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Biochemistry 2010, 49, 8944–8954 DOI: 10.1021/bi1008062
ADP-Ribosylation of Cross-Linked Actin Generates Barbed-End Polymerization-Deficient F-Actin Oligomers† Alexandru A. Perieteanu, Danielle D. Visschedyk, A. Rod Merrill, and John F. Dawson* University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received May 20, 2010; Revised Manuscript Received August 21, 2010 ABSTRACT: Actin filament subunit interfaces are required for the proper interaction between filamentous actin (F-actin) and actin binding proteins (ABPs). The production of small F-actin complexes mimicking such interfaces would be a significant advance toward understanding the atomic interactions between F-actin and its many binding partners. We produced actin lateral dimers and trimers derived from F-actin and rendered polymerization-deficient by ADP-ribosylation of Arg-177. The degree of modification resulted in a moderate reduction in thermal stability. Calculated hydrodynamic radii were comparable to theoretical values derived from recent models of F-actin. Filament capping capabilities were retained and yielded pointed-end dissociation constants similar those of wild-type actin, suggesting native or near-native interfaces on the oligomers. Changes in DNase I binding affinity under low and high ionic strength suggested a high degree of conformational flexibility in the dimer and trimer. Polymer nucleation activity was lost upon ADP-ribosylation and rescued upon enzyme-mediated deADP-ribosylation, or upon binding to gelsolin, suggesting that interactions with actin binding proteins can overcome the inhibiting activities of ADP-ribosylation. The combined strategy of chemical cross-linking and ADP-ribosylation provides a minimalistic and reversible approach to engineering polymerization-deficient F-actin oligomers that are able to act as F-actin binding protein scaffolds.
Actin is a eukaryotic protein that is essential for a myriad of biochemical processes such as cellular mobility, cytokinesis, sarcomere contraction, and protein transport (32, 36). Under physiological conditions, globular actin (G-actin) self-associates to form long double-stranded, right-handed helical actin filaments (F-actin) containing hundreds of actin subunits. In the cell, an array of actin binding proteins (ABPs)1 control and/or take advantage of actin polymerization (5). Two general classes of F-actin-specific ABPs exist: those that bind to the ends of filaments (capping proteins) and those that bind along the length of the filaments (side-binding ABPs). In actin filaments, adjacent actin subunits are responsible for the formation of these critical ABP binding interfaces. The goal of this research is to develop short F-actin oligomers to serve as structural platforms for the elucidation of molecular interactions between F-actin and ABPs. Any strategy to this end involves two aspects: (1) covalent cross-linking of actin subunits in F-actin and (2) modification of the resulting actin oligomers to inhibit actin polymerization. The extent of these modifications must be strategically balanced with maintaining physiological interactions that are integral to F-actin function. Previous work has shown that an actin longitudinal (or longpitch) dimer can be formed by covalently cross-linking Gln-41 to Cys-374 of adjacent protomers using N-(4-azido-2-nitrophenyl)putresceine (ANP) (11). ANP-actin dimers were also shown † This work was supported by NSERC Grant 250188-04 to J.F.D. and grants from HSFP, CIHR, and CCF to A.R.M. *Address correspondence to this author. Tel: (519) 824-4120 ext 58181. Fax: (519) 837-1802. E-mail:
[email protected]. 1 Abbreviations: PBM, N,N0 -p-phenylenebismaleimide; ADPr, ADPribosylated; [NBD], concentration based on nucleotide binding domains; β-ME, 2-mercaptoethanol; DNase I, deoxyribonuclease I; ABPs, actin binding proteins; py-actin, pyrene iodoacetamide labeled actin; ADPr-py-actin, ADP-ribosylated py-actin; Cc, critical concentration; MWCO, molecular weight cutoff.
pubs.acs.org/Biochemistry
Published on Web 08/26/2010
to maintain some affinity for the N-terminal segment of gelsolin, deoxyribonuclease I (DNase I), and myosin subfragment 1 (22). Conversely, lateral (or short-pitch) actin dimers have been produced by covalently cross-linking Cys-374 to Lys-191 of adjacent protomers using N,N0 -p-phenylenebismaleimide (PBM) (18, 25, 34). Similarly, PBM trimers possessing both lateral and longitudinal contacts have been constructed and shown to act as filament nuclei, essentially increasing filament growth kinetics by circumventing slow nucleation events (8, 18). Three general modification strategies have been used to inhibit actin polymerization: translational modifications (14, 29, 41), covalent posttranslational modifications (9, 17, 19), and noncovalent posttranslational modifications (2, 4, 16, 23, 27). Here, we employ a recently discovered mono-ADP-ribosyltransferase from Photorhabdus luminescens to specifically modify PBMcross-linked actin oligomers (39). ADP-ribosyltransferase enzymes (ADPRTs) covalently transfer an ADP-ribose (ADPr) moiety from NADþ to a target protein. ADP-ribosylation at Arg-177 of actin is also catalyzed by a variety of other, classical ADPRTs such as Clostridium perfringens iota toxin, Clostridium botulinum C2 toxin, and Salmonella enterica virulence protein SpvB. In this work we use a combined strategy of PBM cross-linking and ADP-ribosylation to produce and purify polymerizationdeficient short-pitch actin dimers (ADPr dimer) and actin trimers (ADPr trimer). Both ADPr dimer and ADPr trimer were found to possess similar hydrodynamic radii to those calculated from structural data, suggesting that both species adopt a physiological conformation. While the modifications result in slightly decreased thermal stability and altered nucleotide exchange kinetics, the constructs possessed conformational flexibility and retained their ability to cap existing filament barbed ends. Finally, we found that the filament nucleation capabilities of actin dimers and trimers were abolished upon ADP-ribosylation r 2010 American Chemical Society
Article but were rescued upon enzyme-mediated reversal of the ADPribosylation (deADPr) and upon structural remodeling induced by gelsolin binding. This study provides a minimalistic and reversible approach to engineering polymerization-deficient F-actin oligomers able to act as F-actin binding protein scaffolds. EXPERIMENTAL PROCEDURES Reagents. All reagents were purchased from either SigmaAldrich (St. Louis, MO) or Fisher Scientific (Mississauga, Ontario, Canada). Chromatographic columns were supplied by GE Healthcare (Piscataway, NJ). Purifications were performed on an AKTA FPLC system. Protein concentrators (10 kDa MWCO) were purchased from Millipore (Billerica, MA). Protein Preparation. Skeletal muscle acetone powder was prepared from chicken breast as described by Spudich and Watt (37). Actin concentrations were determined from absorbance at 290 nm using an extinction coefficient of 67742 M-1 cm-1 or using the Bio-Rad protein assay (Mississauga, Ontario, Canada). Actin samples in G-buffer (2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM 2-mercaptoethanol, β-ME) were frozen in liquid N2 and stored at -70 °C unless used for crosslinking. Where applicable, we report concentrations based on the presence of high-affinity nucleotide binding domains [NBD], allowing for differentiation between actin subunit concentration and the concentration of total complex. Photox was purified from insoluble inclusion bodies as previously described (39). The resulting protein was dialyzed into 20 mM Tris-HCl, pH 7.5, and 50 mM NaCl, concentrated to 0.2-1.0 mg/mL, and stored at -70 °C. Human gelsolin was prepared as described (26) and stored at -70 °C. ADPr trimer-gelsolin heterocomplexes were formed by incubating 1:1 ADPr trimer and gelsolin, followed by purification on a Superdex S200 column preequilibrated in buffer G-10 (10 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM β-ME, 0.2 mM NaN3, 0.25 mM phenylmethanesulfonyl fluoride) with 50 mM NaCl. Purity and composition were assessed by native PAGE and SDS-PAGE (data not shown). Bovine pancreas deoxyribonuclease I (DNase I) purchased from Worthington Biochemical Corp. (Lakewood, NJ) was purified using a Superdex S200 gel chromatography column to remove traces of a higher molecular weight impurity (data not shown). Samples were flash frozen in liquid N2 and stored at -70 °C. Gelsolin and DNase I were thawed and exchanged into G-buffer using HiTrap desalting columns for native PAGE binding assays. Bio-Rad protein assay was employed for concentration determination of gelsolin and DNase I. ADP-Ribosylation and De-ADP-ribosylation. Actin samples were diluted to 25-100 μM and the G-buffer Tris-HCl, pH 8.0, concentration was increased to 10 mM. Samples were incubated with 20-200 nM photox (0.1-1% v/v), and a 5-10-fold molar excess of NADþ was titrated into the sample mixture with slow stirring over a period of 15 min. Enzymatic activity was monitored upon addition of toxin using fluorescence (excitation 305 nm, emission 405 nm) and the NADþ analogue, etheno-NADþ (ε-NADþ). ADPr-actin samples were exchanged into 20 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5, G-buffer supplemented with 30 nM nicotinamide. ADP-ribosylation reversal was initiated by the addition of 200 nM photox and allowed to proceed overnight at 4 °C while being dialyzed against 30 nM nicotinamide. Samples were then exchanged into G-buffer. Actin Cross-Linking with PBM. Chemical cross-linking of actin with PBM was performed as described (18) with some
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modifications. Actin (3-6 mg/mL) was polymerized at pH 8.0 by the addition of 50 mM KCl and 2 mM MgCl2 under ambient temperature for 1-3 h. Cross-linking of actin was initiated by the addition of 10 mM sodium tetraborate, pH 9.0, and a 1.5-fold molar excess of a 25 mM stock solution of PBM dissolved in DMF and terminated after 30 min at ambient temperature using a 150-fold molar excess of β-ME over PBM. Cross-linked F-actin was harvested by centrifugation at 167424g for 75 min and resuspended in G-buffer to a final concentration of 1-2 mg/mL. The resulting PBM-cross-linked actin mixture was dialyzed extensively over 3-4 days with at least five buffer exchanges to depolymerize cross-linked filaments. ADP-ribosylation was initiated as described above. Remaining F-actin was removed by centrifugation at 167424g for 75 min. Samples were concentrated and applied to a Superdex S200 column preequilibrated in buffer G-10 with 50 mM NaCl. Fractions containing ADPr dimer and ADPr trimer were pooled, and iterations of gel filtration were performed. As a final measure, polymerization was induced over a period of 30 min, and samples were centrifuged at 392000g for 20 min. Supernatants (2-7 mg/mL) were then dialyzed against buffer G-10, flash frozen in liquid N2, and stored at -70 °C. When utilized, samples were desalted into G-buffer and used within 2 days of thawing. Polymerization. Polymerization was monitored using three techniques. (1) Light Scattering Intensity Copolymerization. Actin (0-20 μM) and ADP-ribosylated actin (0-20 μM) were brought to ambient temperature, and polymerization was induced with a 10-fold stock solution of polymerization salts (250 mM Tris-HCl, pH 8.0, 500 mM KCl, 20 mM MgCl2, 2 mM ATP, and 10 mM EGTA), yielding F-buffer. Light scattering at 90° to the incident light beam was monitored using a pulsed-beam Varian Cary Eclipse fluorescence spectrophotometer (Mississauga, Ontario, Canada) at an excitation of 360 nm and emission of 360 nm. All solutions were passed through 0.22 μm filters to minimize background light scattering, and experiments were performed with a replicate number of n g 3. (2) Pyrene Polymerization Assay. Skeletal actin was labeled with pyrene iodoacetamide (py-actin) as previously described (3). Actin samples in G-buffer were diluted and brought to 2.5% py-actin prior to induction of polymerization. Polymerization of 10 μM [NBD] of actin, ADPr-actin, ADPr dimer, or ADPr trimer was initiated by the addition of stock polymerization salts. Fluorescence intensity was monitored using a Cary Eclipse spectrofluorometer at excitation and emission wavelengths of 347 and 407 nm, respectively. Nucleation experiments were similarly performed in triplicate with 5 μM actin (2.5% py-actin) substituted with 0.25 μM [NBD] of the nuclei being examined. Pyrene fluorescence was also used for monitoring the copolymerization of actin and pyrene-labeled ADPr-actin (ADPrpy-actin). Briefly, py-actin was ADP-ribosylated as described above and excess NADþ removed by gel-filtration chromatography. Samples containing 2 μM ADPr-py-actin and increasing concentrations of actin (0-50 μM) were monitored after the introduction of stock polymerization salts. Experiments were also performed using non-ADP-ribosylated py-actin. The ratios of the baseline-corrected steady-state (180 min) change in fluorescence (ΔF) intensities of ADPr-py-actin and py-actin were plotted against the concentration of unmodified actin; a curve representing the proportion of ADPr-py-actin filament incorporation was generated. Assembly curves generated using Sephadex S300 purified actin did not yield different results.
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(3) Polymerization Sedimentation Assay. Polymerization of 20 μM [NBD] actin, ADPr-actin, ADPr dimer, or ADPr trimer was induced by incubating the samples in polymerization salts for 3 h at room temperature. Samples were subjected to centrifugation at 392000g for 20 min. Supernatants and pellets were separated and analyzed by SDS-PAGE. Dual-Color Filament Capping Assay. The dual-color filament capping assay was performed as described by Harris et al. (10). Actin (4 μM) was polymerized for 4 h in F-buffer. Samples were diluted 2-fold with 300 nM ADPr-actin, dimer, or trimer and allowed to incubate at ambient temperature for 5 min. Samples were again diluted 2-fold with equimolar rhodaminephalloidin. Samples were further diluted 10-fold with 400 nM G-actin and 400 nM Alexa 488-phalloidin and rapidly brought to F-buffer conditions using concentrated stock. The solution was allowed to incubate for 10 min before being diluted an additional 5-fold with F-buffer supplemented with 3 mg/mL glucose, 120 μg/mL glucose oxidase, and 18 μg/mL catalase. The final mixture (1 μL) was loaded onto a 0.01% poly-(L-lysine)-coated slide. Images were taken through rhodamine and fluorescein filters. The percent occurrence of two colored events was determined and plotted for each condition. At least six images were analyzed, totaling at least 300 independent filaments for each sample. The standard deviation of the mean percent occurrence was calculated from the variance between images. Inhibition of Actin Nucleation Assay. Polymerization of actin in the presence of filament nuclei was monitored using an Applied Photophysics SX20 stopped-flow spectrofluorometer (Surrey, U.K.) set to an excitation wavelength of 350 nm and an emission cutoff filter of 395 nm. F-Actin (1 μM, 5% py-actin) in 5 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 100 mM KCl, 0.2 mM ATP, and 0.2 mM β-ME was mixed with 1 volume of 3 μM actin and 5% py-actin in 5 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, and 0.2 mM β-ME supplemented with 0.002-2.0 μM ADPr-actin, ADPr dimer, or ADPr trimer. The rate of polymerization between 25 and 50 s was plotted against the concentration of ADP-ribosylated actin, and a binding isotherm was used to extrapolate the apparent dissociation constants (Kapp d ) for ADPr-actin and its oligomers to the barbed end of actin filaments. Assembly curves generated using Sephadex S300 purified actin did not yield different results. Acrylamide Gel Electrophoresis. SDS-PAGE was performed as described previously (20) and visualized by staining with Coomassie Brilliant Blue R250 or under UV light. Native PAGE was performed with 8% nonreducing, nondenaturing acrylamide gels supplemented with 0.2 mM ATP and 0.2 mM CaCl2. Native PAGE electrophoresis was also performed on ice at low voltage (90% incorporation was extrapolated to occur only at ratios of actin to ADPr-py-actin of >75:1. An ADPr trimer modified with only one ADP-ribose would possess an actin to ADPr-actin ratio of 2:1 and, based on our data, would have an efficiency of incorporation
