Autotransporter passenger domain secretion requires a hydrophobic cavity at the extracellular entrance of the β-domain pore

Whooping cough (pertussis) is a highly contagious acute respiratory illness of humans caused by the Gram-negative bacterial pathogen Bordetella pertussis. The AT (autotransporter) BrkA (Bordetella serum-resistance killing protein A) is an important B. pertussis virulence factor that confers serum resistance and mediates adherence. In the present study, we have solved the crystal structure of the BrkA β-domain at 3 ? (1 ?=0.1 nm) resolution. Special features are a hairpin-like structure formed by the external loop L4, which is observed fortuitously sitting inside the pore of the crystallographic adjacent β-domain, and a previously undiscovered hydrophobic cavity formed by patches on loop L4 and β-strands S5 and S6. This adopts a ubiquitous structure characteristic of all AT β-domains. Mutagenesis studies have demonstrated that the hairpin-like structure and hydrophobic cavity are crucial for BrkA passenger domain (virulence effector) translocation. This structure helps in understanding the molecular mechanism of AT assembly and secretion and provides a potential target for anti-pertussis drug design.     

The β-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors

Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central β-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the β-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host.     

DNA-binding specificity of the Lon protease α-domain from Brevibacillus thermoruber WR-249

Lon protease has been well studied in many aspects; however, the DNA-binding specificity of Lon in prokaryotes has not been clearly identified. Here we examined the DNA-binding activity of Lon protease α-domains from Brevibacillus thermoruber (Bt), Bacillus subtilis (Bs), and Escherichia coli (Ec). MALDI-TOF mass spectroscopy showed that the α-domain from Bt-Lon binds to the duplex nucleotide sequence 5′-CTGTTAGCGGGC-3′ (ms1) and protected it from DNase I digestion. Surface plasmon resonance showed that the Bt-Lon α-domain binds with ms1 double-stranded DNA tighter than Bs- and Ec-Lon α-domains, whereas the Bt-Lon α-domain has dramatically lower affinity for double-stranded DNA with 0 and 50% identity to the ms1 binding sequence. Our results indicated that Bt-Lon α-domain plays a critical role with ms1 sequence in the DNA-binding specificity.     

Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain

Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with Fe^II₄-MTα and Co^II₄-MTα species existing in water. Furthermore, the Fe^II₄-MTα species was found to promote the reduction of met-myoglobin and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red with Fe^II₄-MTα (1:1) was found to proceed with a conversion of 98% over a period of 6 h at 25 °C. This indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation and reactivity of the tetranuclear iron cluster in the protein matrix.     

The future of the β-lactams

In the 80 years since their discovery the β-lactam antibiotics have progressed through structural generations, each in response to the progressive evolution of bacterial resistance mechanisms. The generational progression was driven by the ingenious, but largely empirical, manipulation of structure by medicinal chemists. Nonetheless, the true creative force in these efforts was Nature, and as the discovery of new β-lactams from Nature has atrophied while at the same time multi-resistant and opportunistic bacterial pathogens have burgeoned, the time for empirical drug discovery has passed. We concisely summarize recent developments with respect to bacterial resistance, the identity of the new β-lactams, and the emerging non-empirical strategies that will ensure that this incredible class of antibiotics has a future.     

Genetic relationship between β-galactosidase and β-fucosidase in the mouse

Evidence for the identity of β-galactosidase and β-fucosidase enzymes in the house mouse was obtained by examination of the enzyme activities in animals from different crosses between C57BL/Kl mice with high galactosidase and fucosidase activities and DBA/2/Kl mice with low activities. There is a strong correlation between the activities of these two enzymes in different tissues of F₂ animals. A comparison of the fractionation properties of β-galactosidase and β-fucosidase showed that the two activities had a parallel distribution and identical thermostability. These data suggest that the same enzyme catalyzes the hydrolysis of both substrates.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

Effect of α-domain substitution on the structure, property and function of human neuronal growth inhibitory factor

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal β-domain rather than its C-terminal α-domain. However, previous bioassay results have shown that the single β-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the α-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the β(MT3)–β(MT3) mutant and the β(MT3)–α(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd₃S₉ cluster of the β(MT3)–β(MT3) mutant decreased significantly while the Cd₃S₉ cluster of the β(MT3)–α(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the β(MT3)–β(MT3) mutant decreased significantly, while the β(MT3)–α(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the α-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the β-domain by domain–domain interactions, thus influencing the bioactivity of hMT3. Z.-C. Ding and Q. Zheng contributed equally to this work.     

Fungal β-trefoil trypsin inhibitors cnispin and cospin demonstrate the plasticity of the β-trefoil fold

The recently identified fungal protease inhibitors cnispin, from Clitocybe nebularis, and cospin, from Coprinopsis cinerea, are both β-trefoil proteins highly specific for trypsin. The reactive site residue of cospin, Arg27, is located on the β2–β3 loop. We show here, that the reactive site residue in cnispin is Lys127, located on the β11–β12 loop. Cnispin is a substrate-like inhibitor and the β11–β12 loop is yet another β-trefoil fold loop recruited for serine protease inhibition. By site-directed mutagenesis of the P1 residues in the β2–β3 and β11–β12 loops in cospin and cnispin, protease inhibitors with different specificities for trypsin and chymotrypsin inhibition have been engineered. Double headed inhibitors of trypsin or trypsin and chymotrypsin were prepared by introducing a second specific site residue into the β2–β3 loop in cnispin and into the β11–β12 loop in cospin. These results show that β-trefoil protease inhibitors from mushrooms exhibit broad plasticity of loop utilization in protease inhibition.     

Molecular Cloning of the Dog β1 and β2 Adrenergic Receptors

Abstract

We report the isolation of the genes encoding the β1 and β2 adrenergic receptors from dog genomic DNA. Sequence analysis of both genes revealed intronless open reading frames of 473 and 415 amino acid residues, receptively. Heterologous expression of both receptors in CHO cells indicated that both receptors are functionally similar to the human homologs. Comparing the dog β1 and β2 adrenergic receptors, the β1 receptor appears to bind to G proteins more tightly than the β2 receptor. Heterologously expressed receptors provide a convenient system for evaluating novel receptor agonists and antagonists.     

Bacterial β-peptidyl aminopeptidases: on the hydrolytic degradation of β-peptides

The special chemical and biological features of beta-peptides have been investigated intensively during recent years. Many studies emphasize the restricted biodegradability and the high metabolic stability of this class of compounds. beta-Peptidyl aminopeptidases form the first family of enzymes that hydrolyze a variety of short beta-peptides and beta-amino-acid-containing peptides. All representatives of this family were isolated from Gram-negative bacteria. The substrate specificities of the peptidases vary greatly, but the enzymes have common structural properties, and a similar reaction mechanism can be expected. This review gives an overview on the beta-peptidyl aminopeptidases with emphasis on their biochemical and structural properties. Their possible physiological function is discussed. Functionally and structurally related enzymes are compared to the beta-peptidyl aminopeptidases.     

Comparison of the β-lactamases ofBacteroides species

-Lactam antibiotic susceptibility and the presence of -lactamase were examined in clinical strains ofBacteroides species. All strains produced a noninducible, cell-associated cephalosporinase. Based on isoelectric focusing, molecular weight determinations, substrate profiles, and inhibition studies, it was concluded that allBacteroides strains examined produced a very similar, if not identical, -lactamase in terms of these enzymatic and physical characteristics.     

Cloning of the β2-microglobulin gene in the zebrafish

The ₂-microglobulin (₂m) is a protein found in the serum in a free form and on the cell surface in a form noncovalently associated with the chain of the class I major histocompatibility complex (Mhc) molecules. In mammals, the ₂m-encoding gene (B2m) is found on a chromosome different from the Mhc proper. We have isolated and characterized the B2m gene of the zebrafish, Brachydanio rerio, family Cyprinidae. We obtained both cDNA and genomic clones of the Brre-B2m gene. The cDNA clones contained the entire coding sequence, the entire 3 untranslated (UT) region, and at least part of the 5UT region. The genomic clone contained the entire Brre-B2m gene. The coding sequence specifies 97 amino acid residues of the mature protein so that the zebrafish ₂m is two residues shorter than human and one residue shorter than cattle, fowl, or turkey ₂m (codons at positions 85 and 86 have been deleted in the Brre-B2m. gene). The amino acid and nucleotide sequence similarities between zebrafish and human ₂m (B2m) are 45% and 59%, respectively. Approximately 24% of the positions are invariant and an additional 9% show only conservative substitutions in comparisons which include all known 2m sequences (fish, avian, and mammalian). Most of the conserved positions are in the strands (some 47% of the -strand positions are conserved in the three vertebrate classes). The Brre-B2m gene consists of four exons separated by three introns. All of the introns are considerably shorter than the corresponding introns in the mammalian B2m genes. The coding sequences of the cDNA and the genomic clones are almost identical but the sequences of the 3'UT regions differ at 1.7% of the sites, suggesting that the genes borne by these clones might have diverged at least 0.7 million years (my) ago. In contrast to the human B2m gene, the Brre-B2m gene shows no bias in the distribution of the CpG dinucleotides: the dinucleotides are distributed evenly along the entire available sequence. The haploid genome of the zebrafish contains only one copy of the B2m gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L05383 (B2M) and L05384 (B2RG). Correspondence to: J. Klein.     

Lipases in the preparation of β-blockers

β-Adrenergic blocking agents are pharmaceutical products used for the treatment of hypertension and angina pectoris. These β-blockers can be prepared in optically active form by conventional crystallization procedures, by asymmetric chiral synthesis and by using stereoselective enzymes.