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The possible role of the central b-domain~residues 151-287! of streptokinase~SK! was probed by site-specifically altering two charged residues at a time to alanines in a region~residues 230-290! previously identified by Peptide Walking to play a key role in plasminogen~PG! activation. These mutants were then screened for altered ability to activate equimolar "partner" human PG, or altered interaction with substrate PG resulting in an overall compromised capability for substrate PG processing. Of the eight initial alanine-linker mutants of SK, one mutant, viz. SK KK256,257AÃ SK-D1!, showed a roughly 20-fold reduction in PG activator activity in comparison to wild-type SK expressed in Escherichia coli~nSK!. Five other mutants were as active as nSK, with two @SK RE248.249AA and SK EK281.282AA , referred to as SK~C! and SK~H!, respectively# showing specific activities approximately one-half and two-thirds, respectively, that of nSK. Unlike SK~C! and SK~H!, however, SK~D1! showed an extended initial delay in the kinetics of PG activation. These features were drastically accentuated when the charges on the two Lys residues at positions 256 and 257 of nSK were reversed, to obtain SK KK256.257EE @SK~D2!#. This mutant showed a PG activator activity approximately 10-fold less than that of SK~D1!. Remarkably, inclusion of small amounts of human plasmin~PN! in the PG activation reactions of SK~D2! resulted in a dramatic, PN dose-dependent rejuvenation of its PG activation capability, indicating that it required pre-existing PN to form a functional activator since it could not effect active site exposure in partner PG on its own, a conclusion further confirmed by its inability to show a "burst" of p-nitrophenol release in the presence of equimolar human PG and p-nitrophenyl guanidino benzoate. The steady-state kinetic parameters for HPG activation of its 1:1 complex with human PN revealed that although it could form a highly functional activator once "supplied" with a mature active site, the K m for PG was increased nearly eightfold in comparison to that of nSK-PN. SK mutants carrying simultaneous two-and three-site charge-cluster alterations, viz., SK RE248.249AA;EK281.282AA @SK~CH!#, SK EK272.273AA;EK281.282AA @SK~FH!#, and SK RE248.249AA;EK272.273AA;EK281.282AA @SK~CFH!#, showed additive0synergistic influence of multiple charge-cluster mutations on HPG activation when compared to the respective "single-site" mutants, with the "triple-site" mutant @SK~CFH!# showing absolutely no detectable HPG activation ability. Nevertheless, like the other constructs, the double-and triple-charge cluster mutants retained a native like affinity for complexation with partner PG. Their overall structure also, as judged by far-ultraviolet circular dichroism, was closely similar to that of nSK. These results provide the first experimental evidence for a direct assistance by the SK b-domain in the docking and processing of substrate PG by the activator complex, a facet not readily evident probably because of the flexibility of this dom...

Studies of sulfidic springs have provided new insights into microbial metabolism, groundwater biogeochemistry, and geologic processes. We investigated Great Sulphur Spring on the western shore of Lake Erie and evaluated the phylogenetic affiliations of 189 bacterial and 77 archaeal 16S rRNA gene sequences from three habitats: the spring origin (11-m depth), bacterial-algal mats on the spring pond surface, and whitish filamentous materials from the spring drain. Water from the spring origin water was cold, pH 6.3, and anoxic (H 2 , 5.4 nM; CH 4 , 2.70 M) with concentrations of S 2؊ (0.03 mM), SO 4 2؊ (14.8 mM), Ca 2؉ (15.7 mM), and HCO 3 ؊ (4.1 mM) similar to those in groundwater from the local aquifer. No archaeal and few bacterial sequences were >95% similar to sequences of cultivated organisms. Bacterial sequences were largely affiliated with sulfur-metabolizing or chemolithotrophic taxa in Beta-, Gamma-, Delta-, and Epsilonproteobacteria. Epsilonproteobacteria sequences similar to those obtained from other sulfidic environments and a new clade of Cyanobacteria sequences were particularly abundant (16% and 40%, respectively) in the spring origin clone library. Crenarchaeota sequences associated with archaeal-bacterial consortia in whitish filaments at a German sulfidic spring were detected only in a similar habitat at Great Sulphur Spring. This study expands the geographic distribution of many uncultured Archaea and Bacteria sequences to the Laurentian Great Lakes, indicates possible roles for epsilonproteobacteria in local aquifer chemistry and karst formation, documents new oscillatorioid Cyanobacteria lineages, and shows that uncultured, cold-adapted Crenarchaeota sequences may comprise a significant part of the microbial community of some sulfidic environments.

No abstract

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (␣, ␤, and ␥), and also their twodomain combinations, viz. ␣␤ and ␤␥ in Escherichia coli. After purification, ␣ and ␤ showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs ␣␤ and ␤␥ showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in cofactor activity between the two-and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two-and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK⅐HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SKplasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK⅐HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels. Streptokinase (SK)1 is a widely used bacterial thrombolytic protein that is secreted by several species of ␤-hemolytic streptococci (1, 2). It consists of a single polypeptide chain of 414 residues and is organized into three structurally similar, independently folding domains (termed ␣, ␤, and ␥ in order from N to C terminus of the polypeptide) that are separated by coiled coils and small flexible regions at the two ends (3-5). Like several other well known thrombolytic proteins, such as urokinase and tissue-plasminogen activator, SK exerts its effects through the conversion of human plasminogen (HPG) to its proteolytically active form, plasmin (HPN). Thus, during the treatment of various circulatory disorders, e.g. myocardial infarction, deep vein thrombosis, pulmonary embolism, etc., HPN generated by the activation of HPG helps restore blood flow to the afflicted part by proteolytic dissolution of the fibrin in the pathological clot. In contrast to tissue-plasminogen activator and urokinase, which are intrinsically HPG-specific proteases and thus "directly...