Field Analyzer in the Evaluation of the Relationship between the Structure and Function of the Macula

Purpose

This study was conducted in order to compare relationships between the macular visual field (VF) mean sensitivity measured by MAIA^TM (Macular Integrity Assessment), MP-3, or Humphry field analyzer (HFA) and the ganglion cell and inner plexiform layer (GCA) thicknesses.

Methods

This cross-sectional study examined 73 glaucoma patients and 19 normal subjects. All subjects underwent measurements for GCA thickness by Cirrus HD-OCT and static threshold perimetry using MAIA^TM, MP-3, or HFA. VF and OCT in the retinal view were used to examine both the global relationship between the VF sensitivity and GCA thickness, and the superior hemiretina and inferior hemiretina. The relationship between the GCA thickness and macular sensitivity was examined by Spearman correlation analysis.

Results

For each instrument, statistically significant macular VF sensitivity (dB) and GCA thickness relationships were observed using the decibel scale (R = 0.547–0.687, all P < 0.001). The highest correlation for the global (R = 0.682) and the superior hemiretina (R = 0.594) GCA thickness-VF mean sensitivity was observed by the HFA. The highest correlation for the inferior hemiretina (R = 0.687) GCA thickness-VF mean sensitivity was observed by the MP-3. Among the three VF measurement instruments, however, no significant differences were found for the structure-function relationships.

Conclusions

All three VF measurement instruments found similar structure-function relationships in the central VF.     

Structure,Function, and Dynamics of the Gα Binding Domain of Ric-8A

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Differential Nutrient Limitation of Soil Microbial Biomass and Metabolic Quotients (qCO2): Is There a Biological Stoichiometry of Soil Microbes?

Background

Variation in microbial metabolism poses one of the greatest current uncertainties in models of global carbon cycling, and is particularly poorly understood in soils. Biological Stoichiometry theory describes biochemical mechanisms linking metabolic rates with variation in the elemental composition of cells and organisms, and has been widely observed in animals, plants, and plankton. However, this theory has not been widely tested in microbes, which are considered to have fixed ratios of major elements in soils.

Methodology/Principal Findings

To determine whether Biological Stoichiometry underlies patterns of soil microbial metabolism, we compiled published data on microbial biomass carbon (C), nitrogen (N), and phosphorus (P) pools in soils spanning the global range of climate, vegetation, and land use types. We compared element ratios in microbial biomass pools to the metabolic quotient qCO₂ (respiration per unit biomass), where soil C mineralization was simultaneously measured in controlled incubations. Although microbial C, N, and P stoichiometry appeared to follow somewhat constrained allometric relationships at the global scale, we found significant variation in the C∶N∶P ratios of soil microbes across land use and habitat types, and size-dependent scaling of microbial C∶N and C∶P (but not N∶P) ratios. Microbial stoichiometry and metabolic quotients were also weakly correlated as suggested by Biological Stoichiometry theory. Importantly, we found that while soil microbial biomass appeared constrained by soil N availability, microbial metabolic rates (qCO₂) were most strongly associated with inorganic P availability.

Conclusions/Significance

Our findings appear consistent with the model of cellular metabolism described by Biological Stoichiometry theory, where biomass is limited by N needed to build proteins, but rates of protein synthesis are limited by the high P demands of ribosomes. Incorporation of these physiological processes may improve models of carbon cycling and understanding of the effects of nutrient availability on soil C turnover across terrestrial and wetland habitats.     

Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-α-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 ? resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the αD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.     

Probing the Structure–Function relationship and amyloidogenic propensities in natural variants of apolipoprotein A-I

BackgroundApolipoprotein A-I (apoA-I) protects against atherosclerosis and participates in the removal of excess cellular cholesterol from peripheral organs. Several naturally occurring apoA-I mutations are associated with familial systemic amyloidosis, with deposition of amyloid aggregates in peripheral organs, resulting in multiple organ failure. Systematic studies on naturally occurring variants are needed to delineate their roles and involvement in pathogenesis.MethodsWe performed a comparative structure–function analysis of five naturally occurring apoA-I variants and the wild-type protein. Circular dichroism, Fourier-transform infrared spectroscopy, thioflavin T and congo red fluorescence assays, thermal, chemical, and proteolytic stability assays, and 1,2-Dimyristoyl-sn-glycero-3-phosphocholine clearance analyses were used to assess the effects of mutations on the structure, function, stability, aggregation, and proteolytic susceptibility of the proteins to explore the mechanisms underlying amyloidosis and hypercholesterolemia.ResultsWe observed structural changes in the mutants independent of fibril formation, suggesting the influence of the surrounding environment. The mutants were involved in aggregate formation to varying degree; L170P, R173P, and V156E showed an increased propensity to aggregate under different physiological conditions. β sheet formation indicates that L170P and R173P participate in amyloid formation. Compared to WT, V156E and L170P exhibited higher capacity for lipid clearance.ConclusionsThe selected point mutations, including those outside the hot spot regions of apoA-I structure, perturb the physiochemical and conformational behavior of the protein, influencing its function.General significanceThe study provides insights into the structure–function relationships of naturally occurring apoA-I variants outside the hot spot mutation sites.