Abstract
Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation.
Highlights
Cysteine-rich proteins (CRPs; referred to as cysteine and glycine-rich proteins; CSRPs) are muscle cell differentiation related proteins [1,2,3,4,5,6,7] and they consist of two LIM domains, which are double zinc-finger-like structures that mediate protein-protein interactions, and two glycine-rich regions
It was considered that N-terminal LIM domain and glycine-rich region (N-LIM) or C-terminal LIM domain and glycine-rich region (C-LIM) of cysteine-rich protein 2 (CRP2) influences the interaction between CRP2 and Filamentous actin (F-actin), because we previously revealed that N-LIM of CRP2 directly bound to filamentous actin (Factin) [6]
CRP2 directly associates with F-actin through its N-terminal LIM domain and glycine-rich region [6,14]
Summary
Cysteine-rich proteins (CRPs; referred to as cysteine and glycine-rich proteins; CSRPs) are muscle cell differentiation related proteins [1,2,3,4,5,6,7] and they consist of two LIM domains, which are double zinc-finger-like structures that mediate protein-protein interactions, and two glycine-rich regions. CRP1 is expressed in multiple smooth muscle organs and CRP2 is expressed in vascular smooth muscle cells (SMCs) [9,10,11], and these proteins act as cofactors for SMC differentiation [4]. CRP2 forms complex with serum response factor (SRF) and GATA proteins in the nucleus, and this complex strongly activates SMC-.
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