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 low pH The calcium latch is sensitive to calcium but not pH Emeline Lagarrigue 1 , Diane Ternent 2 , Sutherland K. Maciver 2 , Abdellatif Fattoum 3 , Yves Benyamin 1 and Claude Roustan 1 1 UMR 5539 (CNRS) Laboratoire de motilite ´ cellulaire (Ecole Pratique des Hautes Etudes), Universite ´ de Montpellier 2, Montpellier Cedex 5, France; 2 Genes and Development Group, Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, Scotland; 3 Centre 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 pH is 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.], pH values 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 pH overrides the require- ment for calcium for actin-binding within G4–6 to a modest extent so that a K d of 1 l M is measured, compared to 30–40 n M 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 nullmicemovemoreslowly[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) Eur. J. Biochem. 270, 4105–4112 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03803.x Like calcium activation, low pH causes an increase in the hydrodynamic size of the molecule while potentiating actin filament severing and nucleating activities [21]. Here, we show that although gelsolin can be activated by low pH in an apparently similar manner to that induced by calcium, important differences exist. The most striking difference is that the calcium ÔlatchÕ between G2 and 6 that is released by calcium is not released by lowered pH. Also, the actin-binding site in G4 that is high affinity (30 n M )in calcium-activated gelsolin is low affinity (1 l M )in pH-activated gelsolin. It is proposed [20] that calcium binding especially in G4–6, sets off a cascade of exchanged ion-pairs that leads to the disruption of the interdomain bonds most notably those between G2 and G6 [5,22]. We propose that low pH sets 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 G2–6 interface. Methods Proteins and peptides Rabbit skeletal muscle actin was isolated from acetone powder [23] and stored in buffer G (2 m M Tris [pH 7.5], 0.1 m M CaCl 2 ,0.1m M ATP). 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 151–266 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]. G1–3 (residues 1–407) was expressed in BL21(de3) cells and purified as described previously from the soluble fraction of the bacteria [27]. G4–6 (residues 407–755) [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 m M Tris (pH 8.0), 1 m M EGTA, 1 m M sodium azide, 50 m M NaClandaddedtoa DE52 column equilibrated with the same buffer. Pure gelsolin was eluted off the column with 10 m M Tris (pH 8.0), 2 m M CaCl 2 ,1m M sodium azide, 50 m M NaCl [28]. Synthetic peptides derived from gelsolin sequences 159–193 and 203–225 [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 G4–6 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 0.1 M NaHCO 3 buffer pH 8.6. Immunological techniques The ELISA technique [32], was used to monitor interaction of gelsolin domains, peptides to gelsolin domains and to actin as described previously [22,33]. Polyclonal antibodies to domains G4–6 were elicited in rabbits as described previously [33]. The binding parameters (apparent dissoci- ation constant K d and the maximal binding A max )were determined by nonlinear fitting: A ¼ A max ½L=ðK d þ½LÞ ð1Þ where, A is the absorbance at 405 nm and L the ligand concentration, by using the CURVE FIT software devel- 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 K d (apparent dissociation constant) and A max (maximum effect) were calculated by nonlinear fitting of the experi- mental data points as for ELISA (Eqn 1) or by using the following equation: F ¼ 1=2A max ½E À1 ðð½Eþ½Lþ½K d Þ À fð½Eþ½Lþ½K d Þ 2 À 4½E½Lg 0:5 Þ where, [E] is the concentration of the fluorescent protein. The maximum fluorescence change (A max ) at infinite substrate concentration expressed as percentage vari- ation from initial fluorescence: F 8 –F °/F ° · 100 was calculated by the relation F 8 ) F °/F ° ¼ 0. A max /F ° where F ° and F 8 are fluorescence intensities for zero and infinite ligand concentrations, respectively. Collisional quenching of fluorophore such as tryptophan in our study is described by the Stern–Volmer equation, F °/F ¼ 1+KD· [Q]whereF and F ° are the fluorescence intensities in the presence and in the absence of the quencher, Q, respectively, and KD the Stern–Volmer 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 (I – )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 A 280nm (1 cm )1 ) ¼ 15.5 l M for G4–6, 21.0 l M for G1–3, 79 l M for G2 and 8.93 l M for whole gelsolin. These extinction coefficients were calculated by tryptophan, tyrosine and cysteine content [35]. 4106 E. Lagarrigue et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Results 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 pH 6.0 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 pH 7.5 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 pH value is lowered to 6.8 and EGTA is present, we observe no gelsolin binding (data not shown). In contrast, at pH 5.7 we determined a saturation curve for gelsolin interaction in the presence of 5 m M EGTA, which is similar to that found at pH 7.5 in the presence of calcium (Fig. 1). Activation of gelsolin at low pH is 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 pH induced 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 pH 5.7. A tight interaction was observed under these conditions and a similar binding for this domain was also found at pH 7.5 (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 l M [calcium]; second, at % 1 l M 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 pH values (between 5.7 and 6.5). An increase of pH induced 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 pH value of about 6.0 (Fig. 3). In addition, quenching experiments were performed to test the accessibility of the tryptophan at pH 5.7 and 6.8 in the presence of EGTA compared with the conformation at pH 6.8 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 pH 5.7 and 342 at pH 6.8 in EGTA). The results obtained using the less bulky molecule, acrylamide (Fig. 4), show that the tryptophans of domains G4–6 are at pH 5.7 in EGTA or pH 6.8 in calcium and somewhat less shielded from the solvent than at pH 6.8 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 pH 7.5 in 0.15 M NaCl, 0.2 m M ATP, 50 m M Tris buffer containing either 4 m M CaCl 2 (d)or5 m M EGTA (j)orpH 5.7in0.1 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 pH 5.7 (j)or pH 6.8 (h) in the presence of coated G-actin. The interaction was monitored at 405 nm. Ó FEBS 2003 Low pH activation of gelsolin (Eur. J. Biochem. 270) 4107 K d is less for the last condition (K d ¼ 0.025 M )1 ) than for the other two (K d ¼ 0.042 M )1 ) or for a small tryptophan peptide (21 amino acids) used as model (K d ¼ 0.060 M )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 pH value 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 pH sensitivity 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 pH 5.7 and at pH 6.8, 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 K d of 0.5 l M was obtained at pH 5.7, a value similar to that reported previously for experiments at pH 7.5 [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 (pH 9) were added successively to unlabeled G4–6 or labeled G4–6 in 10 m M Mes buffer (pH 5.7) in the presence of 5 m M EGTA to increase pH to 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 pH 5.7 in the presence of 5 m M EGTA (h)orpH 6.8 in the presence of 5 m M EGTA (j)or1m 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. pH of 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. 4108 E. Lagarrigue et al. (Eur. J. Biochem. 270) Ó FEBS 2003 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 pH 5.7 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 K d (Table 1) are similar to those obtained at pH 7.5. It appears that the interaction between G2 and G4–6 is calcium but not pH sensitive. Does G4–6 interact with actin at acidic pH? The above results suggest that the conformation of G4–6 at acidic pH is 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 l M ) were added. Binding was monitored by using specific G4–6 directed antibodies. We observed a tight binding at pH 6.8 in the presence of 1 m M calcium (apparent K d ¼ 30 n M ), and in the presence of 1 m M EGTA, no binding occured. In contrast, at pH 5.7 and with 5 m M EGTA, a weak interaction was observed and a K d of about 1 l M was estimated (Fig. 8). Fig. 6. Binding of G2 domain to coated G4-6 monitored by ELISA. Biotinylated G2 domain (0–0.8 l M )wasincubatedin5m M EGTA, 0.1 M Mes buffer pH 5.7 (h)orpH6.8(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 (pH 5.7) EGTA (pH 6.8) EGTA (pH 7.5) Ca 2+ (pH 7.5) Fluo(OG) ELISA Fluo(OG) ELISA Fluo(FITC) ELISA Fluo(FITC) ELISA G2 0.5 0.15 NS 0.2 0.5 a 0.3 a 3 a 1 a 159–193 0.5 0.7 NS 0.7 1 a 1 a 2–3 a 2 a 197–226 2 ND NS ND 3 a 3 a NI a NI a G-actin 1 1 NS NI NI NI 40 n M 30 n M 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 pH 7.5 in 0.1 m M ATP, 10 m M Tris buffer containing either 1 m M CaCl 2 (d)or5m M EGTA (j)orat 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 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 pH 5.7 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). Ó FEBS 2003 Low pH activation of gelsolin (Eur. J. Biochem. 270) 4109 These data were confirmed by studies in solution using fluorescence measurements. G4–6 domains were labeled by Oregon green ITC and increasing concentrations of G-actin were added (data not shown) we observed a decrease in the fluorescence intensity of labeled G4–6 domains at pH 5.7 in the presence of EGTA. Analysis of these data shows that the fluorescence intensity decrease, extrapolated to infinite concentration, is about 15%. An estimation of the apparent K d (1 l M ) can be obtained (Table 1). The same experiment conducted in the presence of calcium at pH 6.8 shows that the interaction of G-actin with Oregon green labeled G4–6 does not induce any spectral change. Consequently, further analyses, using FITC-labeled G4–6, were carried out at pH 7.5. Labeled G4–6 was incubated with increasing concentrations of G-actin (between 0 and 0.2 l M )andthe changes in fluorescence were monitored. Saturation curves were observed in the presence of calcium and an apparent K d ¼ 40 n M was determined (Table 1). No binding was observed at this pH when 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 pH 5.7 produces only a small inhibition of the polymeriza- tion process when compared with the control condition (monitored at pH 7.5). These results suggest that the conformation induced by lowering pH does 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 pH has 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 pH treatment, a similar unfolding can be recorded using dynamic light scattering [21] and it might be assumed that a low pH also 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 K d being rather large. At low pH, the helical latch could be the rate limiting step in the kinetics of activation. Alternatively, pH induced 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 pH does not affect the Fig. 9. Effect of G4-6 on actin polymerization. Pyrenyl actin (3 l M )was mixedwith0.1mKCl,2m M MgCl 2 (A) in 5 m M EGTA, 10 m M Mes (pH 5.7) or (B) in 0.4 m M CaCl 2 ,10m M Tris buffer (pH 7.5) and the polymerization was followed vs. time in the absence (––) or the pres- ence (- - -) of 3 l M G4–6. 4110 E. Lagarrigue et al. (Eur. J. Biochem. 270) Ó FEBS 2003 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 pH value. Between pH values 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 pH in EGTA), which also suggests similar conformation. pH dependence of gelsolin-actin binding The gelsolin molecule forms a direct complex with two actin monomers (GA 2 ), through three distinct actin binding sites. The GA 2 complex may not be equivalent to the geometry at the barbed end of the capped actin filament, as the actins in GA 2 are 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 pH removes (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 (K d 1m M ) and calcium-activated (K d 30–40 n M ) 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 GA 2 isfastestatlowerpHin thepresenceofcalcium[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, pH changes 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 pH conditions 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, pH is expected to affect calcium binding, as for some sites, low pH should result in ion-pair swapping that would substitute for calcium binding. Although intracellular pH is held close to neutral in most cell types, cell signalling involving significant changes in global intracellular pH are 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 pH transients 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 pH and calcium activated gelsolin. Also it is necessary to determine if low pH affects 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. References 1. dos Remedios, C.G., Chhabra, D., Kekic, M., Dedova, I.V., Tsubakihara, M., Berry, D.A. & Noseworthy, N.J. (2003) Actin binding proteins: regulation of cytoskeletal microfilaments. 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Chem. 270, 15222–15230. 4112 E. Lagarrigue et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . The activation of gelsolin by low pH The calcium latch is sensitive to calcium but not pH Emeline Lagarrigue 1 , Diane Ternent 2 , Sutherland. exposed. Low pH is 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