Mapping of Drebrin Binding Site on F-Actin

Abstract

Drebrin is a filament binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full length protein. Site directed mutagenesis, electron microscopic (EM) reconstruction and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and 1,1-methanediyl bis(methanethiosulfonate) (MTS1). Site directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 Å from C374 of actin protomer 1, and drebrin's native cysteine 308 is in close proximity to C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), can link the N-terminal G-S extension of the recombinant DrABD, to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different length (5.4 -19 Å). These results suggest that the 'core' DrABD is centered on actin's subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of EM reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin binding proteins.

Actin-binding protein drebrin E is involved in junction dynamics during spermatogenesis

“…27,28 Despite the presence of an ADF-H domain in drebrin and the fact that it binds F-actin with high affinity in vitro, drebrin has not been found to possess any F-actin severing, bundling, capping and/ or nucleating activity. 21,[29][30][31] However, drebrin can compete with α-actinin, fascin and tropomyosin for binding to F-actin, 21,24,27 and drebrin E was shown to inhibit the actin-crosslinking, as well as the actin-binding activity, of α-actinin. 24 Thus, even though ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

Drebrin preserves endothelial integrity by stabilizing nectin at adherens junctions

“…In particular, the importance of the N-terminal region of drebrin, including the actin depolymerizing factor homology region [ADF-H (Poukkula et al, 2011;Xu and Stamnes, 2006)], and the actin-binding domain [ABD (Grintsevich et al, 2010;], are well documented. For example, the destabilizing effect of drebrin on dendritic spine morphology in rat hippocampal neurons seems to be based on its ABD (Biou et al, 2008).…”

Actin binding proteins and spermiogenesis

“…Interestingly, drebrins do not possess any F-actin severing, bundling, capping, nucleating or cross-linking activity per se, and they do not have any intrinsic biological activity. 54,[64][65][66] However, drebrins were found to compete with the binding of actin regulatory proteins, such as α-actinin, fascin and tropomyosin to F-actin, 54,55,67,68 thereby regulating the actin network. In short, drebrins regulate actin dynamics largely via their ability to "maintain" the "proper" levels of actin regulatory proteins to specific cellular domains in response to changes in environment, growth and development, pathological conditions and toxicants.…”