Abstract
Profilins are abundant cytosolic proteins that are universally expressed in eukaryotes and that regulate actin filament elongation by binding to both monomeric actin (G-actin) and formin proteins. The atypical profilin Arabidopsis AtPRF3 has been reported to cooperate with canonical profilin isoforms in suppressing formin-mediated actin polymerization during plant innate immunity responses. AtPRF3 has a 37-amino acid-long N-terminal extension (NTE), and its suppressive effect on actin assembly is derived from enhanced interaction with the polyproline (Poly-P) of the formin AtFH1. However, the molecular mechanism remains unclear. Here, we solved the crystal structures of AtPRF3Δ22 and AtPRF3Δ37, as well as AtPRF2 apo form and in complex with AtFH1 Poly-P at 1.5-3.6 Å resolutions. By combining these structures with molecular modeling, we found that AtPRF3Δ22 NTE has high plasticity, with a primary "closed" conformation that can adopt an open conformation that enables Poly-P binding. Furthermore, using molecular dynamics simulation and free-energy calculations of protein-protein binding, along with experimental validation, we show that the AtPRF3Δ22 binds to Poly-P in an adaptive manner, thereby enabling different binding modes that maintain the interaction through disordered sequences. Together, our structural and simulation results suggest that the dynamic conformational changes of the AtPRF3 NTE upon Poly-P binding modulate their interactions to fine-tune formin-mediated actin assembly.
Highlights
Profilins are abundant cytosolic proteins that are universally expressed in eukaryotes and that regulate actin filament elongation by binding to both monomeric actin (G-actin) and formin proteins
The presence of formin AtFH1 with a coexistence of 0.5 M AtPRF3 and AtPRF1 at 1:1 ratio showed an overall negative regulation in AtFH1-mediated actin assembly, which is at a similar level of F-actin polymerization by using 0.5 M AtPRF3 only (Fig. 1)
Formin FH1 domain effectively increases the profilin-actin concentration at the barbed end to support rapid elongation of actin filament by delivering G-actin to barbed end
Summary
G-actin, monomeric actin; NTE, N-terminal extension; Poly-P, polyproline; MST, microscale thermophoresis; H-REMD, Hamiltonian replica-exchange molecular dynamics; CTH, C-terminal helix; PDB, Protein Data Bank; MM-PBSA, molecular mechanics–Poisson Boltzmann surface area; RMS, root mean sqaure. The molecular basis for understanding the potential conformational regulation of the AtPRF3 NTE is unknown. Studies of the dynamic association of AtPRF3 NTE with formin Poly-P is essential to understand how NTE dynamically regulates profilin activities by modulating its binding states toward Poly-P in plant development and defense mechanisms. We performed the molecule simulation of AtPRF3 NTE in the presence and absence of Poly-P, and the results suggest that AtPRF3 adopts various conformations to modulate the Poly-P binding. Our simulation results demonstrated that the NTE evolves dynamically by adopting different interaction modes toward Poly-P, through which an effective Poly-P binding and negative regulation in actin assembly have remained. Our results suggest that the unique ability of the AtPRF3 NTE by adopting various conformations, which enables precise modulations of actin assembly in defense mechanisms
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
