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.
Hertzog
Maud
M
IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy.
Milanesi
Francesca
F
Hazelwood
Larnele
L
Disanza
Andrea
A
Liu
HongJun
H
Perlade
Emilie
E
Malabarba
Maria Grazia
MG
Pasqualato
Sebastiano
S
Maiolica
Alessio
A
Confalonieri
Stefano
S
Le Clainche
Christophe
C
Offenhauser
Nina
N
Block
Jennifer
J
Rottner
Klemens
K
Di Fiore
Pier Paolo
PP
Carlier
Marie-France
MF
Volkmann
Niels
N
Hanein
Dorit
D
Scita
Giorgio
G
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