Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

Abstract

Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin’s involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin.

Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins

“…Cultures were induced with 1 mM IPTG, and incubated at 37 °C in a shaking incubator for 4 h. Cells were pelleted at 4 °C and resuspended in ice-cold buffer containing 10 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), pH 6.8, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 10 mM 2-mercaptoethanol, 1 mM PMSF, 5 mM benzamidine, SIGMAFAST protease inhibitor cocktail (EDTA-free), and lysed using a French press. Cofilins were purified by sequential anion and cation exchange chromatography as described previously [78]. Briefly, cell lysates were passed through DE52 (DEAE cellulose) and suplphopropyl (SP)-sepharose (Sigma-Aldrich, St. Louis, MO, USA) columns connected in tandem (in this order), followed by elution from the SP-sepharose column with a gradient of 50 to 500 mM NaCl in buffer containing 10 mM PIPES, pH 6.8, 0.5 mM EDTA, 10 mM 2-mercaptoethanol, and 0.5 mM PMSF.…”

Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

“…S3D cofilin binds actin filaments ϳ15-fold more weakly than WT cofilin (Table 1). Residues Ser-3, Gly-4, and Val-6 of the WT cofilin-2 N terminus form part of the cofilin-actin filament interface (46,47). The S3D substitution or Ser-3 phosphorylation introduces a steric clash that dissociates and repositions the cofilin N terminus away from actin ( Fig.…”

miR-3189-3p Mimics Enhance the Effects of S100A4 siRNA on the Inhibition of Proliferation and Migration of Gastric Cancer Cells by Targeting CFL2

“…CFL2 encodes cofilin-2 protein, which is a member of the ADF/cofilins family of actin-binding proteins. By analyzing the structure of CFL2, Yehl et al [ 34 ] found that human CFL2 could bind to F-actin and played an essential role in accelerating actin treadmilling. Schwickert et al [ 35 ] confirmed that CFL2 is a target gene of miR-142-3p and is involved in regulating breast cancer invasiveness.…”