Molecular basis for the dual function of Eps8 on actin dynamics: bundling and capping.
Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans.
IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy.
Maud
M
Hertzog
Francesca
F
Milanesi
Larnele
L
Hazelwood
Andrea
A
Disanza
HongJun
H
Liu
Emilie
E
Perlade
Maria Grazia
MG
Malabarba
Sebastiano
S
Pasqualato
Alessio
A
Maiolica
Stefano
S
Confalonieri
Christophe
C
Le Clainche
Nina
N
Offenhauser
Jennifer
J
Block
Klemens
K
Rottner
Pier Paolo
PP
Di Fiore
Marie-France
MF
Carlier
Niels
N
Volkmann
Dorit
D
Hanein
Giorgio
G
Scita
D000199:Actins / Q000378:metabolism / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism / Q000502:physiology*; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D013816:Thermodynamics
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C489989:EPS8 protein, human; D047908:Intracellular Signaling Peptides and Proteins