The influence of truncating the carboxy-terminal amino acid residues of streptococcal enolase on its ability to interact with canine plasminogen.

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexa-his N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to an Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer.

Altering Residue 134 Confers an Increased Substrate Range of Alkylated Nucleosides to the E. coli OGT Protein.

O6-Alkylguanine-DNA alkyltransferases (AGTs) are proteins responsible for the removal of mutagenic alkyl adducts at the O6-atom of guanine and O4-atom of thymine. In the current study we set out to understand the role of the Ser134 residue in the Escherichia coli AGT variant OGT on substrate discrimination. The S134P mutation in OGT increased the ability of the protein to repair both O6-adducts of guanine and O4-adducts of thymine. However, the S134P variant was unable, like wild-type OGT, to repair an interstrand cross-link (ICL) bridging two O6-atoms of guanine in a DNA duplex. When compared to the human AGT protein (hAGT), the S134P OGT variant displayed reduced activity towards O6-alkylation but a much broader substrate range for O4-alkylation damage reversal. The role of residue 134 in OGT is similar to its function in the human homolog, where Pro140 is crucial in conferring on hAGT the capability to repair large adducts at the O6-position of guanine. Finally, a method to generate a covalent conjugate between hAGT and a model nucleoside using a single-stranded oligonucleotide substrate is demonstrated.

The Energetics of Streptococcal Enolase Octamer Formation: The Quantitative Contributions of the Last Eight Amino Acids at the Carboxy-Terminus.

The enolase produced by Streptococcus pyogenes is a homo-octamer whose overall shape resembles that of a donut. The octamer is best described as a tetramer of dimers. As such, it contains two types of interfaces. The first is common to almost all enolases as most enolases that have been studied are dimers. The second is unique to the octamers and includes residues near the carboxy-terminus. The primary sequence of the enolase contains 435 residues with an added 19 as an N-terminal hexahistine tag. We have systematically truncated the carboxy-terminus, individually removing the first 8 residues. This gave rise to a series of eight structures containing respectively, 435, 434, 433, 432, 431, 430, 429 and 427 residues. The truncations cause the protein to gradually dissociate from octamers to enzymatically inactive monomers with very small amounts of intermediate tetramers and dimers. We have evaluated the contributions of the missing residues to the monomer/octamer equilibrium using a combination of analytical ultracentrifugation and activity assays. For the dissociation reaction, octamer <== ==> 8 monomer truncation of all eight C-terminal residues resulted in a diminution in the standard Gibbs energy of dissociation of about 59 kJ/mole of octamer relative to the full length protein. Considering that this change is spread over eight subunits, this translates to a change in standard Gibbs interaction energy of less than 8 kJ/mole of monomer distributed over the eight monomers. The resulting proteins, containing 434, 433, 432, 431, 430, 429 and 427 residues per monomer, showed intermediate free energies of dissociation. Finally, three other mutations were introduced into our reference protein to establish how they influenced the equilibrium. The main importance of this work is it shows that for homo-multimeric proteins a small change in the standard Gibbs interaction energy between subunits can have major physiological effects.

The interaction of canine plasminogen with Streptococcus pyogenes enolase: they bind to one another but what is the nature of the structures involved?

For years it has been clear that plasminogen from different sources and enolase from different sources interact strongly. What is less clear is the nature of the structures required for them to interact. This work examines the interaction between canine plasminogen (dPgn) and Streptococcus pyogenes enolase (Str enolase) using analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), fluorescence polarization, dynamic light scattering (DLS), isothermal titration calorimetry (ITC), and simple pull-down reactions. Overall, our data indicate that a non-native structure of the octameric Str enolase (monomers or multimers) is an important determinant of its surface-mediated interaction with host plasminogen. Interestingly, a non-native structure of plasminogen is capable of interacting with native enolase. As far as we can tell, the native structures resist forming stable mixed complexes.

Synthesis, biophysical and repair studies of O6-2'-deoxyguanosine adducts by Escherichia coli OGT.

Oligonucleotides containing modified 2'-deoxyguanosines bearing a seven carbon linker at the O(6)- atom with either a terminal hydroxyl or 2'- deoxyguanosine group have been synthesized as potential intermediates formed during repair of interstrand cross-linked DNA. Repair of these substrates with Escherichia coli OGT was investigated with an assay involving cleavage of the unmodified duplex with the restriction endonuclease PvuII followed by analysis of the products by denaturing polyacrylamide gel electrophoresis. Duplexes containing these modifications were repaired by OGT suggesting that direct repair may play a role, in combination with other repair pathways, in reversing interstrand crosslink DNA damage.

Use of hydrostatic pressure to produce 'native' monomers of yeast enolase.

The effects of hydrostatic pressure on yeast enolase have been studied in the presence of 1 mm Mn(2+). When compared with apo-enolase, and Mg-enolase, the Mn-enzyme differs from the others in three ways. Exposure to hydrostatic pressure does not inactivate the enzyme. If the experiments are performed in the presence of 1 mm Mg(2+), or with apo-enzyme, the enzyme is inactivated [Kornblatt, M.J., Lange R., Balny C. (1998) Eur. J. Biochem 251, 775-780]. The UV spectra of the high pressure forms of the Mg(2+)- and apo-forms of enolase are identical and distinct from the spectrum of the form obtained in the presence of 1 mm Mn(2+); this suggests that Mn(2+) remains bound to the high pressure form of enolase. With Mn-enolase, the various spectral changes do not occur in the same pressure range, indicating that multiple processes are occurring. Pressure experiments were performed as a function of [Mn(2+)] and [protein]. One of the changes in the UV spectra shows a dependence on protein concentration, indicating that enolase is dissociating into monomers. The small changes in the UV spectrum and the retention of activity lead to a model in which enolase, in the presence of high concentrations of Mn(2+), dissociates into native monomers; upon release of pressure, the enzyme is fully active. Although further spectral changes occur at higher pressures, there is no inactivation as long as Mn(2+) remains bound. We propose that the relatively small and polar nature of the subunit interface of yeast enolase, including the presence of several salt bridges, is responsible for the ability of hydrostatic pressure to dissociate this enzyme into monomers with a native-like structure.

The fate of the prion protein in the prion/plasminogen complex.

The cellular prion protein (PrP(c)) forms complexes with plasminogen. Here, we show that the PrP(c) in this complex is cleaved to yield fragments of PrP(c). The cleavage is accelerated by plasmin but does not appear to be dependent on it.

The effects of osmotic and hydrostatic pressures on macromolecular systems.

Osmotic pressure and hydrostatic pressure can be used effectively to probe the behavior of biologically important macromolecules and their complexes. Using the two techniques requires a theoretical framework as well as knowledge of the more common pitfalls. Both are discussed in this review in the context of several examples.