Partially folded bovine pancreatic trypsin inhibitor analogues attain fully native structures when co-crystallized with S195A rat trypsin.

Crystal structures, at 1.7 A resolution, were solved for complexes between each of two chemically synthesized partially folded analogues of bovine pancreatic trypsin inhibitor (BPTI) with the proteolytically inactive rat trypsin mutant S195A. The BPTI analogue termed [14-38](Abu) retains only the disulfide bond between Cys14 and Cys38, while Cys5, Cys30, Cys51, and Cys55 are replaced by isosteric alpha-amino-n-butyric acid residues. The analogue K26P,A27D[14-38](Abu) contains two further replacements, by statistically favored residues, in the type I beta-turn that has been suggested to be a main site for initiation of BPTI folding. As a control, the structure of the complex between S195A trypsin and wild-type BPTI was also solved. Despite significant differences in the degree of structure detected among these three BPTIs in solution by several biophysical techniques, their tertiary folds once bound to S195A trypsin in a crystalline lattice are essentially superimposable.

Structure and biological activities of beta toxin from Staphylococcus aureus.

Beta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-A resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.

Structure of the streptococcal cell wall C5a peptidase.

The structure of a cell surface enzyme from a gram-positive pathogen has been determined to 2-A resolution. Gram-positive pathogens have a thick cell wall to which proteins and carbohydrate are covalently attached. Streptococcal C5a peptidase (SCP), is a highly specific protease and adhesin/invasin. Structural analysis of a 949-residue fragment of the [D130A,S512A] mutant of SCP from group B Streptococcus (S. agalactiae, SCPB) revealed SCPB is composed of five distinct domains. The N-terminal subtilisin-like protease domain has a 134-residue protease-associated domain inserted into a loop between two beta-strands. This domain also contains one of two Arg-Gly-Asp (RGD) sequences found in SCPB. At the C terminus are three fibronectin type III (Fn) domains. The second RGD sequence is located between Fn1 and Fn2. Our analysis suggests that SCP binding to integrins by the RGD motifs may stabilize conformational changes required for substrate binding.

Structure of peptide sex pheromone receptor PrgX and PrgX/pheromone complexes and regulation of conjugation in Enterococcus faecalis.

Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone binding destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.

Roles of the equatorial tyrosyl iron ligand of protocatechuate 3,4-dioxygenase in catalysis.

The active site Fe(III) of protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas putida is ligated axially by Tyr447 and His462 and equatorially by Tyr408, His460, and OH(-). Tyr447 and OH(-) are displaced as protocatechuate (3,4-dihydroxybenzoate, PCA) chelates the iron and appear to serve as in situ bases to promote this process. The role(s) of Tyr408 is (are) explored here using mutant enzymes that exhibit less than 0.1% wild-type activity. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with the iron through solvents. Moreover, PCA binds as a monodentate rather than a bidentate ligand, and Tyr447 fails to dissociate. Although the new residues at position 408 do not directly bind to the iron, large changes in the spectroscopic and catalytic properties are noted among the mutant enzymes. Resonance Raman features show that the Fe-O bond of the monodentate 4-hydroxybenzoate (4HB) inhibitor complex is significantly stronger in the mutants than in wild-type 3,4-PCD. Transient kinetic studies show that PCA and 4HB bind to 3,4-PCD in a fast, reversible step followed by a step in which coordination to the metal occurs; the latter process is at least 50-fold slower in the mutant enzymes. It is proposed that, in wild-type 3,4-PCD, the Lewis base strength of Tyr408 lowers the Lewis acidity of the iron to foster the rapid exchange of anionic ligands during the catalytic cycle. Accordingly, the increase in Lewis acidity of the iron caused by substitution of this residue by solvent tends to make the iron substitution inert. Tyr447 cannot be released to allow formation of the usual dianionic PCA chelate complex with the active site iron, and the rate of electrophilic attack by O(2) becomes rate limiting overall. The structures of the PCA complexes of these mutant enzymes show that hydrogen-bonding interactions between the new solvent ligand and the new second-sphere residue in position 408 allow this residue to significantly influence the spectroscopic and kinetic properties of the enzymes.

Biophysical analyses of designed and selected mutants of protocatechuate 3,4-dioxygenase1.

The catechol dioxygenases allow a wide variety of bacteria to use aromatic compounds as carbon sources by catalyzing the key ring-opening step. These enzymes use specifically either catechol or protocatechuate (2,3-dihydroxybenozate) as their substrates; they use a bare metal ion as the sole cofactor. To learn how this family of metalloenzymes functions, a structural analysis of designed and selected mutants of these enzymes has been undertaken. Here we review the results of this analysis on the nonheme ferric iron intradiol dioxygenase protocatechuate 3,4-dioxygenase.