Specific cleavage of the DNase-I binding loop dramatically decreases the thermal stability of actin Anastasia V Pivovarova1, Sofia Yu Khaitlina2 and Dmitrii I Levitsky1,3 A N Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia A N Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Keywords actin; differential scanning calorimetry; DNase-I binding loop; proteolytic cleavage; thermal unfolding Correspondence D I Levitsky, A N Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, 119071 Moscow, Russia Fax: +7 495 954 2732; Tel: +7 495 952 1384 E-mail: levitsky@inbi.ras.ru (Received 10 June 2010, revised 14 July 2010, accepted 16 July 2010) doi:10.1111/j.1742-4658.2010.07782.x Differential scanning calorimetry was used to investigate the thermal unfolding of actin specifically cleaved within the DNaseI-binding loop between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases, subtilisin or ECP32 ⁄ grimelysin (ECP), respectively The results obtained show that both cleavages strongly decreased the thermal stability of monomeric actin with either ATP or ADP as a bound nucleotide An even more pronounced difference in the thermal stability between the cleaved and intact actin was observed when both actins were polymerized into filaments Similar to intact F-actin, both cleaved F-actins were significantly stabilized by phalloidin and aluminum fluoride; however, in all cases, the thermal stability of the cleaved F-actins was much lower than that of intact F-actin, and the stability of ECP-cleaved F-actin was lower than that of subtilisin-cleaved F-actin These results confirm that the DNaseI-binding loop is involved in the stabilization of the actin structure, both in monomers and in the filament subunits, and suggest that the thermal stability of actin depends, at least partially, on the conformation of the nucleotidebinding cleft Moreover, an additional destabilization of the unstable cleaved actin upon ATP ⁄ ADP replacement provides experimental evidence for the highly dynamic actin structure that cannot be simply open or closed, but rather should be considered as being able to adopt multiple conformations Structured digital abstract l MINT-7980274: Actin (uniprotkb:P68135) and Actin (uniprotkb:P68135) bind (MI:0407) by biophysical (MI:0013) Introduction Actin is one of the most abundant and highly conserved cell proteins It is involved in many different cellular processes that are essential for growth, differentiation and motility Actin is found in two main states: as monomers (G-actin) and as a helical polymer (F-actin) Polymerization of G-actin into F-actin is accompanied by hydrolysis of tightly bound ATP followed by a slower release of Pi; as a result, protomers of F-actin contain tightly bound ATP, ADP or ADP-Pi The atomic resolution structures of G-actin revealed that it is divided into two easily distinguishable domains by a deep cleft containing the tightly bound nucleotide and cation [1] The nucleotide-binding cleft was suggested to exist in two main states, closed and open [2,3], and solution studies on nucleotide exchange and susceptibility of the cleft to limited proteolysis appear to be consistent with the opening of the cleft upon the transition from the ATP- to ADP-G-actin Abbreviations D-loop, DNase I-binding loop; DSC, differential scanning calorimetry; DHcal, calorimetric enthalpy; Tm, thermal transition temperature 3812 FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS A V Pivovarova et al [4–6] By contrast, in the numerous crystal structures published to date, including those of ADP-G-actin [7,8], the open nucleotide-binding cleft has been observed only in profilin-bound actin crystals [9] Only the closed conformation was also revealed by molecular dynamics simulations of the crystal actin structures [10–12], whereas metadynamics simulations have demonstrated that the closed conformation of the nucleotide-binding cleft is the most stable state only when ATP is bound, and the ADP-bound state favors a more open conformation of the cleft [13] It is possible therefore that crystallization favors a closed state for G-actin even though the state of the cleft in solution may vary [14,15] The other nucleotide-dependent conformational transitions in actin crystals involve so-called DNase I-binding loop (D-loop) in subdomain of the actin molecule [7] In tetramethylrhodamine-modified ADPG-actin, the D-loop was found to be in an a-helical conformation, whereas this region was disordered in the ATP-bound actin, suggesting that the D-loop folds on ATP hydrolysis [7,16] This transition within the D-loop was supported by the results of metadynamic simulations demonstrating a distinct allosteric relationship between the conformation of the D-loop (ordered or disordered) and the state of the nucleotide-binding cleft (open or closed) [13] These data are consistent with the results of the biochemical observations indicating that proteolytic modifications of the D-loop affect the state of the interdomain cleft [5,17] The D-loop of actin can be specifically cleaved with two bacterial proteases One of them is subtilisin, which cleaves the D-loop between Met47 and Gly48 [18] The other protease, which specifically cleaves actin at the only site between Gly42 and Val43 (Fig 1), was initially isolated and characterized as a Fig 3D atomic structure of G-actin The four subdomains are indicated by the encircled numbers Arrows show the cleavage sites in the D-loop, between Gly42 and Val43 by ECP32 ⁄ grimelysin, and between Met47 and Gly48 by subtilisin Thermal unfolding of cleaved actin minor protein of lactose-negative Escherichia coli A2 strain and referred to as protease ECP32 [19,20] More recently, it was found that the A2 strain producing protease ECP32 is identical to Serratia grimesii, and therefore this enzyme was named grimelysin [21] Although ECP32 and grimelysin were suggested to be identical enzymes [21], both names are used in the literature In accordance with previous studies [5,17], we refer to this protease as ECP in the present study The nucleotide-binding cleft in both subtilisin-cleaved and ECP-cleaved G-actin is clearly in a more open conformation compared to intact actin, as demonstrated by the increased nucleotide exchange rate in solution [17,22] and their higher susceptibility to limited proteolysis [5,17] By contrast, the crystal structure of ECPcleaved G-actin showed the nucleotide-binding cleft to be in a typical closed conformation, probably as a result of crystallization preferentially trapping actin in only one of its possible conformations [14] Taking into account the ambiguity of the nucleotide-binding cleft conformation and its relationship with the D-loop, the present study aimed to determine whether the specific cleavage of the D-loop affects the structural properties of the entire actin molecule and, in particular, conformational transitions of the nucleotide-binding cleft For this purpose, we studied the effects of the D-loop cleavage on the thermal unfolding of G- and F-actin Previously, the thermal unfolding of G-actin containing different nucleotides was indirectly studied with the DNase-I inhibition assay [23] and by monitoring the change in absorbance of tetramethylrhodamine-actin [24] The results obtained showed that replacement of the tightly bound ATP by ADP led to a significant decrease in the thermal stability of G-actin [23,24] In the present study, we applied differential scanning calorimetry (DSC), which is the most direct and effective method for studying the thermal unfolding of proteins Previous studies have shown that DSC can be successfully used to reveal the changes in the thermal unfolding of actin induced by interaction of G-actin with actin-binding proteins [25– 27], G–F transformation of actin, and stabilization of F-actin by phalloidin and Pi analogs [28] Moreover, the effects of nucleotides on the thermal unfolding of F-actin have been studied by this method and a ‘dissociative’ mechanism for the thermal denaturation of F-actin has been proposed [28,29] In the present study, we used DSC to characterize the thermal unfolding of actin specifically cleaved within the D-loop by ECP or subtilisin The results obtained show for the first time that the cleavage strongly decreases the thermal stability of G-actin and especially that of F-actin, both in the absence and the FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS 3813 Thermal unfolding of cleaved actin A V Pivovarova et al presence of phalloidin and Pi analogs These results are discussed with regard to a more open conformation of the interdomain cleft in the cleaved actin compared to intact actin, both in the monomers and in the filament subunits Table Calorimetric parameters obtained from the DSC data for intact, ECP-cleaved and subtilisin-cleaved G-actins The parameters were extracted from Figs and The error of the given values of Tm did not exceed ±0.2 °C The relative error of the given values of DHcal did not exceed ±10% G-actin Results Effects of the D-loop cleavage on the thermal unfolding of G-actin The excess heat capacity curves obtained for intact, ECP-cleaved, and subtilisin-cleaved ATP-Ca-G-actins are presented in Fig It is seen that the G-actin species cleaved within the D-loop are clearly less thermostable than noncleaved G-actin The thermal transitions of both ECP-cleaved and subtilisin-cleaved G-actin are shifted to a lower temperature, by 4–5 °C, compared to that of intact G-actin, and the values of calorimetric enthalpy, DHcal, determined for the cleaved G-actins are $ 57–63% of those for noncleaved G-actin (Table 1) Thus, both cleavages within the D-loop strongly decrease the thermal stability of ATP-Ca-G-actin, with no significant difference between the effects of ECP and subtilisin It is important to note that heating cleaved G-actins in the Fig Temperature dependences of the excess heat capacity (Cp) of intact (curve 1), ECP-cleaved (curve 2) and subtilisin-cleaved (curve 3) ATP-Ca-G-actins The actin concentration was 24 lM Other conditions: mM Hepes (pH 7.6), 0.2 mM CaCl2 and 0.2 mM ATP The inset shows representative SDS ⁄ PAGE patterns of intact (lanes and 1¢), ECP-cleaved (lanes and 2¢) and subtilisin-cleaved (lanes and 3¢) G-actin before (lanes 1, and 3) and after heating in the calorimetric cell up to 80 °C (lanes 1¢, 2¢ and 3¢) Note that the positions of actin (lanes and 1¢) and its C-terminal fragments produced by ECP (36 kDa) (lanes 2, 2¢) or by subtilisin (35 kDa) (lanes and 3¢) remain unchanged after the heating–cooling procedure 3814 Nucleotide Cation Tm (°C) DHcal (kJỈmol)1) Intact Intact Intact ECP-cleaved ECP-cleaved ECP-cleaved Subtilisin-cleaved ATP ATP ADP ATP ATP ADP ATP Ca2+ Mg2+ Mg2+ Ca2+ Mg2+ Mg2+ Ca2+ 61.2 59.9 48.8 55.9 53.6 44.5 57.0 585 570 340 370 390 145 335 calorimeter cell did not lead to any further proteolysis of the proteins (Fig 2, inset) We also compared the thermal unfolding of intact and ECP-cleaved G-actins in the different states, with the tightly bound Ca2+ replaced by Mg2+ and with the tightly bound ATP replaced by ADP (Fig 3) The replacement of Ca2+ by Mg2+ in ATP-G-actin had no appreciable effect on the thermal unfolding of either intact or ECP-cleaved G-actin: in both cases, it only slightly decreased the maximum thermal transition temperature (Tm), by 1–2 °C, with no effect on the DHcal value (Table 1) By contrast, the replacement of bound ATP by ADP caused a dramatic decrease in the thermal stability of G-actin Intact ADP-Mg-G-actin demonstrated the thermal transition with Tm of 48.8 °C (Fig 3A) (i.e 11 °C less than that of ATP-Mg-G-actin) and its calorimetric enthalpy (340 kJỈmol)1) was much less than that of ATP-Mg-G-actin (570 kJỈmol)1) (Table 1) Figure 3A shows that the sample contains only ADP-actin because no peak at 60 °C (corresponding to the thermal transition of ATP-Mg-actin) was seen on the thermogram A similar effect was observed on ECP-cleaved Mg-G-actin with ATP replaced by ADP (Fig 3B) In this case, the nucleotide replacement decreased the Tm by °C and led to a more than two-fold decrease in the DHcal value (Table 1) Thermal unfolding of F-actin with the cleaved D-loop Previous studies have shown that ECP-cleaved actin is unable to polymerize unless its tightly bound Ca2+ is replaced with Mg2+, and that the Mg2+-bound form has higher critical concentration and polymerizes more slowly than Mg-G-actin cleaved with subtilisin [17,20,30] In agreement with these data, in the present study, ECP-cleaved Mg-G-actin polymerized more FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS A V Pivovarova et al Thermal unfolding of cleaved actin Fig DSC curves of Mg-F-actin assembled from intact (curve 1), ECP-cleaved (curve 2) and subtilisin-cleaved (curve 3) ATP-Mg-Gactin The actin concentration was 24 lM Other conditions: 20 mM Hepes (pH 7.3), 0.1 M KCl, mM MgCl2 and 0.7 mM ADP The inset shows time courses of polymerization of intact (curve 1), ECP-cleaved (curve 2) and subtilisin-cleaved (curve 3) actins Polymerization was monitored by recording light-scattering intensity at 350 nm upon the addition of 0.1 M KCl and mM MgCl2 to ATPMg-G-actins Fig DSC curves of intact G-actin (A) and ECP-cleaved G-actin (B) with different tightly bound nucleotide and cation: ATP-Ca-Gactin, ATP-Mg-G-actin and ADP-Mg-G-actin The actin concentration was 24 lM Other conditions: mM Hepes (pH 7.6), 0.2 mM CaCl2 or MgCl2, and 0.2 mM ATP or ADP slowly than subtilisin-cleaved Mg-G-actin, which, in turn, demonstrated slower polymerization than intact, noncleaved Mg-G-actin Nevertheless, light-scattering measurements showed complete polymerization of all the Mg-G-actin species to Mg-F-actin after 1.5 h of incubation with 100 mm KCl and mm MgCl2 in the presence of mm ATP (Fig 4, inset) Figure shows that Mg-F-actin obtained from the cleaved Mg-G-actin is much less thermostable than noncleaved Mg-F-actin, and a decrease in the thermal stability is even more pronounced than in the case of G-actin The thermal transitions of ECP-cleaved and subtilisin-cleaved F-actin are shifted to a lower temperature, by 11.3 and 8.8 °C, respectively, compared to that of intact F-actin (Table 2) Importantly, a pronounced difference is observed between the thermal transitions of ECP-cleaved and subtilisin-cleaved F-actin (Fig 4) ECP-cleaved F-actin unfolds not only Table Calorimetric parameters obtained from the DSC data for Mg-F-actin assembled from intact, ECP-cleaved and subtilisincleaved Mg-G-actin The parameters were extracted from Figs and The error of the given values of Tm did not exceed ± 0.2 °C The relative error of the given values of DHcal did not exceed ±10% Mg-F-actin Stabilizer Tm (°C) DHcal (kJỈmol)1) Intact Intact Intact Intact ECP-cleaved ECP-cleaved ECP-cleaved ECP-cleaved Subtilisin-cleaved Subtilisin-cleaved Subtilisin-cleaved Subtilisin-cleaved – Phalloidin AlF4À Phalloidin + AlF4À – Phalloidin AlF4À Phalloidin + AlF4À – Phalloidin AlF4À Phalloidin + AlF4À 69.9 82.5 83.4 90.8 58.6 68.5 70.3 81.7 61.1 76.5 76.4 84.4 650 1065 800 1080 525 655 415 690 415 635 720 780 at lower temperature (58.6 versus 61.1 °C), but also with a much lower cooperativity The width at the half-height of the thermal transition, which can serve as a relative measure for cooperativity of the transition, was equal to 8.5 °C for ECP-cleaved Mg-F-actin and 4.3 °C for subtilisin-cleaved Mg-F-actin Thus, FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS 3815 Thermal unfolding of cleaved actin A V Pivovarova et al although both cleaved G-actins unfold similarly (Fig 2), a pronounced difference in the thermal unfolding between ECP-cleaved and subtilisin-cleaved actin is revealed when these actins are polymerized into filaments Stabilization of the cleaved F-actin by phalloidin and aluminum fluoride It is well known that cyclic heptapeptide phalloidin binds to F-actin with very high affinity at the interface of three adjacent actin protomers [31,32] and stabilizes actin filaments (i.e it significantly increases the thermal stability of F-actin) [25,28,33–35] A very similar stabilizing effect was observed in the presence of Pi analogs, aluminum fluoride (AlFÀ ) or beryllium fluoride (BeFx) [25,28,34,36], which form complexes with F-actin subunits that mimic their ADP-Pi state The stabilizing effects of phalloidin and AlF4À (or BeFx) were similar but independent of each other because simultaneous addition of both stabilizers caused an additional increase in the thermal stability of F-actin [28,34] The subsequent experiments were designed to investigate the effects of the two F-actin stabilizers, phalloidin and AlF4À , on the thermal unfolding of F-actin specifically cleaved within the D-loop In agreement with previous studies [25,28,29,34], the binding of phalloidin or AlF4À significantly increased the thermal stability of Mg-F-actin Both stabilizers shifted the maximum of the F-actin thermal transition from 69.9 °C to 82–83 °C (Table 2), and their simultaneous addition increased the Tm up to $ 91 °C (Fig 5A) Similar to intact F-actin, both cleaved F-actin species are significantly stabilized by phalloidin and AlF4À (Fig 5B,C) Each of these stabilizers increased the Tm of the cleaved F-actin, by 10–12 °C for ECP-cleaved F-actin and by $ 15 °C for subtilisin-cleaved F-actin (Table 2), and their simultaneous addition resulted in an additive effect that is expressed in the further increase of the Tm value by $ 12–13 °C (Fig 5B) or °C (Fig 5C) However, in all these stabilized states, the Tm value for the cleaved F-actin was significantly lower than that of intact F-actin, by 9–14 °C for ECPcleaved F-actin and by 6–7 °C for subtilisin-cleaved F-actin (Table 2) This means that ECP-cleaved F-actin is less thermostable than subtilisin-cleaved F-actin not only in the absence of stabilizers (Fig 4), but also in the presence of phalloidin and AlF4À (Fig 5B,C) There are also other distinct differences between ECP-cleaved F-actin and subtilisin-cleaved F-actin, whose thermal denaturation is more similar to that of intact F-actin First, along with the main transition at 68.5 °C, the DSC profile of the phalloidin-stabilized 3816 Fig DSC curves of intact Mg-F-actin (A), ECP-cleaved Mg-Factin (B) and subtilisin-cleaved Mg-F-actin (C) stabilized by phalloidin or AlF4À , or simultaneously by both stabilizers Concentrations of stabilizers: 24 lM phalloidin and mM AlF4À (5 mM NaF and mM AlCl3) Other conditions were as described in Fig ECP-cleaved F-actin demonstrated a pronounced shoulder at $ 60 °C (Fig 5B) Second, in the presence of AlF4À , this cleaved F-actin demonstrated, along with the main thermal transition at 70 °C, a clear peak at $ 57 °C corresponding to the thermal unfolding of this protein in the absence of AlF4À (Fig 5B) This suggests a much lower affinity of ECP-cleaved F-actin for phalloidin and AlF4À than that in intact and subtilisincleaved F-actin To test this assumption, we investigated the thermal unfolding of ECP-cleaved F-actin in the presence of different concentrations of phalloidin and AlF4À (Fig 6) At relatively low phalloidin ⁄ actin molar ratio of : 4, two peaks are observed on the DSC profile FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS A V Pivovarova et al Thermal unfolding of cleaved actin Thus, a much higher concentration of AlF4À (more than mm) is required to achieve complete thermal stabilization of ECP-cleaved F-actin compared to intact F-actin, for which full stabilization was observed even in the presence of 50 lm AlF4À [36] This reflects at least an order of magnitude lower affinity of the cleaved F-actin to AlF4À Importantly, upon simultaneous addition of AlF4À and phalloidin, we observed neither the peak of nonstabilized actin protomers, nor the shoulder characteristic of phalloidin-stabilized ECP-cleaved F-actin (Fig 6B) These results suggest that the binding of AlF4À to ADP-F-actin substantially modifies the structural state of cleaved actin subunits stabilized by phalloidin or phalloidin increases the affinity of the cleaved actin subunits to AlF4À Discussion Fig DSC curves for ECP-cleaved Mg-F-actin (24 lM) either in the presence of phalloidin (Ph) at different concentrations (6, 12 or 72 lM) (A), or in the presence of 0.1 mM AlF4À in the absence or in the presence of 24 lM Ph, and in the presence of 0.5 mM AlF4À + 24 lM Ph (B) Other conditions were as described in Fig (Fig 6A), and the large peak with Tm at 58.6 °C corresponds to the nonstabilized ECP-cleaved F-actin (i.e it reflects the thermal unfolding of those actin protomers, which are not affected by phalloidin) This means that effect of phalloidin on the thermal stability of ECPcleaved F-actin is much less cooperative than in the case of intact F-actin, when one bound phalloidin was shown to stabilize up to seven neighboring protomers in the actin filament [37] The peak of nonstabilized actin disappeared with an increase in the phalloidin ⁄ actin molar ratio (Figs 5B and 6A) However, the pronounced shoulder at $ 61–65 °C was observed on the DSC profile of ECP-cleaved F-actin even in the presence of a three-fold molar excess of phalloidin (Fig 6A), thus suggesting that protomers of phalloidin-stabilized F-actin exist in two structural states with different thermal stability At a low concentration of AlF4À (0.1 mm), we again observed a pronounced peak at 58.6 °C corresponding to the nonstabilized ECP-cleaved F-actin (Fig 6B) The data reported in the present study show that cleavage of actin between Gly42-Val43 or Met47-Gly48 within the D-loop strongly decreases the thermal stability both of monomers and polymers According to previous studies, these cleavages increased the rate of the nucleotide exchange on the cleaved G-actin and its susceptibility to limited proteolysis, probably as a result of the transition of the nucleotide-binding cleft to a more open conformation [5,17,22] The relationship between the conformation of the D-loop and the nucleotide-binding cleft was recently demonstrated in metadynamic simulations experiments [13] We assume therefore that the decrease in the thermal stability observed by DSC on actin species cleaved within the D-loop is associated with opening of the cleft Does the thermal stability of G-actin reflect the conformational state of the nucleotide-binding cleft? An intact actin structure is maintained by the presence of high-affinity cation and nucleotide tightly bound in the interdomain cleft; removal of the nucleotide or cation results in actin denaturation Therefore, the stability of actin depends on the affinity of the tightly bound cation and nucleotide that involves both protein–ligand interaction and conformation of the interdomain cleft Upon heating, irreversible unfolding of G-actin is preceded by reversible loss of the nucleotide–cation complex [23] Obviously, the more tightly nucleotide and cation are bound in the interdomain cleft and the more ‘closed’ is the cleft, the higher the temperature needed to remove them from the cleft and to induce thermal unfolding of G-actin The relative FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS 3817 Thermal unfolding of cleaved actin A V Pivovarova et al affinity of G-actin for ATP is much higher than for ADP [4], and the interdomain cleft is suggested to be in a more open conformation in the ADP-bound state than in the ATP-bound state [5,12,13] In agreement with this and with previous studies [23,24], the results obtained in the present study show that ADP-G-actin is much less thermostable than ATP-G-actin (Fig 3A and Table 1) ATP-G-actin is less thermostable with bound Mg2+ than with Ca2+ [23] (Table 1), and this reduction in stability may be explained by the lower affinity of ATP-G-actin for Mg2+ than for Ca2+ [4,38] Thus, the ligand-dependent thermal stability of actin monomer can be accounted for by the different affinity of these ligands to actin However, the thermal stability of the cleaved actins is lower than the corresponding stability of non-modified actin both in the ATP- and ADP-states This cannot be explained by the different affinity but suggests that the thermal stability of G-actin may depend on the conformation of the nucleotide-binding cleft This suggestion is supported by the studies on the effects of actin-binding proteins on actin structure Actin-binding proteins profilin and cofilin, when bound to G-actin between subdomains and 3, have antagonistic effects on the conformation of the nucleotide-binding cleft Profilin stabilizes the ‘open’ conformation of the cleft [7,39,40], whereas cofilin appears to lock the cleft in its ‘closed’ conformation [39–42] Previous studies on the thermal unfolding of G-actin showed that profilin binding decreased the actin thermal stability [23], whereas significant stabilization of G-actin was observed in its complexes with cofilin [25,26] Stabilization of G-actin was also observed in the complexes of G-actin with thymosin b4 [27] and gelsolin segment [24], which appear to induce conformational transitions closing the nucleotide-binding cleft [6,27,43,44] Thus, the increased thermal stability of G-actin appears to correspond to the closed conformation of the nucleotide-binding cleft, whereas the decreased thermal stability is a feature of the actin with the open cleft conformation The cleavage within the D-loop enhances the nucleotide exchange [17] and increases accessibility of the cleft to limited proteolysis [5], which characterizes the cleft opening It is therefore likely that the decreased thermal stability of G-actin cleaved within the D-loop also results from the opening of the nucleotide-binding cleft in these actin species It is noteworthy that the replacement of tightly bound ATP by ADP in ECP-cleaved G-actin induces an additional decrease in the thermal stability of this actin species already destabilized by the cleavage within the D-loop (Fig 3B and Table 1) This suggests 3818 that the nucleotide-binding cleft is highly dynamic and cannot be simply open or closed but rather should be considered as being more open or more closed In these terms, by analogy with the ‘superclosed’ state recently revealed in ATP-G-actin by molecular dynamics simulations [12], the nucleotide-binding cleft of ADP-G-actin cleaved within the D-loop appears to adopt the extra open conformation Comparison of the effects produced by the cleavage of the D-loop with ECP and subtilisin Although the cleavages of the D-loop between Gly42Val43 and Met47-Gly48 decreased the thermal stability of G-actin to a similar extent (Fig 2), the effects of the cleavages became quite different when the cleaved actins were polymerized into filaments The thermal stability of F-actin assembled from ECP-cleaved actin was noticeably less than that of subtilisin-cleaved F-actin (Fig 4) These results are consistent with the earlier observed effects of these cleavages on the susceptibility of the nucleotide-binding cleft to limited proteolysis with trypsin [5,17] In the cleaved G-actins, susceptibility of trypsin cleavage sites at Arg62 and Lys68 in the nucleotide-binding cleft was increased similarly [17] After polymerization, these sites became almost inaccessible for trypsin in intact F-actin and only slightly accessible for trypsin in subtilisin-cleaved F-actin By contrast, F-actin assembled from ECPcleaved G-actin was easily fragmented by trypsin These observations indicate that the open conformation of ECP-cleaved actin was preserved upon polymerization, whereas F-actin assembled from subtilisincleaved monomers more closely resembled intact F-actin than ECP-cleaved F-actin [17] Thus, the lower thermal stability of ECP-cleaved versus subtilisincleaved F-actin corresponds to a more open nucleotide-binding cleft According to the recent model of actin filament [45], the N-terminal part of the D-loop is located at the inter-monomer interface, participating both in the intra-strand contacts between actin subunits along the filament and in the lateral contacts stabilizing the inter-strand interaction, whereas the C-terminal part of the loop is not involved in the inter-strand contacts Recently, this structural difference was supported in mutational cross-linking experiments showing that the N-terminal part of the D-loop (residues 41–45) is in close proximity to residue 265 of the actin subunit in the opposite strand and can be easily cross-linked to this residue, whereas the rate and extent of the crosslinking reaction strongly declined for the C-terminal residues of the D-loop [46] Therefore, the inter-strand FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS A V Pivovarova et al contacts of the N-terminal part of the D-loop appear to play a crucial role in stabilization of the actin filament [17,30,47] The cleavage of the D-loop between Gly42 and Val43 impairs these contacts [47], and this may explain why the cleavage of the D-loop in its N-terminal part with ECP more strongly destabilizes F-actin than cleavage by subtilisin between Met47 and Gly48 in the C-terminal part of the loop It is also important that the cleavage with ECP did not affect the filament length but more strongly enhanced the turnover rate of polymer subunits than the cleavage with subtilisin [17,30] Thus, the low thermal stability of F-actin assembled from ECP-cleaved monomers strongly correlates with the high dynamics of this actin species [17], supporting the idea of the monomer dissociation being the first step of thermal inactivation of F-actin [28,29] Although both cleaved actins are stabilized with phalloidin and AlF4À , stabilization of ECP-cleaved F-actin demonstrates specific features that are not characteristic of subtilisin-cleaved or intact actin The most interesting features are the extremely low affinity of ECP-cleaved F-actin to AlF4À and the pronounced shoulder observed on the DSC profile of this actin species even in the presence of a three-fold molar excess of phalloidin (Figs 5B and 6) This suggests that protomers of phalloidin-stabilized cleaved F-actin exist in two different structural states Phalloidin binds to F-actin at the interface of three adjacent actin protomers [31] and appears to stabilize actin filament in two inter-related ways: by stabilizing lateral interactions between the two filament strands and by inducing conformational changes in actin subunits resulting in the state of the nucleotide-binding cleft being similar to that in ATP-actin filaments without phalloidin [32] It is plausible that the shoulder on the DSC profile of phalloidin-stabilized ECP-cleaved F-actin belongs to a population of the protomers in which the conformational effect of phalloidin is not completed This explanation, although requiring further examination with independent approaches, is supported by the disappearance of the shoulder after the addition of AlF4À (Fig 6B) This Pi analog (as well as another analog, BeFx) is known to bind to Pi site in the nucleotidebinding cleft and mimic ADP-Pi or ATP actin filaments [48], thus stabilizing the filament by closing the cleft in actin subunits [49,50] Hence, the increase in the thermal stability of ECP-cleaved F-actin and the disappearance of the shoulder on the DSC profile can be accounted for by the combined effect of phalloidin and AlF4À on the nucleotide-containing cleft Accordingly, the phalloidin-induced effect may increase the affinity of AlF4À to actin, whereas AlF4À -induced clo- Thermal unfolding of cleaved actin sure of the cleft diminishes the population of the subunits remaining nonstabilized by phalloidin via its effect on the cleft conformation This interpretation is consistent with recently published DSC data showing that cooperative effect of phalloidin on the thermal stability of F-actin becomes noncooperative in the presence of AlF4À [51] Phalloidin can stabilize F-actin with a very high cooperativity, with the half-maximal effect being observed at a phalloidin ⁄ actin molar ratio of : 20 [52] In the DSC experiments on intact actin [37], only 10–15% of actin protomers remained unaffected by phalloidin at a phalloidin ⁄ actin molar ratio of : By contrast, more than half of subunits of ECP-cleaved F-actin remained nonstabilized by phalloidin under the same conditions (Fig 6A), consistent with a reduced cooperativity in the effect of phalloidin on the steady-state ATPase activity of ECP-cleaved actin [17] Taken together with the evidence concerning the critical role of the lateral contacts for stabilization of filaments assembled from ECP-actin monomers [47], these data allow us to assume that only the effect of phalloidin on the conformation of the nucleotide-binding cleft is cooperative; it is propagated along the filament by allosteric interactions between phalloidin-bound and free protomers By contrast, the stabilizing effect of phalloidin on the lateral inter-strand interactions is noncooperative; it requires direct binding of phalloidin to actin protomers According to this interpretation, an explanation for the appearance of the pronounced shoulder on the DSC profile of the ECP-cleaved F-actin stabilized by phalloidin (Fig 6A) can be proposed This shoulder appears to reflect the thermal unfolding of the actin protomers whose cleft remains open, and therefore they are stabilized only by lateral inter-strand interactions induced by the direct binding of phalloidin On the other hand, the main transition at 68.5 °C (Fig 6A) most likely corresponds to the thermal unfolding of actin subunits that are stabilized not only by the inter-strand interactions, but also by phalloidininduced closing of the nucleotide-binding cleft In conclusion, the results obtained in the present study suggest that the thermal stability of actin, regardless of whether it is modified by limited proteolysis or by stabilizers, depends on the conformation of the interdomain nucleotide-binding cleft Accordingly, the lower thermal stability of subtilisin- or ECP-cleaved actin compared to intact actin supports the idea [5,13] and also provides additional experimental evidence for a distinct allosteric relationship between conformation of the D-loop and the state of the nucleotide-binding cleft FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS 3819 Thermal unfolding of cleaved actin A V Pivovarova et al Experimental procedures Reagents Subtilisin (type VIII bacterial protease), ATP, ADP, EGTA, Hepes, phenylmethylsulfonyl fluoride, KCl, CaCl2, MgCl2, AlCl3, NaF and phalloidin were purchased from Sigma Chemical Co (St Louis, MO, USA); hexokinase was kindly provided by Dr N Yu Goncharova (Department of Biochemistry, School of Biology, Moscow State University, Russia) Protein preparations Rabbit skeletal muscle actin was prepared from acetonedried muscle powder according to the method of Spudich and Watt [53] G-actin was stored in buffer containing mm Tris-HCl (pH 8.0), 0.2 mm ATP, 0.2 mm CaCl2, 0.5 mm b-mercaptoethanol and 0.03% NaN3 (buffer G) The actin molar concentration was determined by monitoring A290 using an E1% of 6.3 cm)1 [54] and a molecular mass of 42.3 kDa ECP-cleaved G-actin was obtained as described previously [17,30] Ca-G-actin (3.0 mgỈmL)1) was digested at an enzyme ⁄ protein mass ratio of $ : 100 for h at 25 °C and then overnight at °C Because actin cleaved with ECP between Gly42 and Val43 is fairly resistant to further proteolysis by this protease, it was not necessary to use any protease inhibitor The cleaved actin was used within 8–10 h Subtilisin-cleaved actin was prepared essentially as described by Schwyter et al [18] Ca-G-actin (3 mgỈmL)1) was digested for h at an enzyme ⁄ protein mass ratio of : 500 at 25 °C, and the proteolysis was stopped with mm phenylmethylsulfonyl fluoride The cleaved actin preparations were analyzed by SDS ⁄ PAGE [55] Usually, more than 85% of actin was cleaved It is important that the main part of the noncleaved actin appears to correspond to small aggregates of unfolded (socalled ‘inactivated’) G-actin [56], in which the D-loop becomes almost inaccessible to proteolytic cleavage [57] ATP-Ca-G-actin was transformed into ATP-Mg-G-actin by a 3–5 of incubation with 0.2 mm EGTA ⁄ 0.1 mm MgCl2 at 25 °C To obtain ADP-Mg-G-actin, the actinbound ATP was converted into ADP by incubation of ATP-Mg-G-actin with 0.8 mm ADP, mm glucose and hexokinase (8 mL)1) for h at °C [5] It is known that, under similar conditions, only $ 0.4% of ATP was determined in the actin samples after h of incubation with glucose and hexokinase [58] Intact, ECP-cleaved and subtilisin-cleaved Mg-G-actins (3 mgỈmL)1) were polymerized by the addition of 100 mm KCl and mm MgCl2 in the presence of mm ATP Polymerization was monitored by an increase in intensity of light scattering at 90° measured at 350 nm on a Cary Eclipse fluorescence spectrophotometer (Varian Australia Pty Ltd, Mulgrave, Victoria, Australia) 3820 Stabilization of F-actin (24 lm) by phalloidin or by aluminum fluoride (AlF4À ) was performed as described previously [25,28], by the addition of 6–72 lm phalloidin or 0.1– 1.0 mm AlCl3 in the presence of mm NaF and 0.7 mm ADP DSC DSC experiments were performed on a DASM-4M differential scanning microcalorimeter (Institute for Biological Instrumentation, Pushchino, Russia) as described previously [25,28,29,36] All measurements were carried out at a scanning rate of KỈmin)1 The experiments with G-actin were performed in mm Hepes, pH 7.6, containing 0.2 mm CaCl2 or MgCl2 and 0.2 mm ATP (or 0.2 mm ADP in the case of ADP-Mg-G-actin), whereas the thermal unfolding of F-actin was studied in 20 mm Hepes (pH 7.3), 0.1 m KCl, mm MgCl2 and 0.7 mm ADP The final concentration of actin was 24 lm The reversibility of the thermal transitions was assessed by reheating of the sample immediately after cooling from the previous scan The thermal denaturation of all actin samples was fully irreversible Calorimetric traces were corrected for instrumental background and possible aggregation artifacts by subtracting the scans obtained from the reheating of the samples The temperature dependence of the excess heat capacity was further analyzed and plotted using Origin software (MicroCal, Northampton, MA, USA) The thermal stability of actin was described by the Tm, and DHcal was calculated as the area under the excess heat capacity function DSC experiments with different actin species were performed at least twice with very good reproducibility, and the representative curves are shown Acknowledgements We are grateful to Dr Alevtina Morozova for providing us with protease ECP32 ⁄ grimelysin This work was supported by the Russian Foundation for Basic Research (grants 09-04-00266 to D.I.L and 08-0400408 to S.Yu.Kh), the Program ‘Molecular and Cell Biology’ of the Russian Academy of Sciences, and by the grant from the President of Russian Federation (grant MK 2965.2009.4 to A.V.P.) 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and polymerization properties of monomeric ADP-actin Biochim Biophys Acta 995, 109–115 FEBS Journal 277 (2010) 3812–3822 ª 2010 The Authors Journal compilation ª 2010 FEBS ... transitions of the nucleotide -binding cleft For this purpose, we studied the effects of the D -loop cleavage on the thermal unfolding of G- and F -actin Previously, the thermal unfolding of G -actin containing... assume therefore that the decrease in the thermal stability observed by DSC on actin species cleaved within the D -loop is associated with opening of the cleft Does the thermal stability of G -actin. .. that the thermal stability of G -actin may depend on the conformation of the nucleotide -binding cleft This suggestion is supported by the studies on the effects of actin -binding proteins on actin