Figure 1.
Structure of the octameric enolase.
The identical subunits are arranged as a tetramer of A-B pairs. The color coding is: “A” subunits are green and “B” subunits are yellow. The two putative plasminogen binding sites are shown. The first: only one atom of the C-terminal lysine-433 is visible in the X-ray structure. It is coloured red. The adjacent leucine-432 is also coloured red for convenience of viewing. The second: the putative site consists of residues 248–256. It is coloured orange. Figure 1A. Top down view showing the positions of the two sites. On the A subunits, the second site is easily seen; it shows up on the B subunits when the molecule is flipped through 180°. Figure 1B. An end on view of the octamer. The orange second site is clearly exposed whereas the red C-terminal is mostly buried.
Figure 2.
Analytical ultracentrifugation of Str enolase, dPgn, and a mixture containing a ratio of 1 Str octamer∶1 dPgn.
The solid black line is the combined Str enolase, dPgn mixture. The red line is the Str enolase, and the green line is the dPgn. There is almost perfect overlap between the red line and the Str enolase portion of the mix. There is almost perfect overlap between the green line and the dPgn portion of the mix. All proteins were ca 0.010 mM. The centrifuge speed was 32, 000, 20°C. The data were analyzed using Sedfit.
Table 1.
Stokes' Radii of Str enolase, dPgn and their mixture.
Table 2.
Fluorescence polarization of Str enolase interacting with IAF-Pg S741C-hPgn.
Figure 3.
Binding of plasminogen and enolase determined by ITC.
(A.) Native Str enolase was titrated into native dPgn. There was no detectable binding. (B.) Str enolase was brought to pH 9. It precipitated and was dialyzed to bring the pH back to pH 6.9. The protein loss was about 50%. The remaining protein retained 30% enolase activity. Enolase was titrated into dPgn. There were 0.18 sites available for binding on the average enolase monomer. (C.) Str enolase was brought to pH 4.5. It precipitated. The precipitate was dialyzed back to pH 6.9 and then dissolved in 27% urea (final) and then dialyzed again at pH 6.9. Approximately 30% of the original enolase was recovered and that retained about 30% of its specific activity. Enolase was titrated with dPgn. This protein bound 0.34 dPgn per average monomer of Str enolase.
Figure 4.
The influence of Str enolase on the precipitation kinetics of dPgn.
Each sample contained 0.2 M DTT. The bottom trace (dash dot) contained 0.019 mM Str enolase but no dPgn. The solid trace contained 0.002 mM dPgn but no Str enolase. The dotted trace contained 0.002 mM dPgn and 0.0019 mM Str enolase. The top trace (dash) contained 0.002 mM dPgn and 0.019 mM Str enolase. Under the reducing conditions shown here, Str enolase enhances the rate at which dPgn precipitates. In the absence of reductant, there is no precipitation of the two proteins over a period of days.
Figure 5.
Str enolase and dPgn co-precipitate in the presence of reductant.
SDS-PAGE of pellets and supernates from an experiment similar to that described in Figure 4. The mixtures containing the proteins and the reductant were centrifuged and the phases separated. The upper and lower lanes 4 contain standards; 1∶1 dPgn and Str enolase (monomers); the top band is the 90 kDa dPgn and the bottom is the 45 kDa Str enolase monomer. The lanes 1 show that dPgn precipitates almost quantitatively in the presence of reductant. Lanes 2 shows that the precipitating dPgn pulls down enolase from a 1∶1 mix of dPgn and Str enolase monomer but the precipitation is not quantitative. Lanes 3 show that the mixture which contains a 1∶3 ratio of dPgn to Str enolase monomer will pull down a significant fraction of the Str enolase.
Figure 6.
Screening of amine coupled Str enolase with different proteins.
The F137L/E363G Str enolase coated chip was tested for binding with 500 nM of each of the following proteins: (from top to bottom) yeast enolase, Str enolase, dPgn, BSA and MBP.
Figure 7.
Titration of the amine coupled Str enolase with dPgn (A) and the amine coupled dPgn with Str enolase(B).
7A. In the top curve, the dPgn concentration was 500 nM. It was diluted 1∶1 in each successive descending curve. The bottom trace is the baseline. The reaction appears to be totally reversible. 7B. Titration of amine coupled dPgn with Str enolase The dPgn clearly binds enolase in a multiphase reaction. The initial phase is very rapid and does not appear to be reversible on the time scale of the experiment. The second phase appears to be reversible. In the top curve, the Str enolase concentration was 500 mM. It was diluted 1∶1 in each successive descending curve.
Figure 8.
Binding of proteins to his-tagged Str enolase coupled to Ni-NTA chips.
Figure 8A. Different proteins binding to Ni-NTA immobilized F137L/E363G Str enolase. The enolase was titrated with dPgn (red), wt-Str enolase with no tag (black) and MBP (green). Each set represents titration with 15 nM, 62 nM, 125 nM, 500 nM and 2000 nM protein followed by washout. At the lower concentrations of titrating protein there is some specificity for dPGN which disappears at higher concentrations. Figure 8B. The wt-Str enolase was immobilized on Ni-NTA via the carboxyterminal deca-his-tag. dPgn (red), wt-Str enolase with no tag (black) and MBP (green) were flowed over the immobilized protein at concentrations of 15 nM, 62 nM, 125 nM, 500 nM and 2000 nM. There is some specificity at the lower concentrations but not at the higher. The two titrations are very similar but differ in the total response and the response to low concentrations of titrant protein.
Table 3.
Quaternary Structure of Enolase in SPR binding buffer as determined by AUC.
Figure 9.
Enolase adsorbed to the surface of phospholipid micelles will bind dPgn.
Azolectin (2 mg/mL) was suspended in 10 mM potassium phosphate, 100 mM NaCl, pH 7 and sonicated until clear. Str enolase, when present, was added to 300 nM; dPgn when present was 1000 nM. The first protein addition (Experiment 3: Str enolase, experiment 4, dPgn) was made to the azolectin micelles and the solution sonicated for another two minutes. The second protein addition was made was made and the solutions incubated at 4°C for three days (convenience timing). The samples were centrifuged at 20 000 rpm at 4°C for 2 hours. The phospholipids were extracted with isopropanol, chloroform and the remaining material dissolved in SDS sample buffer. The SDS-PAGE patterns of the pellets and supernates is shown. Lane 1 contained Str enolase only. The micelles pull down the enolase. Lane 2 contained dPgn only. The micelles do not pull down the dPgn. Lanes 3/4 contained Str enolase and dPgn. The micelles pull down the enolase which pulls down the dPgn.