Kinetics Control Preferential Heterodimer Formation of Platelet-derived Growth Factor from Unfolded A- and B-chains

Carsten Müller,Susanne Richter,Ursula Rinas

doi:10.1074/jbc.m212317200

Abstract

The folding and assembly of platelet-derived growth factor (PDGF), a potent mitogen involved in wound-healing processes and member of the cystine knot growth factor family, was studied. The kinetics of the formation of disulfide-bonded dimers were investigated under redox reshuffling conditions starting either from unfolded and reduced PDGF-A- or B-chains or an equimolar mixture of both chains. It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step. The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics, indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways. In contrast, the formation of the heterodimer occurred three times more rapidly compared with the formation of the homodimers. As both monomeric forms revealed similar renaturation kinetics, it can be concluded that the first-order rate-determining folding step does not occur during monomer folding but must be attributed to conformational rearrangements of the dimerized, not yet disulfide-bonded protein. These structural rearrangements allow a more rapid formation of intermolecular disulfide bonds between the two different monomers of a heterodimer compared with the formation of the disulfide bonds between two identical monomers. The preferential formation of disulfide-bonded heterodimers from an equimolar mixture of unfolded A- and B-chains is thus a kinetically controlled process. Moreover, similar activation enthalpies for the formation of all different isoforms suggest that faster heterodimerization is controlled by entropic factors.

Highlights

Platelet-derived growth factor (PDGF)1 is a potent mitogen for cells of mesenchymal origin, i.e. smooth muscle cells, connective tissue cells, or blood cells (1Ϫ3)
It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step
The kinetic data are well described either by assuming a first- or a second-order rate-determining reaction and the putative rate constants extracted from the slopes of the linearized kinetic equations were determined to be k1 ϭ 1.5 10Ϫ5 sϪ1 or k2 ϭ 2.5 molϪ1 l sϪ1, respectively (Fig. 2B)

Summary

EXPERIMENTAL PROCEDURES

PDGF-BB Structure Visualization—PDGF-BB structure data were obtained from the Protein Data Bank (www.rcsb.org/pdb/; accession number 1pdg) and visualized using the program RasMol, Version 2.7.2.1 Standard renaturation conditions by SEC were: 1 mol lϪ1 Tris-HCl (pH 7.8), 0.5 mol lϪ1 guanidinium hydrochloride (GdnHCl), 10 mmol lϪ1 glutathione reduced (GSH), 0.25 mmol lϪ1 glutathione oxidized (GSSG). Under these conditions, the eluted PDGF monomers were able to dimerize in the eluate fraction to yield the dimeric, disulfide-bonded, and biologically active growth factor [14]. A, time course data of the relative concentrations of PDGF-A and -B monomers (XI) and disulfide-bonded dimers (f) after subjecting an equimolar mixture of unfolded and reduced PDGF-A and -B chains to SEC under renaturing conditions. If there is linearity for the temperature dependence of the rate constant, the enthalpy and entropy of activation can be determined from the slope and the y intercept of Equation 5, respectively

RESULTS

Final yield

DISCUSSION