Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris.

The crystal structures of carboxypeptidase T (CpT) complexes with phenylalanine and arginine substrate analogs - benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid - were determined by the molecular replacement method at resolutions of 1.57 Å and 1.62 Å to clarify the broad substrate specificity profile of the enzyme. The conservative Leu211 and Leu254 residues (also present in both carboxypeptidase A and carboxypeptidase B) were shown to be structural determinants for recognition of hydrophobic substrates, whereas Asp263 was for recognition of positively charged substrates. Mutations of these determinants modify the substrate profile: the CpT variant Leu211Gln acquires carboxypeptidase B-like properties, and the CpT variant Asp263Asn the carboxypeptidase A-like selectivity. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 was shown to be responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S1' subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis. This is a novel insight into the substrate selectivity of metallocarboxypeptidases that demonstrates the importance of interactions between the S1' subsite and the catalytic center.

Antimicrobial activity of different proteins and their fragments from Bacillus thuringiensis parasporal crystals against clostridia and archaea.

Proteins of parasporal crystals (Cry proteins) from entomopathogenic bacterium Bacillus thuringiensis (subspecies kurstaki, galleriae, tenebrionis) as well as some fragments of these proteins, obtained by limited proteolysis, are capable of antimicrobial action against anaerobic bacteria and archaea-Clostridium butyricum, Clostridium acetobutylicum and Methanosarcina barkeri. The MICs are 45-150 microg/mL. Electron microscopy showed that lysis of M. barkeri cells in the presence of 49kDa fragment of Cry3Aa toxin is generally similar to the bacterial cell lysis, which has been previously detected in the presence of Cry11A, Cry1Ab and other Cry proteins. The Cry1D-like toxin from crystals of B. thuringiensis subsp. galleriae has been put forward as an example of the supposition that cell wall and some of its components like teichoic acid and N-acetylgalactosamine have possible influence on Cry toxins, enhancing their antimicrobial activity. The possible ecological role of the antimicrobial activity of Cry proteins is also discussed.

Novel antibacterial proteins from entomocidal crystals of Bacillus thuringiensis ssp. israelensis.

Proteins with molecular masses of 36 and 34 kDa (Bti36 and Bti34) were isolated from entomocidal crystals formed by Bacillus thuringiensis ssp. israelensis cells. The samples of Bti36 contained the admixture of a protein with a molecular mass of 33 kDa (Bti33), apparently a product of proteolysis of Bti36. These 3 proteins are significantly different in N-terminal sequences from known delta-endotoxins of B. thuringiensis and show antibacterial activity toward Micrococcus luteus. The combination of Bti36 and Bti33 also suppresses the growth of some other microorganisms including Streptomyces chrysomallus. The effects of the mixture of Bti36 and Bti33 on the M. luteus cell surface and on the surface of S. chrysomallus cells and exospores are similar, but they are different from the effect of endotoxin Cry11A on micrococcal cells.

In vitro refolding of carboxypeptidase T precursor from Thermoactinomyces vulgaris obtained in Escherichia coli as cytoplasmic inclusion bodies.

Carboxypeptidase T precursor from Thermoactinomyces vulgaris, which fails to contain its own leader peptide, has been expressed in Escherichia coli as insoluble cytoplasmic inclusion bodies. The yield of a washed recombinant protein from 1 L of culture liquid was about 60 mg. The obtained inclusion bodies were denatured in 6 M guanidine-HCl and then renatured by a rapid dilution. The important role of calcium for the complete stabilization of the refolded carboxypeptidase T precursor was established. After removal of minor admixture proteins by gel-filtration through Superdex 75, an electrophoretically homogeneous preparation of the native precursor of carboxypeptidase T was obtained. Processing of the resulting protein by subtilisin led to the formation of the mature carboxypeptidase T in which N-terminal sequence, molecular size, thermal stability, and catalytic properties were comparable to those of the natural enzyme.

The crystal structure of glutamyl endopeptidase from Bacillus intermedius reveals a structural link between zymogen activation and charge compensation.

Extracellular glutamyl endopeptidase from Bacillus intermedius (BIEP) is a chymotrypsin-like serine protease which cleaves the peptide bond on the carboxyl side of glutamic acid. Its three-dimensional structure was determined for C222(1) and C2 crystal forms of BIEP to 1.5 and 1.75 A resolution, respectively. The topology of BIEP diverges from the most common chymotrypsin architecture, because one of the domains consists of a beta-sandwich consisting of two antiparallel beta-sheets and two helices. In the C2 crystals, a 2-methyl-2,4-pentanediol (MPD) molecule was found in the substrate binding site, mimicking a glutamic acid. This enabled the identification of the residues involved in the substrate recognition. The presence of the MPD molecule causes a change in the active site; the interaction between two catalytic residues (His47 and Ser171) is disrupted. The N-terminal end of the enzyme is involved in the formation of the substrate binding pocket. This indicates a direct relation between zymogen activation and substrate charge compensation.

Antibacterial activity of Cry- and Cyt-proteins from Bacillus thuringiensis ssp. israelensis.

Mosquitocidal endotoxins Cry4B, Cry11A, and CytA from Bacillus thuringiensis ssp. israelensis as well as the products of their limited proteolysis display antibacterial activity relative to Micrococcus luteus. The endotoxin Cry11A also induces the lysis of the micrococcus protoplasts. Potassium and sodium ions and N-acetylgalactosamine increased the antibacterial effect of Cry11A, whereas glucose and N-acetylglucosamine inhibited it. The endotoxin Cry11A displays the antibacterial effect on some other microorganisms.

An alternative processing of integrin alpha(v) subunit in tumor cells by membrane type-1 matrix metalloproteinase.

Membrane type-1 matrix metalloproteinase (MT1-MMP) and alpha(v)beta(3) integrin are both essential to cell invasion. Maturation of integrin pro-alpha(v)chain (pro-alpha(v)) involves its cleavage by proprotein convertases (PC) to form the disulfide-bonded 125-kDa heavy and 25-kDa light alpha chains. Our report presents evidence of an alternative pathway of pro-alpha(v) processing involving MT1-MMP. In breast carcinoma MCF7 cells deficient in MT1-MMP, pro-alpha(v) is processed by a conventional furin-like PC, and the mature alpha(v) integrin subunit is represented by the 125-kDa heavy chain and the 25-kDa light chain commencing from the N-terminal Asp(891). In contrast, in cells co-expressing alpha(v)beta(3) and MT1-MMP, MT1-MMP functions as an integrin convertase. MT1-MMP specifically cleaves pro-alpha(v), generating a 115-kDa heavy chain with the truncated C terminus and a 25-kDa light chain commencing from the N-terminal Leu(892). PC-cleavable alpha(3) and alpha(5) but not the PC-resistant alpha(2) integrin subunit are also susceptible to MT1-MMP cleavage. These novel mechanisms involved in the processing of integrin alpha subunits underscore the significance and complexity of interactions between MT1-MMP and adhesion receptors and suggest that regulation of integrin functionality may be an important role of MT1-MMP in migrating tumor cells.