PROFILIN INTERACTS WITH THE GLY-PRO-PRO-PRO-PRO-PRO SEQUENCES OF VASODILATOR-STIMULATED PHOSPHOPROTEIN (VASP) - IMPLICATIONS FOR ACTIN-BASED LISTERIA MOTILITY

Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequ ences: first, the tightly bound bacterial surface protein ActA uses it s multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E )E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the b acterial surface; and second, VASP then deploys its own multiple GPPPP P (or GP(5)) registers to localize the actin-regulatory protein profil in to promote actin polymerization, We now report that fluorescence ti tration showed that GP(5)GP(5)GP(5) peptide binds to profilin (K-D of 84 mu M), and the peptide weakly inhibits exchange of actin-bound nucl eotide in the absence or presence of profilin. Microinjection of synth etic GPPPPP triplet into Listeria-infected PtK2 cells promptly arreste d motility at an intracellular concentration of 10 mu M. This inhibiti on was completely neutralized when equimolar concentrations of profili n and GP(5)GP(5)GP(5) were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-L-proline [Bjorkegren, C., Rozy cki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEES Lett, 333, 123-126], exhibits little or no evidence of saturable GP(5)GP(5) GP(5) binding, When an equimolar concentration of this [His-133-Ser]-p rofilin mutant was co-injected with GP(5)GP(5)GP(5), the peptide's inh ibitory action remained completely unaffected, indicating that GP(5)GP (5)GP(5) binding to wild-type profilin represents a key step in actin- based pathogen motility. We also present a model that shows how the fo cal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.