Tài liệu Báo cáo Y học: Binding of gelsolin domain 2 to actin An actin interface distinct from that

Binding of gelsolin domain 2 to actin

An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin

Celine Renoult1

, Laurence Blondin1

, Abdellatif Fattoum2

, Diane Ternent3

, Sutherland K. Maciver3

,

Fabrice Raynaud1

, Yves Benyamin1 and Claude Roustan1

1

UMR 5539 (CNRS) Laboratoire de Motilite´ Cellulaire (Ecole Pratique des Hautes Etudes), Universite´ de Montpellier, France; 2

Centre de Recherches de Biochimie Macromole´culaire, Montpellier, France; 3

Genes and Development Group,

Department of Biomedical Sciences, University of Edinburgh, Scotland

It is generally assumed that of the six domains that comprise

gelsolin, domain 2 is primarily responsible for the initial

contact with the actin filament that will ultimately result in

the filament being severed. Other actin-binding regions

within domains 1 and 4 are involved in gelsolin’s severing

and subsequent capping activity. The overall fold of all

gelsolin repeated domains are similar to the actin

depolymerizing factor (ADF)/cofilin family of actin-binding

proteins and it has been proposed that there is a similarity in

the actin-binding interface. Gelsolin domains 1 and 4 bind

G-actin in a similar manner and compete with each other,

whereas domain 2 binds F-actin at physiological salt

concentrations, and does not compete with domain 1. Here

we investigate the domain 2 : actin interface and compare

this to our recent studies of the cofilin : actin interface. We

conclude that important differences exist between the

interfaces of actin with gelsolin domains 1 and 2, and with

ADF/cofilin. We present a model for F-actin binding of

domain 2 with respect to the F-actin severing and capping

activity of the whole gelsolin molecule.

Keywords: actin; actin-binding proteins; cofilin; gelsolin.

The organization of the actin microfilaments in cells is

dynamic and is quickly rearranged in response to extra￾cellular signals. Gelsolin is one of the members of a family

of proteins (e.g. severin, villin), that is essential for

microfilament remodelling [1–3]. There are two forms of

gelsolin which differ in their N-terminal extremities. One is

specifically located in the blood and acts with vitamin

D-binding protein to accelerate clearing of actin from the

circulation [4], while the other form is intracellular. In vitro,

gelsolin interacts with G- and F-actins, promotes nucleation

and both severs and caps actin filaments. Cofilin belongs to

another family of actin-binding proteins that also severs

actin filaments and increases polymerization dynamics [5].

Despite a lack of sequence homology between the cofilin

and gelsolin families the fold adopted by each of gelsolin’s

130 amino-acid subdomains [2] is similar to the actin

depolymerizing factor (ADF)/cofilin family fold [6]. In

contrast with cofilin, gelsolin does not appear to be essential

for viability in the organisms where this has been tested,

probably due to the expression of related genes such as

adseverin/scinderin [7], but gelsolin is specifically required

for rapid movement of various dynamic cells [8]. Thus,

gelsolin over-expression in fibroblasts leads to enhanced cell

motility [9,10].

Domains 1–3 (S1–3) are sufficient for capping and

severing, while the C-terminal half of the molecule is

directly implicated in calcium regulation. In particular,

gelsolin domain 1 (S1) interacts both with monomeric actin,

and with the barbed end of the actin filaments inhibiting

polymerization.

S2, in contrast, preferably binds to the side of the actin

filament. Severing activity seems to require the binding of

S2 to the filament, followed by interaction of S1 between

two adjacent actins along the filament axis [11].

The tertiary structure of whole gelsolin in the inactive

Ca21 free state has been determined [2], as has S1 in

complex with actin [11], gelsolin S4–6 [12], severin

domain 2 [13,14] and villin domain 2 [15]. The structure of

each gelsolin domain shows a surprising similarity to the

cofilin fold [6]. Therefore it is possible to hypothesize that

S2 binds actin in the same manner as cofilin [16].

The solution of the gelsolin structure [2], showed that

when S1 is in position according to the G-actin–S1 model

[11], S2 is not in contact with actin. This might suggest a

reorientation of S1 : S2 interfaces so that S2 could contact

both of the binding sites on the same actin unit in the

filament to which S1 is joined [12]. In addition, by studying

the S2–6 interaction with F-actin, McGough et al. [17]

showed that the S2–3 position on F-actin is similar to the

actin-binding domain of a-actinin. Robinson et al. [12]

presented a model for gelsolin interaction based on the

Note: web pages are available at http://www.ephe.univ-montp2.fr, and

http://www.bms.ed.ac.uk/research/smaciver/index.htm.

Note: A gelsolin amino-acid numbering system based on the plasma

human gelsolin [1], in which S1 is defined as extending from Pro39 to

Tyr133 and S2 as being Gly137 to Leu247 [2], is used is this report.

Correspondence to C. Roustan, UMR 5539(CNRS) UM2 CC107,

Place E. Bataillon 34095 Montpellier Cedex 5, France.

Fax: 133 04 67 14 49 27,

E-mail: [email protected]

(Received 14 June 2001, revised 28 September 2001, accepted

4 October 2001)

Abbreviations: S1–6, the six repeated segments of gelsolin; ADF,

Actin depolymerizing factor; 1,5-I-AEDANS, N,-iodoacetyl-N0

-(sulfo￾1-naphthyl)-ethylenediamine; ELISA, enzyme-linked immunosorbant

assay; FITC, fluorescein 5-isothiocyanate; G-actin, monomeric actin;

F-actin, filamentous actin; EEDQ, N-ethoxycarbonyl-2-ethoxy￾1,2-dihydroquinoline.

Eur. J. Biochem. 268, 6165–6175 (2001) q FEBS 2001