The control of the urokinase-catalyzed activation of human glutamic acid 1-plasminogen by positive and negative effectors

The urokinase-catalyzed activation of human Glu1-plasminogen (Glu1Pg) has been found to be inhibited by monovalent anions in the following order of effectiveness: I- greater than SCN- greater than Cl- greater than IO3- greater than HCOO- greater than F- greater than OAc-. The inhibition is reversed by epsilon-aminocaproic acid, with its effectiveness in this capacity generally inversely proportional to the strength of the binding of the anion. The physical basis for the anion inhibition and epsilon-aminocaproic acid stimulation lies in the ability of these effectors to cause measurable opposite alterations in the conformation of Glu1Pg, which are revealed through study of the sedimentation velocity of the protein under various conditions. The kinetic mechanism of the chloride inhibition of Glu1Pg activation has been examined in detail. It has been found that the Glu1Pg.Cl complex serves as an alternate substrate to Glu1Pg for urokinase, with a greatly increased Km (25 +/- 3 and 2.2 +/- 0.3 microM, respectively) for activation. The kcat for the urokinase.Glu1Pg.Cl complex is approximately the same as that for urokinase.Glu1Pg (1.6 +/- 0.2 - 2.0 +/- 0.2/s). Similarly, the stimulation by epsilon-aminocaproic acid also results from effects on the Km of the activation, which is reduced to 1.8 +/- 0.2 microM for the Glu1Pg.Cl.epsilon-aminocaproic acid complex. The kcat for the urokinase.Glu1Pg.Cl.epsilon-aminocaproic acid of 2.4 +/- 0.3/s complex is not greatly different from that for urokinase.Glu1Pg.Cl. Nuclear magnetic resonance studies of the Glu1Pg-induced line broadening of the 35Cl- spectra in the presence and absence of epsilon-aminocaproic acid suggest that Cl- and epsilon-aminocaproic acid simultaneously bind to the protein and that each of these effectors displays its effects through separate binding sites.     

The protein Id: a negative regulator of helix-loop-helix DNA binding proteins

We have isolated a cDNA clone encoding a novel helix-loop-helix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodimeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal differentiation, Id RNA levels decrease upon induction. Transfection experiments indicate that over-expression of Id inhibits the trans-activation of the muscle creatine kinase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a basic region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimeric complexes.     

À la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors

To investigate the activation mechanism of the enhancer-binding protein XylR encoded by the TOL plasmid of Pseudomonas putida mt-2, a combinatorial library was generated composed of shuffled N-terminal A domains of the homologous regulators DmpR, XylR and TbuT, reassembled within the XylR structure. When the library was screened in vivo for responsiveness to non-effectors bulkier than one aromatic ring (such as biphenyl) or bearing an entirely different distribution of electronegative groups (e.g. nitrotoluenes), protein variants were found that displayed an expanded inducer range including the new effectors. Although the phenotypes endowed with the corresponding changes were largely similar, the modifications involved different sites within the A domain. The positions of the mutations within a structural model of the A domain suggest that expansion of the inducer profile can be brought about not only by changes in the effector pocket of the protein but also by unlocking steps of the signal transmission mechanism that follows effector binding. These results provide a rationale for evolving in vitro regulators à la carte that are responsive to predetermined, natural or xenobiotic chemical species.     

Regulators of the Bacillus subtilis cydABCD operon: identification of a negative regulator, CcpA, and a positive regulator, ResD

The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions -58 and -107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3'-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions -4 to -33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.