doi:10.1016/j.jmb.2007.04.056 J. Mol. Biol. (2007) 370, 331–348 Molecular and Structural Basis for Redox Regulation of β-Actin Ingrid Lassing1†, Florian Schmitzberger2†, Mikael Björnstedt3 Arne Holmgren2, Pär Nordlund2, Clarence E. Schutt4 and Uno Lindberg1⁎ 1Department of Microbiology, An essential consequence of growth factor-mediated signal transduction is the Tumor Biology, and Cell generation of intracellular H2O2. It operates as a second messenger in the Biology, Karolinska Institutet, control of actin microfilament dynamics, causing rapid and dramatic changes SE-171 77 Stockholm, Sweden in the morphology and motile activity of stimulated cells. Little is understood about the molecular mechanisms causing these changes in the actin system. 2Department of Medical Here, it is shown that H O acts directly upon several levels of this system, and Biochemistry and Biophysics, 2 2 some of the mechanistic effects are detailed. We describe the impact of Karolinska Institutet, SE-171 77 oxidation on the polymerizability of non-muscle β/γ-actin and compare with Stockholm, Sweden that of muscle α-actin. Oxidation of β/γ-actin can cause a complete loss of 3Division of Pathology F46, polymerizability, crucially, reversible by the thioredoxin system. Further, Department of Laboratory oxidation of the actin impedes its interaction with profilin and causes Medicine, Karolinska University depolymerization of filamentous actin. The effects of oxidation are critically Hospital, Huddinge, SE-141 86 dependent on the nucleotide state and the concentration of Ca2+.Wehave Stockholm, Sweden determined the crystal structure of oxidized β-actin to a resolution of 2.6 Å. 4 The arrangement in the crystal implies an antiparallel homodimer connected Department of Chemistry, by an intermolecular disulfide bond involving cysteine 374. Our data indicate Princeton University, that this dimer forms under non-polymerizing and oxidizing conditions. We Princeton, NJ 08544-1009, USA identify oxidation of cysteine 272 in the crystallized actin dimer, likely to a cysteine sulfinic acid. In β/γ-actin, this is the cysteine residue most reactive towards H2O2 in solution, and we suggest plausible structural determinants for its reactivity. No other oxidative modification was obvious in the structure, highlighting the specificity of the oxidation by H2O2. Possible consequences of the observed effects in a cellular context and their potential relevance are discussed. © 2007 Elsevier Ltd. All rights reserved. β Keywords: ADP- -actin; antiparallel dimer; cysteine sulfinic acid; H2O2; *Corresponding author thioredoxin sincereactiveoxygenspecies(ROS)quenching Introduction 1 abolishes it. Intracellular H2O2 is transiently gen- In many eukaryotic cells redox effects appear to erated upon activation of receptors for peptide growth factors and cytokines as well as integrin- have central roles in signal transduction, and H2O2 is essential for growth factor-induced signaling, mediated cell adhesion. It acts as a rapidly produced and effective second messenger, whose spatiotem- poral presence in cells often correlates with changes in the microfilament system.2,3 † I.L. and F.S. contributed equally to this work. Growth factor-stimulated H2O2 production is also Abbreviations used: DTNB, closely interrelated with increases in the intracellular 5,5′-dithiobis(2-nitrobenzoic acid); DTT, 1,4-dithiothreitol; concentration of Ca2+,4 and appears to correspond to Gdn-HCl, guanidine hydrocloride; ROS, reactive oxygen the kinetics of actin polymerization-dependent out- species; Trx, thioredoxin; TR, thioredoxin reductase. growth of lamellipodia and filopodia.5,6 E-mail address of the corresponding author: Actins are highly conserved eukaryotic proteins [email protected] existing in different cellular isoforms. Non-muscle 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. 332 Redox Regulation of β-Actin β-actin and γ-actin, amongst the most abundant of assembly/disassembly and related changes of proteins in many cell types, are pivotal in maintain- actin during redox conditions as well as its regula- ing and mediating the infrastructure of the cellular tion by associated proteins. matrix. They form an essential part of chemo- Most of the research on actin has been dedicated mechanical transduction systems in non-muscle to characterizing its attributes under reducing cells, where they, together with actin-binding pro- conditions. Equally important is an understanding teins, generate force for various activities involved of its properties in an oxidizing environment, in translocation and cell shape change. representative of the circumstances during signal The constant and rapid reorganization of the actin transduction by H2O2. Experiments to study an microfilament system during cell motility and effect of oxidation on actin in vitro have been done migration, mitogenesis, and phagocytosis depends with muscle α-actin,13 whereas the non-muscle β/γ- on nucleation, elongation, and depolymerization of actins would seem to be the more relevant isoform actin filaments as elementary processes.6,7 The with respect to cell shape and motility. Indeed, one dynamic reorganization of cellular actin is highly of the few significant differences between α-actin regulated, and ROS appear to be one vital regula- and β/γ-actin is the number of cysteine residues, the tory element.2 Experiments have indicated that actin thiol groups of which are the protein moieties could constitute a direct target for oxidative mod- generally most reactive towards H O . Non-muscle – 2 2 ification in vivo.8 10 Moreover, actin was shown to be β and γ-actins have six cysteine residues (Figure 1), oxidatively modified in pathophysiological states and α-actin has five. suggestive of oxidation as a cause of mechanical Here, we have examined the biochemical effects of 11,12 dysfunction. Thus, an explanation of the mole- oxidation by H2O2 on the capacity of the actin iso- cular basis of redox-regulated microfilament pro- formstoformfilamentsandcharacterizedthe cesses requires an understanding of the mechanism structural consequences of oxidation in β-actin. α Figure 1. Illustration of the position of cysteine residues and the profilin and DNase I interaction sites in actin. The C atom cartoon represents the β-actin structure.17 The color scheme for this and the following model illustrations, unless stated otherwise, is as follows. Ball-and-stick representation for the atoms: carbon, grey; nitrogen, blue; oxygen, red; phosphorus, orange; sulfur, yellow. Amino acid residues involved in the interaction with DNase I (derived from the α- actin–DNase I complex104) are colored magenta and those in contact with profilin are colored light-blue. Redox Regulation of β-Actin 333 The results show that oxidation by H2O2 has Table 2. Effect of calcium on oxidation of cysteine profound and differential effects on both mono- residues in ADP-β/γ-actin meric and filamentous non-muscle actin, and can lead to the formation of disulfide-linked antiparallel Oxidized cysteine 2+ residues homodimers. [Ca ]free Oxidation conditions (μM) Number Identity 5mMH2O2, 10 min, 25 °C 1 2 Cys272/Cys374 Results 5mMH2O2, 15 min, 10 0.6 Cys272 at 25 °C 5mMH2O2, 15 min, 37 °C 10 2.3 Cys272/Cys374 Actin thiol groups and their reactivity towards 5mMH2O2, 15 min, 37 °C 100 1 Cys272 ′ 20 mM H2O2, 40 min, 100 2 Cys272/Cys374 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) 37 °C and H2O2 Number of oxidized SH-groups after incubation with H2O2 in the presence of different concentrations of Ca2+. The number of accessible SH-groups in α-actin and β/γ-actin in the presence of ATP or ADP (see Materials and Methods), is given in Tables 1 and 2. the lag phase or the steady-state level of polymer- At high concentrations (0.1 mM) of Ca2+, mono- α β γ ization significantly (Figure 2(a)). In contrast, oxida- meric -actin and / -actin in the presence of both tion of ADP-β/γ-actin at 1 μMCa2+ (two cysteine ATP and ADP had one and two accessible cysteine residues oxidized) led to a complete loss of poly- residues, respectively. At submicromolar concentra- β γ 2+ merizability (Figure 2(b)). Incubation of ADP- / - tions of Ca , an additional DTNB-reactive SH- actin with H O with 0.1 mM Ca2+ (one cysteine group appeared in α-actin-ADP, as shown earlier.14 2 2 β γ oxidized; Table 2) resulted in an actin which Similarly, the ADP-form of / -actins exposed an polymerized more slowly but reached a steady- extra DTNB-reactive cysteine residue at 1 μMCa2+. β γ state level comparable to that of the non-oxidized In the ATP-form of / -actin, the additional SH- control (data not shown). Similarly, oxidation of α- group was only partially accessible and, in the 2+ 2+ actin in the ADP form at 0.1 mM Ca , for a presence of 0.1 mM Ca , its appearance required a prolonged period, led to decreased polymerizability higher concentration of H2O2 or prolonged incuba- (data not shown). tion time. Thus, one of the three cysteine residues could not be oxidized by H2O2, even though it was Effect of the thioredoxin system on oxidized accessible to DTNB. Notably, for the extra cysteine ADP-β/γ-actin to become unavailable, it was sufficient to increase the concentration of Ca2+ to 10 μM, under otherwise The reversibility of oxidation by H2O2 and its similar conditions (Table 2). effect on the polymerizability of ADP-β/γ-actin at 1 μMCa2+ are illustrated in Figure 2. Oxidized Effect of oxidation on actin polymerizability protein was incubated with the thioredoxin system, β γ which reduces disulfide bonds and cysteine sulfenic Oxidation of ATP- / -actin with 5 mM H2O2 at acids, products of oxidation of cysteine residues 1 μMofCa2+ (one cysteine oxidized) did not affect with H2O2. Indeed, thioredoxin rapidly reduced the oxidized cysteine residues in ADP-β/γ-actin. All Table 1. Number of DTNB reactive SH-groups/actin components of the thioredoxin (Trx) system molecule before and after oxidation with H2O2 (NADPH, thioredoxin reductase (TR), and Trx; see SH groups in Materials and Methods) were required for the Total SH non-denatured actin reduction of the cysteine residues and subsequent 2+ restoration of the capacity of the protein to form A.