Báo cáo khoa học: The activation of gelsolin by low pH The calcium latch is sensitive to calcium but not pH docx

The activation of gelsolin by calcium is a multistep process involving many calcium binding sites that act to unfold the molecule from a tight structure to a more loose form in which thr

The activation of gelsolin by low pH

The calcium latch is sensitive to calcium but not pH

Emeline Lagarrigue1, Diane Ternent2, Sutherland K Maciver2, Abdellatif Fattoum3, Yves Benyamin1 and Claude Roustan1

1

UMR 5539 (CNRS) Laboratoire de motilite´ cellulaire (Ecole Pratique des Hautes Etudes), Universite´ de Montpellier 2, Montpellier Cedex 5, France;2Genes and Development Group, Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, Scotland;3Centre de Recherches de Biochimie Macromole´culaire, UPR 1086 (CNRS), Montpellier Cedex 5, France

Gelsolin is a multidomain and multifunction protein that

nucleates the assembly of filaments and severs them The

activation of gelsolin by calcium is a multistep process

involving many calcium binding sites that act to unfold the

molecule from a tight structure to a more loose form in

which three actin-binding sites become exposed Low pHis

also known to activate gelsolin, in the absence of calcium and

this too results in an unfolding of the molecule Less is

known how pH-activation occurs but we show that there are

significant differences in the mechanisms that lead to

acti-vation Crucially, while it is known that the bonds between

G2 and G6 are broken by co-operative occupancy of calcium

binding sites in both domains [Lagarrique, E., Maciver,

S K., Fattoum, A., Benyamin, Y & Roustan, C (2003)

Eur J Biochem 270, 2236–2243.], pHvalues that activate gelsolin do not result in a weakening of the G2-G6 bonds

We report the existence of pH-dependent conformational changes within G2 and in G4–6 that differ from those induced by calcium, and that low pHoverrides the require-ment for calcium for actin-binding within G4–6 to a modest extent so that a Kd of 1 lM is measured, compared to 30–40 nM in the presence of calcium Whereas the pH-dependent conformational change in G2 is possibly different from the change induced by calcium, the changes measured

in G4–6 appear to be similar in both calcium and low pH Keywords: gelsolin; actin-binding protein; cytoskeleton; microfilament

Actin microfilaments are responsible for much of the

structure of cells and many aspects of their motility Actin

microfilaments in cells are regulated by a host of

actin-binding proteins [1] that act together to model them into a

large variety of structures Gelsolin is one of these; it is a

calcium-activated microfilament severing and actin filament

nucleating protein that is expressed widely in vertebrates [2]

The protein is composed of six repeated segments that are

similar in both sequence [3], and structure [4,5] The actin

binding functions of gelsolin result from three independent

actin-binding sites [6], two of which (G1 and G2) bind the

same actin monomer [7], and a third within G4 [8]

Although the primary function of gelsolin seems likely to

be actin modulation, the protein has a number of seemingly

unconnected functions such as acting as a crystallin in the eye of fish [9], regulation of phospholipase D [10], and it is thought to be involved in apoptosis [11] While the clearest function of gelsolin is to scavenge actin and actin filaments from the serum [12,13], its involvement in cell motility is most vividly illustrated by the fact that its expression levels determine the rate of cell locomotion [14], that motility is reduced when cells are treated with specific gelsolin inacti-vating peptides [15], and fibroblasts isolated from gelsolin null mice move more slowly [16]

In order for the various actin-binding activities of gelsolin to become apparent, the molecule has to be activated This involves the transformation of the com-pactly folded molecule to a more open conformation Activation of gelsolin by calcium has most often been studied and this occurs by the binding of six or more calcium ions The C-terminal half (G4–6) of gelsolin endows the whole molecule with calcium sensitivity [17,18], through two high affinity sites [8] one in G5 and the other in G6 [19,20] The structure of the inactive (calcium free) gelsolin molecule has been solved [4] but only the C-terminal half (G4–6) has been solved both in the presence [19] and absence [20] of bound actin in the active (plus calcium) configuration Strikingly, the structures of calcium-bound G4–6 are very similar in the presence or the absence of actin, meaning that this C-terminal half adopts

an actin-binding compatible shape as soon as it binds the calcium ions [20] In addition to the widely characterized calcium activation, gelsolin is also activated by low pH[21]

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

Place E Bataillon, 34095 Montpellier Cedex 5, France.

Fax: + 33 0467144927; E-mail: roustanc@crit.univ-montp2.fr

Abbreviations: G1–6, The six repeated domains of gelsolin; IPTG,

isopropyl thio-b- D -galactoside; FITC, fluorescein isothiocyanate;

1,5-I-AEDANS,

N,-iodoacetyl-N¢-(sulfo-1-naphthyl)-ethylenedi-amine; G-actin, monomeric actin; F-actin, filamentous actin.

Note: web pages are available at

http://www.dbs.univ-montp2.fr/umr5539/

http://www.ephe.univ-montp2.fr

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

(Received 30 May 2003, revised 19 August 2003,

accepted 22 August 2003)

propose that low pHsets off a similar but distinct set of

ion-pair exchanges, presumably initiated at histidine residues,

that also disrupts interdomain bonds but not those formed

through the G2Ờ6 interface

Methods

Proteins and peptides

Rabbit skeletal muscle actin was isolated from acetone

powder [23] and stored in buffer G (2 mM Tris [pH7.5],

0.1 mMCaCl2, 0.1 mMATP) Actin was labeled at Cys374

by pyrenyl iodoacetamide [24] All recombinant proteins in

this study were expressed in Escherichia coli BL21

deriva-tives using the pMW172 vector [25] Human gelsolin

domain 2 (G2) (residues 151Ờ266 of human serum gelsolin)

was produced in E coli BL21(pLysS) following induction of

expression with isopropyl thio-b-D-galactoside (IPTG) from

the soluble fraction of the bacteria [26] G1Ờ3 (residues

1Ờ407) was expressed in BL21(de3) cells and purified as

described previously from the soluble fraction of the

bacteria [27] G4Ờ6 (residues 407Ờ755) [27] was produced

in BL21(de3) cells and purified from inclusion bodies

Whole gelsolin was produced in E coli BL21(de3) Soluble

protein was dialyzed against 10 mM Tris (pH8.0), 1 mM

EGTA, 1 mMsodium azide, 50 mMNaCl and added to a

DE52 column equilibrated with the same buffer Pure

gelsolin was eluted off the column with 10 mM Tris

(pH8.0), 2 mMCaCl2, 1 mMsodium azide, 50 mMNaCl

[28]

Synthetic peptides derived from gelsolin sequences

159Ờ193 and 203Ờ225 [29] were prepared on a solid

phase support using a 9050 Milligen PepSynthesizer

(Millipore) according to the Fmoc/tBu system The crude

peptides were deprotected and thoroughly purified by

preparative reverse-phase HPLC The purified peptides

were shown to be homogenous by analytical HPLC

Electrospray mass spectra, carried out in the positive ion

mode using a Trio 2000 VG Biotech mass spectrometer

(Altrincham, UK), were in line with the expected

structures

Gelsolin G4Ờ6 domain was labeled by FITC or Oregon

green 488 isothiocyanate as described elsewhere [30]

Biotinylation of the G2 domain by biotinamidocaproate

N-hydroxyl-succinimide ester was performed as reported

previously [31] Excess reagents were eliminated by

chro-matography on a PD10 column (Pharmacia) in 0.1M

NaHCO buffer pH8.6

oped by K Raner, Mt Waverley, Victoria, Australia Details on the different experimental conditions are given in the figure legends

Fluorescence measurements Fluorescence experiments were conducted with a LS 50 Perkin-Elmer luminescence spectrometer Spectra for FITC

or Oregon green isothiocyanate labeled proteins were obtained with the excitation wavelength set at 470 nm Fluorescence changes were deduced from the area of the emission spectra between 510 and 530 nm Emission spectra for the intrinsic tryptophan chromophore were obtained with a wavelength of excitation at 280 nm The parameters

Kd (apparent dissociation constant) and Amax(maximum effect) were calculated by nonlinear fitting of the experi-mental data points as for ELISA (Eqn 1) or by using the following equation:

FỬ 1=2Amax ơE1đđơE ợ ơL ợ ơKdỡ

 fđơE ợ ơL ợ ơKdỡ2 4ơE  ơLg0:5ỡ where, [E] is the concentration of the fluorescent protein The maximum fluorescence change (Amax) at infinite substrate concentration expressed as percentage vari-ation from initial fluorescence: F8ỜF ồ/F ồở 100 was calculated by the relation F8) F ồ/F ồ Ử 0 Amax/F ồ where F ồ and F8are fluorescence intensities for zero and infinite ligand concentrations, respectively

Collisional quenching of fluorophore such as tryptophan

in our study is described by the SternỜVolmer equation, F/F Ử 1 + KD ở [Q] where F and F  are the fluorescence intensities in the presence and in the absence of the quencher, Q, respectively, and KD the SternỜVolmer constant [34] The constant, KD, depends upon the lifetime

of fluorescence without quencher, and the bimolecular rate constant for the quencher In this study iodine (IỜ) and acrylamide were chosen as the quenchers

Analytical methods Protein concentrations were determined by UV absor-bency using a Varian MS 100 spectrophotometer Gelsolin domain concentrations were determined spectro-photometrically using values of A280nm (1 cm)1)Ử 15.5 lMfor G4Ờ6, 21.0 lM for G1Ờ3, 79 lM for G2 and 8.93 lM for whole gelsolin These extinction coefficients were calculated by tryptophan, tyrosine and cysteine content [35]

Effect of pH on the gelsolin binding to G-actin

A detailed study [21] reported that the calcium

concentra-tion required for gelsolin activity is reduced when the pH

value is lowered below 7.0 At less than pH6.0 and in the

absence of calcium, the authors showed that gelsolin severed

actin filaments, nucleated actin polymerization and bound

G-actin

In a previous paper [36], we showed by ELISA that the

binding of gelsolin to G-actin was similar for various actin

iso-forms [rabbit alpha skeletal, bovine alpha cardiac,

bovine aortic and scallop (Pecten) muscles], and

estab-lished conditions under which the ELISA assay faithfully

reflected actin binding The binding of gelsolin to coated

G-actin at pH7.5 was abolished in the presence of excess

EGTA (Fig 1) This finding illustrates the specificity of

binding of gelsolin under the conditions of our ELISA

system If the pHvalue is lowered to 6.8 and EGTA is

present, we observe no gelsolin binding (data not shown)

In contrast, at pH5.7 we determined a saturation curve

for gelsolin interaction in the presence of 5 mM EGTA,

which is similar to that found at pH7.5 in the presence of

calcium (Fig 1)

Activation of gelsolin at low pHis expected to

involve an opening of the molecule as it does during

calcium activation, allowing the N-terminal half (G1–3)

to interact with actin A pHinduced increase in

hydrodynamic size of gelsolin has been found in support

of the notion that the molecules Ôopens upÕ [21] The

assumption that G1–3 could bind actin at low pH

values after the site became available was tested by

incubating G1–3 with coated G-actin in the presence of

EGTA at pH5.7 A tight interaction was observed

under these conditions and a similar binding for this

domain was also found at pH7.5 (Fig 2) in agreement

with earlier studies [27]

Conformational changes in gelsolin induced

by lowering the pH Binding of calcium to gelsolin induces large conformational changes [17] The C-terminal half of the molecule in particular is implicated in this regulation [18,22,37] We have reported previously [22] that the environment of an extrinsic chromophore (FITC) is calcium sensitive Two transitions exist, first, a fluorescence quenching at 0.1 lM [calcium]; second, at 1 lM an increase in fluorescence intensity These conformational changes can be correlated with the occurrence of the two constitutive binding sites in G5 and G6 [22] that are involved in the calcium induced activation of this half of gelsolin

We tested the possible pH-induced conformational changes in G4–6 required for the activation as there seemed

to be similarities between calcium- and pH- induced activation These changes were tested as below First, the intrinsic tryptophan fluorescence of G4–6 domain was measured at various pHvalues (between 5.7 and 6.5) An increase of pHinduced a rise in fluorescence intensity (Fig 3) correlating with a red-shift of the maximum wavelength These spectral effects are compatible with changes in the ionization of amino acids in the vicinity of tryptophan residues In a second experiment, conforma-tional changes were detected by the extrinsic fluorescence measurements of Oregon green-labeled G4–6 domains In both experiments, maximum fluorescence changes were observed for a pHvalue of about 6.0 (Fig 3)

In addition, quenching experiments were performed to test the accessibility of the tryptophan at pH5.7 and 6.8 in the presence of EGTA compared with the conformation at pH6.8 in the presence of calcium No effect was observed using iodine as a quenching molecule in accordance with the poor accessibility of the tryptophan suggested by the position of the maximum wavelength of the fluorescence (336 nm at pH5.7 and 342 at pH6.8 in EGTA) The results obtained using the less bulky molecule, acrylamide (Fig 4), show that the tryptophans of domains G4–6 are at pH5.7

in EGTA or pH6.8 in calcium and somewhat less shielded from the solvent than at pH6.8 in EGTA as the apparent

Fig 1 Effect of pH on the interaction of gelsolin with coated G-actin

monitored by ELISA Gelsolin was incubated at pH7.5 in 0.15 M

NaCl, 0.2 m M ATP, 50 m M Tris buffer containing either 4 m M CaCl 2

(d) or 5 m M EGTA (j) or pH 5.7 in 0.1 m M ATP/100 m M Mes buffer

containing 5 m M EGTA (h) in the presence of coated G-actin The

gelsolin interaction was monitored at 405 nm.

Fig 2 Effect of pH on the interaction of gelsolin G1-3 domain with coated G-actin monitored by ELISA G1–3 domains were incubated in 0.1 m M ATP, 5 m M EGTA, 100 m M Mes buffer at pH5.7 (j) or pH6.8 (h) in the presence of coated G-actin The interaction was monitored at 405 nm.

Kdis less for the last condition (Kd¼ 0.025M )1) than for

the other two (Kd¼ 0.042M )1) or for a small tryptophan

peptide (21 amino acids) used as model (Kd¼ 0.060M )1)

A conformational change due to a pH-shift in the G2

domain was also observed As shown in Fig 5, an

enhancement of tryptophan fluorescence was observed

when the pHvalue increased from 5.8 to 6.5 A small

red-shift (about 4 nm) was also linked to this conformational

transition (not shown)

Interaction of the G2 segment with G4–6 domains

Previously, we have shown that calcium binding to gelsolin

domains G2, 5 and 6 induced conformational changes in

these domains that altered the interaction between domains

G2 and G4–6 [22] Therefore, we investigated the possibility

of a pHsensitivity in the interaction of the G4–6 domains

with the G2 domain by two independent methods

In ELISA experiments, G4–6 domains were coated onto

plastic and the binding of biotinylated G2 domain was

revealed by using alkaline phosphatase labeled streptavidin

Figure 6 shows that binding occurs with similar affinity at

pH5.7 and at pH6.8, in the presence of EGTA These data were confirmed by studies in solution using fluorescence measurements (Table 1) The G4–6 domains were labeled

by Oregon green isothiocyanate and increasing concentra-tions of G2 were added An apparent Kdof 0.5 lM was obtained at pH5.7, a value similar to that reported previously for experiments at pH7.5 [22] These values and those obtained from ELISA, reported above, show a similar interaction of G2 with G4–6 domains in the presence

of EGTA at the acidic or neutral pH The differences in the absolute values observed between the two methods are probably explained by the heterogeneous phases used in ELISA

Fig 3 pH-induced changes in the G4-6 tryptophan and Oregon green

isothiocyanate labeled G4-6 fluorescence emission Aliquots of a

phos-phate solution (pH9) were added successively to unlabeled G4–6 or

labeled G4–6 in 10 m M Mes buffer (pH5.7) in the presence of 5 m M

EGTA to increase pHto a value of 6.25 (A) Log of fluorescence

intensities corresponding to tryptophan emission (j) or Oregon green

emission (h) is plotted vs pH (B) The maximum wavelength of

tryptophan fluorescence emission is plotted vs pH.

Fig 4 Quenching of tryptophan fluorescence of G4–6 domain by acrylamide Stern–Volmer plot for the quenching of G4–6 domain in 0.1 M Mes buffer pH5.7 in the presence of 5 m M EGTA (h) or pH 6.8

in the presence of 5 m M EGTA (j) or 1 m M calcium (d) Quenching

of a small peptide that contains one tryptophan (sequence 355–375 of actin) was reported as a control (n) F /F were determined as des-cribed in experimental procedure section The excitation wavelength was set at 280 nm.

Fig 5 pH induced changes in the G2 tryptophan fluorescence emission pHof G2 in 10 m M Mes buffer in the presence of 5 m M EGTA was varied between 5.7 and 6.50 Log of fluorescence intensities corres-ponding to tryptophan emission (h) are plotted vs pH.

The model of the closed state of gelsolin shows that two

segments of the G2 domain are located in the G2/G4–6

interface [4] The interaction of these two fragments (159–

193 and 203–225 sequences) was tested by fluorescence

using Oregon green labeled G4–6 domains At pH5.7 and

in the presence of EGTA, changes in the fluorescence

intensity (Fig 7) were obtained with the two fragments A

maximum fluorescence change of)15% and )17% were

calculated for 159–193 and 203–225 peptides, respectively

The apparent Kd(Table 1) are similar to those obtained at

pH7.5 It appears that the interaction between G2 and

G4–6 is calcium but not pHsensitive

Does G4–6 interact with actin at acidic pH?

The above results suggest that the conformation of G4–6 at

acidic pHis significantly different from that induced by

calcium binding Therefore, we tested the binding of G4–6

with G-actin by ELISA and fluorescence G-actin was

coated onto plastic and increasing concentrations of G4–6

(between 0 and 0.6 lM) were added Binding was monitored

by using specific G4–6 directed antibodies We observed a

tight binding at pH6.8 in the presence of 1 mM calcium

(apparent Kd¼ 30 nM), and in the presence of 1 mM

EGTA, no binding occured In contrast, at pH5.7 and

with 5 mMEGTA, a weak interaction was observed and a

Kdof about 1 lMwas estimated (Fig 8)

Fig 6 Binding of G2 domain to coated G4-6 monitored by ELISA.

Biotinylated G2 domain (0–0.8 l M ) was incubated in 5 m M EGTA,

0.1 M Mes buffer pH5.7 (h) or pH 6.8 (j) in the presence of coated

G4–6 The interaction was monitored at 405 nm.

Table 1 Interaction of G4-6 with G-actin, G2 and derived fragments ND, not determined; NI, no interaction; NS, no spectrum.

K d (l M )

EGTA (pH5.7) EGTA (pH6.8) EGTA (pH7.5) Ca2+(pH7.5)

Fluo(OG) ELISA Fluo(OG) ELISA Fluo(FITC) ELISA Fluo(FITC) ELISA

a

[22].

Fig 8 Effect of pH on the binding of G4-6 to coated G-actin monitored

by ELISA G4–6 was incubated at pH7.5 in 0.1 m M ATP, 10 m M Tris buffer containing either 1 m M CaCl 2 (d) or 5 m M EGTA (j) or at pH5.7 in 0.1 m M ATP, 100 m M Mes buffer containing 5 m M EGTA (h) in the presence of coated G-actin The interaction was monitored

at 405 nm.

Fig 7 Binding of G4–6 domain with 159–193 and 197–226 fragments derived from gelsolin G2 domain monitored by fluorescence measure-ments Interaction of Oregon green-labeled G4–6 domain (0.7 l M ) with Synthetic peptides 159–193 and 197–226 was carried out in 10 m M

Mes buffer pH5.7 in the presence of 5 m M EGTA Change in fluor-escence emission spectra of Oregon green was recorded at various peptides concentrations: 0–7 l M for the 159–193 peptide (j) and 0–11 l M for the 197–226 peptide (h).

concentrations of G-actin (between 0 and 0.2 lM) and the

changes in fluorescence were monitored Saturation curves

were observed in the presence of calcium and an apparent

Kd¼ 40 nM was determined (Table 1) No binding was

observed at this pHwhen EGTA was present These values

and those obtained from ELISA show a more pronounced

interaction of G-actin at neutral pH

Studying the inhibitory effect of G4–6 on actin

polymeri-zation substantiated this important result As shown in

Fig 9A and B, equimolar addition of G4–6 to actin at

pH5.7 produces only a small inhibition of the

polymeriza-tion process when compared with the control condipolymeriza-tion

(monitored at pH7.5) These results suggest that the

conformation induced by lowering pHdoes not allow a

tight interaction of G4–6 with G-actin

Discussion

The pH-dependence of the G2/G4–6 interface

Activation of gelsolin by calcium and/or low pHhas been

found to be necessary for the binding of actin [21] and

similarly for the binding of tropomyosin [38] Solving the

crystallographic solution of the whole gelsolin molecule in

its inactive state [4] led to the Ôhelix latchÕ hypothesis [5] in

which it is suggested that the C-terminal helix of gelsolin’s

G6 domain binds G2 and that this contact is released upon

the binding of calcium We have further shown that residues

203–225 and 159–193 within G2 form the G6 binding site,

and that occupancy of a calcium binding site in G2 induces

conformational changes through this interface with a

calcium binding site in G6 that results in the release of the

latch [22] A general unfolding of the molecule concomitant

with the release of the ÔlatchÕ between G2 and G4–6 is seen

as gelsolin is activated by calcium During activation of

gelsolin by low pHtreatment, a similar unfolding can be

recorded using dynamic light scattering [21] and it might be

assumed that a low pHalso detaches the G2 to G4–6 latch

The helix constitutes only a part of the switch that results in

full activation of gelsolin Deletion of the last 23 residues

from the C-terminus of gelsolin for example, reduces the

requirement for calcium in activation but does not abolish it

totally [39] Also, it is known that adseverin, a gelsolin

family member that naturally lacks the C-terminal helix has

a similar calcium requirement as the gelsolin mutant lacking

the helix [40] However, both adseverin and gelsolin mutants

lacking the C-terminal 23 residues are equally activated by

lowered pH Together, these observations suggest that the

calcium induced release of the helix latch may occur with other rearrangements between domains caused either by the occupancy by other calcium ions, or by the presence of protons We have shown that the helix latch and the G2 to G4–6 interaction in general is not reduced by low pH It is possible therefore that in pH-activated gelsolin, the helical latch may be the last event to occur being triggered not by direct disruption of the interface but as a consequence of the Kd being rather large At low pH, the helical latch could be the rate limiting step in the kinetics of activation Alternatively, pHinduced conformational changes within

or between other domains of gelsolin may strain the G2/G4–6 interaction

pH-dependent conformational changes in G2 Using tryptophan fluorescence as a probe we have been able

to show calcium dependent conformational changes with G2 [22] Using this same probe, we have now found a pH-dependent conformational change in G2, however, this change is in the opposite direction! This indicates that although a pH-sensitive conformational change does occur

in G2 it is probably different than that induced by calcium This is in line with our finding that pHdoes not affect the

Fig 9 Effect of G4-6 on actin polymerization Pyrenyl actin (3 l M ) was mixed with 0.1m KCl, 2 m M MgCl 2 (A) in 5 m M EGTA, 10 m M Mes (pH5.7) or (B) in 0.4 m M CaCl 2 , 10 m M Tris buffer (pH7.5) and the polymerization was followed vs time in the absence (––) or the pres-ence (- - -) of 3 l M G4–6.

G2/G4–6 interface, as we have previously found evidence

for a connectivity between the G2 calcium binding site

and the G6 site through the interface Low pH-induced

conformational changes within G2 possibly reflect altered

association with G1 and/or G3, rather than G6 as occurs

in the presence of calcium

pH-dependent conformational changes in G4–6

Some experimental data indicate that the calcium and

pH-induced conformational changes in G4–6 are similar

The tryptophan fluorescence of G4–6 is lowered by

increasing pHvalue Between pHvalues 5.7 and 6.2

qualitatively similar data were found across the same range

of pCa values [22] Also, an increase in fluorescence of

FITC-labelled G4–6 [22] and of Oregon green-labelled

G4–6 was found in this range Quenching experiments

(Fig 4) show that tryptophans are similarly accessible in the

presence of calcium and at low pH(while tryptophans were

less exposed at neutral pHin EGTA), which also suggests

similar conformation

pH dependence of gelsolin-actin binding

The gelsolin molecule forms a direct complex with two actin

monomers (GA2), through three distinct actin binding sites

The GA2complex may not be equivalent to the geometry at

the barbed end of the capped actin filament, as the actins in

GA2are antiparallel [41] We [29] and others [4,5,7,20], have

proposed various models for this interaction in which the

N-terminal gelsolin half (G1–3) binds a monomer through

sites in G1 and G2 and the C-terminal half (G4–6) binds a

second monomer in an analogous manner to G1 [42] In

agreement with earlier work [43], we find that G4–6 inhibits

actin polymerization In the presence of calcium, the

structure of G4–6 alone and G4–6 plus actin are so similar

[20] that it is suggested that calcium primes G4–6 for actin

binding, we have found that low pHremoves (to some

extent) the requirement for calcium and so predict that at

low pH, G4–6 will adopt a similar structure to that found in

both the G4–6-actin structure and G4–6 in calcium alone

The large difference in affinity for actin binding by

pH-activated (Kd1 mM) and calcium-activated (Kd30–40 nM)

gelsolin is probably due to the type I calcium site

coordi-nated by both G4 and actin monomer [19] These findings

explain why the formation of GA2is fastest at lower pH in

the presence of calcium [44]

A model for pH activation

Calcium binding by gelsolin domains generally breaks

interdomain salt bridge structures along the connecting

b-sheets and this results in domains moving a small

distance from each other having the overall effect of

enlarging the space occupied by the molecule [20] In some

instances, pHchanges may affect changes in the domain

through similar ion-pair swapping cascades proposed for

calcium binding [20], thereby activating gelsolin in a

similar manner to calcium We suspect that one or more

of the many histidines in the core of the gelsolin molecules

may sense low pHconditions and initiate an ion-pair

swapping cascade We have found however, that pH changes cannot substitute for every calcium site, as important differences exist between pH- and calcium-induced conformational changes If this model is correct, pHis expected to affect calcium binding, as for some sites, low pHshould result in ion-pair swapping that would substitute for calcium binding

Although intracellular pHis held close to neutral in most cell types, cell signalling involving significant changes in global intracellular pHare well documented [45,46], and these are often exaggerated in cell subdomains [47] In addition to gelsolin, other actin-binding proteins such as the ADF/cofilins [48] and EF1a [49] are strongly pH-dependent

so it seems probable that pHtransients that occur in cells may act in part through the actin cytoskeleton

We have shown that there is a clear difference between calcium- and pH-induced activation of gelsolin Future challenges are to determine how low pH-induced conform-ational change compares to calcium-induced conforma-tional change and if there are differences in the properties of pHand calcium activated gelsolin Also it is necessary to determine if low pHaffects the various calcium binding sites

in gelsolin

Acknowledgements

This research was supported by grants from AFM We thank

Dr Paul McLaughlin ICMB, University of Edinburgh for very helpful discussion.

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