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1
1 A crosslinked and ribosylated actin trimer does not interact
2 productively with myosin
3
4 Navneet Sidhu and John F. Dawson
5 Contact: [email protected]
6 Department of Molecular and Cellular Biology, University of Guelph, Guelph Ontario, N1G
7 2W1
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8 Abstract
9 A purified F-actin derived actin trimer that interacts with end-binding proteins did not
10 activate or bind the side-binding protein myosin under rigor conditions. Remodeling of the actin
11 trimer by the binding of gelsolin did not rescue myosin binding, nor did the use of different
12 means of inhibiting the polymerization of the trimer. Our results demonstrate that ADP-
13 ribosylation on all actin subunits of an F-actin derived trimer inhibits myosin binding and that
14 the binding of DNase-I to the pointed end subunits of a crosslinked trimer also remodels the
15 myosin binding site. Taken together, this work highlights the need for a careful balance between
16 modification of actin subunits and maintaining protein-protein interactions to produce a
17 physiologically-relevant short F-actin complex.
18
19 Keywords: actomyosin, conformational change, protein crosslinking, polymerization inhibition,
20
21 Abbreviations: GS: gelsolin, ANP: N-(4-azido-2-nitrophenyl) putrescine, pPBM: para-
22 phenylenebismaleimide, S1: myosin subfragment-1, ADPr-; ADP-ribosylated
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23 Introduction
24 Actin is a highly regulated cytoskeletal protein. Rarely do actin proteins exist on their
25 own in cells, unbound by an actin binding protein (ABP) modulating where and when actin
26 functions (for reviews, see (Dos Remedios et al. 2003, Winder and Ayscough 2005)). Actin
27 binding proteins fall into two main categories: monomer-binding or filament-binding. We call
28 these G-ABPs and F-ABPs to follow the tradition of G-actin and F-actin in the field. Capping
29 proteins bind filament ends, either the pointed end (e.g. tropomodulin) or the barbed-end (e.g.
30 gelsolin) while side-binding proteins interact along the side of the filament (e.g. myosin or
31 tropomyosin) (Fig 1).
32 Current technology is approaching atomic resolution of F-actin complexes using electron
33 microscopy; however, the resolution and quality from this work still lacks important atomic
34 details, such as local conformational differences between neighbouring subunits and flexible
35 regions of the actin molecules when interacting with F-ABPs. X-ray crystallography remains a
36 mainstay for determining atomic resolution interactions between proteins. Our work has been
37 aimed at developing a short F-actin-derived structure by combining chemical crosslinking with
38 polymerization inhibition to serve as a platform for determining the atomic structure of F-ABP
39 complexes. We previously produced an ADP-ribosyl (ADPr)-modified and para-
40 phenylenebismaleimide (pPBM)-crosslinked actin trimer derived from F-actin, referred to as the
41 ADPr-trimer (Perieteanu et al. 2010). We found that DNase-I binds the pointed end and gelsolin
42 binds the barbed end of the ADPr-trimer. Our next step is to study side-binding F-ABPs with the
43 ADPr-trimer, using myosin as an example.
44 Covalent modification of F-actin has different effects on actin function depending on the
45 chemical nature and location of the modification (reviewed in (Kumar and Mansson 2017)). The
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46 interactions between inter- or intrastrand crosslinked F-actin and myosin has been studied (Kim
47 et al. 2002; Mannherz et al. 2008; Morrison et al. 2010). Filaments made up of purified
48 intrastrand crosslinked actin dimers produced 50% myosin activity (Mannherz et al. 2008).
49 When these dimers were bound by G-ABPs to inhibit polymerization, no myosin activity was
50 observed; however, the stoichiometry of the ABP to the subunits may have inhibited myosin
51 activity. Previously, we produced an intrastrand actin dimer containing an actin mutant that was
52 blocked on its pointed end by DNase-I or by ADP-ribosylation on both subunits. We found that
53 these dimers interacted with myosin (Morrison et al. 2010), confirming previous work showing a
54 ternary complex of myosin subfragment-1, actin and DNase-I can be formed (Blanchoin et al.
55 1995).
56 Directly relevant to our work, the impact of pPBM crosslinking of F-actin on myosin
57 activity has been studied previously (Kim et al. 2002). In their work, Kim et al crosslinked F-
58 actin randomly, resulting in crosslinking of half of the subunits in the filaments. The crosslinked
59 F-actin inhibited myosin-mediated filament movement in in vitro motility assays; however,
60 myosin binding and ATPase activity remained largely unchanged (Kim et al. 1998, 2002).
61 Specifically, the Vmax of myosin ATPase activity was comparable between crosslinked and
62 unmodified F-actin, suggesting that crosslinked F-actin subunits retain the ability to bind myosin.
63 ADPr modification of actin on Arg-177 has been reported to inhibit actin polymerization
64 (Wegner and Aktories 1988) and the structure of ADPr-actin has been solved (Margarit et al.
65 2006). In their work, Ballweber et al (Ballweber et al. 2001) examined the impact of ADP-
66 ribosylation of actin monomers on the binding of actin binding proteins, including myosin. They
67 found that G-actin binding proteins and cofilin and gelsolin all interacted with ADPr-monomers.
68 Significantly, the fluorescence of pyrene-labeled ADPr-actin increased in the presence of myosin
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69 and electron micrographs revealed short filaments of ADPr-actin in the presence of myosin,
70 confirming an interaction of myosin with ADPr-actin.
71 Recent electron microscopy reconstructions have produced high resolution structures of
72 the myosin bound to F-actin (Behrmann et al. 2012; Wulf et al. 2016; Banerjee et al. 2017; Fujii
73 and Namba 2017a; Gurel et al. 2017; Mentes et al. 2018). These structures suggest that the
74 location of the pPBM crosslink and the position of ADP-ribosylation on Arg-177 of actin do not
75 sterically block the myosin binding site (Fig 1).
76 Based on the previous biochemical work with pPBM-crosslinked F-actin, ADPr-actin and
77 electron microscopy-based structures of actomyosin, we tested the hypothesis that a pure ADPr-
78 trimer will bind myosin. Our results show that myosin does not interact with the ADPr-trimer,
79 ADPr-trimer capped with gelsolin, or DNase-I capped trimer. Comparing with the recent work
80 of Qu et al (Qu et al. 2018), our results suggest that complete ribosylation of the actin trimer
81 remodels the myosin binding site to inhibit myosin interactions and that the capping of the
82 pointed end of a crosslinked actin trimer with DNase-I also blocks myosin binding. Future work
83 will involve determining a balance of actin modifications that support myosin activity and
84 studying the variety of mechanisms for different F-ABP binding to short F-actin complexes.
85
86
87 Materials and methods
88 Reagents
89 Unless otherwise stated, all buffer reagents and media were obtained from Fisher
90 Scientific (Mississauga, ON) or Sigma–Aldrich (St. Louis, MO). All chromatography columns
91 were obtained from GE Healthcare (GE Healthcare, Piscataway, NJ).
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92
93 Protein Purification
94 Actin protein was purified from turkey breast tissue as described (Perieteanu et al. 2010).
95 Photox ADP-ribosyltransferase enzyme was purified from inclusion bodies from E. coli Rosetta
96 cells as described (Visschedyk et al. 2010). E. coli cells expressing recombinant full-length
97 gelsolin (GS) were lysed in a French Press and the clarified lysate was developed on an Affigel
98 Blue column with a linear gradient of 1 M NaCl in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA)
99 as described (Morrison and Dawson 2007). Fractions containing GS were pooled and dialysed
100 against TE buffer with β-mercaptoethanol (β-ME). Pure myosin and myosin subfragment-1 (S1)
101 was obtained from rabbit soleus muscle following established methods (Margossian and Lowey
102 1982).
103 ADPr-oligomers – Chemical crosslinking of F-actin with p-phenylenebismaleimide
104 (pPBM), depolymerization and ADP-ribosylation (ADPr-) catalyzed by Photox enzyme was
105 performed as described (Perieteanu et al. 2010). F-actin-derived ADPr-oligomers of crosslinked
106 actin were purified with iterative Superdex-200 (26/600) chromatography in G-10 buffer (10 mM
107 Tris-HCl, pH8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM β-ME, 0.2 mM NaN3, 0.25 mM
108 phenylmethanesulfonyl fluoride (PMSF)) with 50 mM NaCl, following the previously published
109 method (Perieteanu et al. 2010). This previously published method results in a stoichiometry of
110 one ADP-ribosylation on Arg-177 of each actin subunit in the crosslinked trimer. Mass
111 spectrometry confirmed the presence of the chemical crosslink between Lys-191 and Cys-374
112 (data not shown).
113 GS:ADPr-trimer – Gelsolin-bound ADPr-trimer (GS:ADPr-trimer) was produced by
114 incubating GS and ADPr-trimer in 1.4 molar ratio overnight at 4˚C. The resulting reaction
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115 mixture was purified with Superdex-200 (10/300) column chromatography developed in G-10
116 buffer with 50 mM NaCl. The elution fractions were run on a native gel with GS alone or ADPr-
117 trimer alone as controls. The fractions containing GS:ADPr-trimer were pooled, stored at 4˚C
118 and studied within a week of purification.
119 D:trimer – To produce DNase-I capped pPBM-crosslinked trimer (D:trimer), F-actin
120 was crosslinked with pPBM using the conditions described above for making ADPr-trimer.
121 Crosslinked F-actin was incubated with DNase-I (Worthington) at a 1:1 actin subunit:DNase-I
122 molecular ratio in F-buffer (25 mM Tris-HCl, pH 8.0, 50 mM KCl, 2 mM MgCl2, 1 mM EGTA,
123 0.1 mM ATP, 1 mM β-ME) overnight at 4˚C. The reaction was centrifuged at 392 000 g for 20
124 min and the resulting supernatant was resolved on a Superdex-200 (10/300) column in G-10
125 buffer.
126
127 Dynamic Light Scattering
128 ADPr-trimer and myosin S1 were clarified through 0.22 um filters. Molar concentrations
129 were adjusted using filtered G-10 buffer with 50 mM NaCl. Dynamic light scattering
130 measurements were performed on Nano S Zetasizer (Malvern, U.K.) equipped with a 633 nm red
131 laser and a 173˚ backscatter detector. Experimental Stokes radii of the samples were determined
132 by a normal distribution fit of volume distribution measurements.
133
134 Acrylamide Gel electrophoresis
135 SDS-PAGE was performed as described previously (Laemmli, 1970) with acrylamide
136 gels comprised of 10% resolving and 5% stacking gel. Native PAGE was performed with non-
137 denaturing acrylamide gels comprised of 8% resolving and 5% stacking gel supplemented with
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138 0.2 mM ATP and 0.2 mM CaCl2. Native PAGE was run at a low voltage (< 60V) on ice. SDS-
139 PAGE and Native gels were visualized with either Coomassie Brilliant Blue R-250 (Sigma
140 Aldrich) or Silver Stain (PierceTM Silver Stain Kit).
141
142 Myosin Actin-activated ATPase assay
143 The myosin ATPase activity assay used was based on a colorimetric assay based on the
144 production of a blue by-product between released inorganic phosphate (Pi) and molybdate,
145 modified from Trybus (2000) for a 96-well format as described (Liu et al. 2017). Single
146 concentration assays (5 uM actin on a per-subunit basis for ADPr-trimer) in the presence of 0.25
147 mg/mL myosin were performed in triplicate with released Pi measured at 10-minute time
148 intervals for 30 minutes. Owing to small amounts of pure GS:ADPr-trimer and D:trimer, myosin
149 ATPase assays were performed with a single concentration of 1.5 uM actin on a per-subunit
150 basis with released Pi measured at 40 and 60 minutes for GS:ADPr-trimer and 20, 40 and 60
151 minutes for D:trimer. The average ATPase activity was plotted using Prism 7.0c software
152 (GraphPad, LaJolla, Calif.).
153
154 Results
155 Myosin does not bind ADPr-trimer under rigor conditions
156 Since the ADPr-trimer contains one potential myosin site per complex (Mentes et al.
157 2018) (Fig 1), we tested the binding of myosin subfragment-1 (S1) to ADPr-trimer under rigor
158 conditions and found little evidence of interaction. No significant increase in hydrodynamic
159 radius was seen between protein alone controls and a mixture of ADPr-trimer and myosin S1
160 (Fig 2) and no shift in the mobility of ADPr-trimer was observed in the presence of myosin S1
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161 on native PAGE gels (Fig 3A). The presence of a single band in the trimer sample on SDS-
162 PAGE shows that all subunits in the trimers are covalently crosslinked to each other (Fig 3B).
163 Since myosin S1 alone was not observed on the native gels, a new band might not run on the gel,
164 or the myosin S1 might be found in the band present on the gel. We performed mass
165 spectrometric analysis on the ADPr-trimer-containing bands from the native PAGE and found no
166 myosin peptides (data not shown). Likewise, no shift was seen in analytical gel filtration
167 chromatography peak elution volumes for ADPr-trimer in the absence or presence of myosin S1
168 (Fig 4); rather, a peak of myosin S1 protein eluted in different fractions than the ADPr-trimer
169 protein.
170 To confirm that S1 does not bind ADPr-trimer, we found no significant increase in
171 myosin ATPase activity in the presence of ADPr-monomer, -dimer, or –trimer (Fig 3C), whereas
172 myosin ATPase activity is activated in the presence of F-actin, as expected.
173
174 Remodeling by gelsolin does not restore myosin binding.
175 Previously, we showed that ADPr-modified actin oligomers do not nucleate actin
176 polymerization (Perieteanu et al. 2010). The binding of gelsolin (GS) to ADPr-trimer restored
177 nucleation of polymerization from the pointed end, suggesting that GS remodels the ADPr-trimer
178 into a native-like state. We therefore tested whether gelsolin-bound ADPr-trimer (GS:ADPr-
179 trimer) would bind myosin S1 under rigor conditions.
180 Our results showed no significant interactions between GS:ADPr-trimer and myosin.
181 Native PAGE (Fig 5) and analytical gel filtration chromatography (Fig 6) analyses of mixtures
182 of purified GS:ADPr-trimer and myosin S1 showed no significant shifts in either band mobility
183 or peak elution volumes. The minimal myosin ATPase activity measured in the presence of
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184 GS:ADPr-trimer was not significantly different than the activity seen with ADPr-monomer as
185 substrate (p = 0.18) (Fig 5C). Therefore, GS remodeling does not restore myosin binding to the
186 ADPr-trimer.
187
188 DNase-I capped pPBM-trimers do not bind myosin
189 While myosin has been shown to bind free ADPr-monomer (Ballweber et al. 2001), the
190 inhibition of S1 binding may be due to the presence of the ADPr moiety on each actin subunit,
191 inhibiting native intersubunit interactions needed for productive myosin binding. Since earlier
192 work reported that DNase-I does not block myosin binding to actin (Blanchoin et al. 1995), we
193 produced pPBM-crosslinked actin trimers capped at the pointed end by DNase-I (D:trimer) using
194 gel filtration chromatography (Fig 7) to test the possibility that ADP-ribosylation inhibits
195 actomyosin interactions. We found no evidence of interactions between D:trimer and myosin in
196 native gel electropherograms or myosin ATPase assays (Fig 8).
197
198 Discussion and conclusions
199 The ADPr-trimer interacts with barbed-end and pointed-end F-ABPs. Perhaps overlooked
200 as an F-ABP, actin monomers bind ADPr-trimer at its pointed end when gelsolin is present on
201 the trimer (Perieteanu et al. 2010). In this paper, we asked whether the ADPr-trimer provides a
202 binding surface for myosin as an example of a side-binding F-ABP. Our work shows that myosin
203 does not bind the ADPr-trimer, nor does the ADPr-trimer activate myosin actin-activated
204 ATPase. We also found that neither gelsolin-bound ADPr-trimer nor crosslinked actin trimer
205 capped with DNase-I (D:trimer) interacts with myosin.
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206 Since previous work shows that pPBM crosslinking inhibits force generation and
207 movement but does not inhibit myosin binding to F-actin and ATPase activity (Knight and Offer
208 1980; Kim et al. 2002), we reasoned that the crosslinking of the ADPr-trimer would permit
209 myosin binding. Similarly, crosslinking F-actin with glutaraldehyde does not inhibit myosin
210 binding (Prochniewicz and Yanagida 1990), but results in a conformational state of actin that
211 does not generate movement (Kozuka et al. 2007).
212 Past research shows that myosin binds to ADPr-monomers (Ballweber et al. 2001);
213 however, the presence of an ADP-ribose on each subunit of the F-actin-derived trimer might
214 disrupt intersubunit contacts necessary for myosin binding. To test this model, we blocked the
215 polymerization of F-actin-derived trimer by binding DNase-I to the pointed end rather than by
216 ADP-ribosylation. We found that myosin also did not bind DNase-I bound crosslinked actin
217 trimer, suggesting that either ADP-ribosylation on each subunit does not inhibit myosin binding,
218 or that DNase-I binding to the pointed end remodels the trimer into a state that blocks myosin.
219 A body of work shows that flexibility and conformational changes in F-actin are essential
220 for function (reviewed in (Blanchoin et al. 2014)). At the single filament level, different
221 conformations within actin subunits depend on the bound nucleotide (Murakami et al. 2010) and
222 blocks of different subunit states can exist along a filament (Galkin et al. 2010). Different
223 subunit states might be the result of, or be bound by, F-ABPs that lead to changes in the twist of
224 F-actin (Mochida et al. 2002; Kozuka et al. 2007; Gurel et al. 2017). Despite the requirement for
225 conformational change in F-actin for function, the literature is clear that pPBM crosslinking
226 permits the binding of myosin to F-actin (Knight and Offer 1980; Kim et al. 2002) and, by
227 extension, the necessary conformational changes in F-actin for binding.
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228 During final preparation of our manuscript, Qu et al reported the isolation of a
229 crosslinked and ADP-ribosylated actin trimer similar to ours that activates myosin ATPase (Qu
230 et al. 2018). To produce their actin trimer, Qu et al first purified PBM-crosslinked actin trimer
231 and then Iota toxin to ADP-ribosylate actin on Arg-177. The ADP-ribosylation was not
232 complete, however, since the resulting trimers were partially polymerization competent. To
233 completely inhibit polymerization, gelsolin segment-1 (GS1) was incubated at a 1:1 molecular
234 ratio to the trimer.
235 The primary difference between the ADPr-trimer we report on here and that described by
236 Qu et al is the stringency of the modifications employed to inhibit polymerization. Our work
237 demonstrates that complete ribosylation of the trimer inhibits myosin binding, while our earlier
238 work shows that binding of all subunits in the trimer with GS1 disrupts F-actin contacts (Dawson
239 et al. 2003). Therefore, our requirement for a homogeneous non-polymerzing actin trimer
240 resulted in treatments that inhibit some F-actin activities, whereas the combination of two
241 modifications at sub-stoichiometric levels likely results in a heterogeneous actin trimer
242 preparation that activates myosin activity.
243 Comparison of our work and that of Qu et al suggests that ribosylation of all subunits in
244 the PBM-trimer remodels the myosin binding site on actin. Moreover, these finding suggests
245 that capping of trimer with DNase-I must also remodel the trimer such that myosin cannot bind,
246 despite the finding that DNase-I can form a ternary complex with actin and myosin. One
247 speculation is that the binding of DNase-I to both pointed end subunits repositions the two
248 molecules and disrupts the myosin binding site.
249 Some of the interactions between actin and myosin are being revealed through advances
250 in high-resolution cryo-EM (Behrmann et al. 2012; Ropars et al. 2016; Wulf et al. 2016;
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251 Banerjee et al. 2017; Fujii and Namba 2017b; Gurel et al. 2017); however, not all of the
252 actomyosin states critical for lever arm movement have been captured (Houdusse and Sweeney
253 2016), and surface loops on different forms of myosin interact with different groups on actin.
254 Moreover, myosin binding changes the flexibility, twist and structural states of actin filaments
255 (discussed in (Mochida et al. 2002; Kozuka et al. 2007; Gurel et al. 2017)), suggesting that
256 myosin binds to F-actin in a cooperative manner through structural changes communicated
257 through the actin subunits in the filament. Indeed, the binding of myosin to F-actin displays
258 cooperative behaviour under specific conditions (Orlova and Egelman 1997; Tokuraku et al.
259 2009; Ngo et al. 2016). However, the work of Qu et al suggests that an actin trimer, with only
260 one myosin binding site located between two along-strand actin subunits, is the minimal
261 structure required for myosin ATPase activity.
262 Requiring actin to function as a polymer with only a few subunits is a challenging design
263 goal. The body of work using chemical crosslinking and different means of controlling actin
264 polymerization illustrates the need for careful balancing between the needs of the design strategy
265 and the activity of actin. In this work, the complete ADP-ribosylation of crosslinked actin trimer
266 and capping of the trimer on the pointed end with DNase-I both result in no activation of myosin
267 ATPase activity. At the same time, other F-ABPs bind to the ADPr-trimer, highlighting the
268 variety of actin binding modes among the F-ABPs. Future work can study the binding of other
269 F-ABPs to the ADPr-trimer to test different modes of actin binding and examine the
270 contributions of conformational flexibility and longer-range contacts along the actin filament for
271 side-binding F-ABPs. Moreover, it will be important to confirm the activity of
272 substoichiometrically-modified crosslinked actin trimers and develop further tests to determine
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273 whether an actin trimer can support lever arm movement and force generation coupled with
274 myosin ATPase activity.
275
276
277 Acknowledgements
278 This work was funded by an NSERC Discovery Grant to J.F.D.
279
280 Author Contributions
281 J.F.D. supervised the research, obtained the funding, and wrote and revised the
282 manuscript. N.S. performed all the experiments for the research and revised the manuscript.
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283 References
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398 Winder, S.J., and Ayscough, K.R. 2005. Actin-binding proteins. J. Cell Sci. 118(4): 651–654.
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401 Sweeney, H.L., Houdusse, A.M., and Schröder, R.R. 2016. Force-producing ADP state of
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403 doi:10.1073/pnas.1516598113.
404
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405 Figure captions
406 Figure 1. An Actin Trimer. (A) An actin trimer consisting of two subunits from one strand of
407 the two-start long pitch F-actin helix (green and pink subunits labelled A3 and A1) and one
408 subunit of the counterpart strand (light blue, A2) with the A3 subunit at the pointed end. Circled
409 in red are the rigour-state myosin interaction sites on the surface of the actin trimer, adapted from
410 (Fujii and Namba 2017a) and the outline of myosin (dotted violet line) in its bound position from
411 the actomyosin structure of Mentes et al (Mentes et al. 2018). (B) Butterfly representation of the
412 trimer with subunit A2 in the same position as in panel A and subunits A1 and A3 rotated 180˚ to
413 show the internal ADP-ribosylated Arg-177 residues (in blue) and the location of the residues
414 participating in the interstrand pPBM crosslinks indicated by a dotted black line: Lys-191 (in
415 red) and Cys-374 (in orange). Note that Cys-374 of subunits A3 and A2 are located behind the
416 surface representation shown. These structures were generated with chains B – D and P from
417 PDB 6C1D (Mentes et al. 2018) and visualized using PyMol.
418
419 Figure 2: Dynamic Light Scattering measurement of ADPr-trimer and myosin S1. The
420 hydrodynamic radii of different samples of ADPr-trimer (4 uM trimer = side binding sites) and
421 myosin S1 (1.5 uM) were measured using a Nano S Zetasizer. The derived radii of the different
422 samples were: (A) ADPr-trimer alone, 4.33 nm; (B) S1 alone, 4.34 nm; and (c) ADPr-trimer and
423 S1, 4.62 nm. Error bars represent the standard deviation for three readings per sample. No
424 significant increase in the radii seen in the reaction mix with ADPr-trimer and S1.
425
426 Figure 3. Subfragment-1 binding and myosin ATPase assays in the presence of purified
427 ADPr-actin oligomers. (A) Native gel of reactions containing 2 uM of ADPr-monomer, ADPr-
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428 dimer, or ADPr-trimer incubated for 20 min on ice in the presence of 0.87 uM myosin S1 in G-
429 10 buffer containing 50 mM NaCl with 60 ug/mL each of antipain, leupeptin, TLCK, and TPCK
430 protease inhibitors. Note that 6.1 ug of S1 was run in lane as a standard, while only 0.95 ug of
431 S1 was run in the reaction mixtures. Since S1 tends to run as a smear on native gels, the
432 resulting diffuse signal at lower S1 concentrations is difficult to visualize with Coomassie
433 staining. (B) SDS-PAGE of the samples in A above, showing the presence of S1 in all reactions.
434 (C) The ATPase hydrolase activity of 0.25 mg/ml myosin was measured in the presence of 5 uM
435 F-actin or purified ADPr-actin oligomers. The average activity for triplicate measurements is
436 plotted ± SEM. The ADPr-actin-containing complex activities were significantly different than
437 that in the presence of F-actin (p-values; ADPr-monomer: 0.003, ADPr-dimer: 0.005, ADPr-
438 trimer: 0.001) ADPr-trimer did not activate myosin ATPase activity beyond that seen with
439 ADPr-monomer (p = 0.679).
440
441 Figure 4. Analytical size exclusion chromatography of ADPr-trimer and myosin S1
442 reactions. (A) A mixture of 2 uM ADPr-trimer and 0.87 uM myosin S1 in G-10 buffer
443 containing 50 mM NaCl or 2 uM ADPr-trimer alone was incubated for 1 hour at room
444 temperature and then developed on a Superdex-200 (10/300) analytical gel filtration column
445 developed in G-10 buffer containing 50 mM NaCl. The bar above the absorbance profile
446 indicates the fractions analyzed in panel (B). (B) Silver-stained SDS-PAGE of 10 uL sample of
447 fractions the chromatographic analysis of the mixture of ADPr-trimer and S1 in (A). The peaks
448 of S1 and ADPr-trimer do not appear to in the same fractions, suggesting no interaction.
449
450 Figure 5. Subfragment-1 binding and myosin ATPase assays in the presence of purified
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451 GS:ADPr-trimer. (A) Native gel of reactions containing 0.23 uM of ADPr-monomer, or
452 Gelsolin:ADPr-trimer complex (GS:ADPr-trimer) incubated for 15 min on ice in the presence of
453 0.20 uM myosin S1 in G-10 buffer containing 50 mM NaCl with 24ug/mL each of antipain,
454 leupeptin, TLCK, and TPCK protease inhibitors. (B) SDS-PAGE of the samples in A, showing
455 the presence of all components in the reactions. Some contaminating full-length myosin is
456 present in the S1 preparation. (C) The ATPase hydrolysis activity of 0.25 mg/ml myosin was
457 measured in the presence of 1.5 uM F-actin, purified ADPr-monomer, GS:ADPr-trimer. The
458 average activity for triplicate measurements is plotted ± SEM. GS:ADPr-trimer did not activate
459 myosin ATPase activity beyond that seen with ADPr-monomer (p = 0.18).
460
461 Figure 6. Analytical size exclusion chromatography analysis of GS:ADPr-trimer-S1
462 binding reactions. (A) Chromatograms of a GS:ADPr-trimer-S1 binding reaction containing 0.9
463 umol of GS:ADPr-trimer and 0.8 umol S1 (dotted line) and 0.9 umol GS:ADPr-trimer alone
464 (solid line) run on a Superdex-200 (10/300) column. Both samples also contained 150 ug/mL
465 antipain, 150 ug/mL leupeptin, 60 ug/mL TLCK, and 60 ug/mL TPCK. A major peak at an
466 elution volume of about 11.8 mL is observed, along with a minor peak eluting at just after 15
467 mL. (B) SDS-PAGE of fractions from the GS:ADPr-trimer-S1 reaction in (A). Fractions B4 to
468 C2 are across the major peak indicated by the top bar in (A) while fraction D9 is from the minor
469 peak. While S1 is present in the loaded sample (Load), S1 is not present as a major band in the
470 peak fractions of the elution profile. (C) Silver stained native gel of major peak fractions
471 indicated by the bottom bar in (A). Note that S1 is not a major band in the peak fractions.
472
473 Figure 7. Purification of DNase I-bound PBM-trimer (D:trimer). (A) Chromatogram of a
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24
474 sample of crosslinked F-actin incubated with DNase-I in a 1:1 molecular ratio developed on a
475 Superdex S-200 (10/300) column as described in Methods and Materials. The identity of DNase-
476 I bound actin oligomers is indicated on the chromatogram. The range of elution fractions
477 analyzed by SDS-PAGE (B) or Native PAGE (C) is indicated by the box above the
478 chromatogram.
479
480 Figure 8: DNase-I bound crosslinked actin trimer (D:trimer) interactions with Myosin S1.
481 (A) Silver stained Native PAGE of binding reactions including 0.42 uM DNase-I capped actin
482 oligomers in the presence or absence of 0.37 uM S1 and 33 ug/mL anitpain, leupeptin, TLCK
483 and TPCK protease inhibitors. DNase-I was also run alone to identify that band. No new bands
484 were seen suggesting S1 bound to the DNase-I bound actin oligomers. (B) Myosin ATPase
485 activity assays including 0.25 mg/mL myosin in the presence of 1.5 uM F-actin or DNase-I
486 bound crosslinked actin oligomers. The average activity for triplicate measurements is plotted ±
487 SEM. No significant activity was observed for DNase-I bound actin trimers.
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Figure 1: An Actin Trimer. (A) An actin trimer consisting of two subunits from one strand of the two-start long pitch F-actin helix (green and pink subunits labelled A3 and A1) and one subunit of the counterpart strand (light blue, A2) with the A3 subunit at the pointed end. Circled in red are the rigour-state myosin
interaction sites on the surface of the actin trimer, adapted from (Fujii and Namba 2017) and the outline of myosin (dotted violet line) in its bound position from the actomyosin structure of Mentes et al (Mentes et al.
2018). (B) Butterfly representation of the trimer with subunit A2 in the same position as in panel A and subunits A1 and A3 rotated 180˚ to show the internal ADP-ribosylated Arg-177 residues (in blue) and the
location of the residues participating in the interstrand pPBM crosslinks indicated by a dotted black line: Lys-191 (in red) and Cys-374 (in orange). Note that Cys-374 of subunits A3 and A2 are located behind the
surface representation shown. These structures were generated with chains B – D and P from PDB 6C1D (Mentes et al. 2018) and visualized using PyMol.
173x158mm (300 x 300 DPI)
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Figure 2: Dynamic Light Scattering measurement of ADPr-trimer and myosin S1. The hydrodynamic radii of different samples of ADPr-trimer (4 uM trimer = side binding sites) and myosin S1 (1.5 uM) were measured using a Nano S Zetasizer. The derived radii of the different samples were: (A) ADPr-trimer alone, 4.33 nm; (B) S1 alone, 4.34 nm; and (c) ADPr-trimer and S1, 4.62 nm. Error bars represent the standard deviation for three readings per sample. No significant increase in the radii seen in the reaction mix with ADPr-trimer
and S1.
99x176mm (300 x 300 DPI)
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Figure 3. Subfragment-1 binding and myosin ATPase assays in the presence of purified ADPr-actin oligomers. (A) Native gel of reactions containing 2 uM of ADPr-monomer, ADPr-dimer, or ADPr-trimer
incubated for 20 min on ice in the presence of 0.87 uM myosin S1 in G-10 buffer containing 50 mM NaCl with 60 ug/mL each of antipain, leupeptin, TLCK, and TPCK protease inhibitors. Note that 6.1 ug of S1 was run in lane as a standard, while only 0.95 ug of S1 was run in the reaction mixtures. Since S1 tends to run as a smear on native gels, the resulting diffuse signal at lower S1 concentrations is difficult to visualize with Coomassie staining. (B) SDS-PAGE of the samples in A above, showing the presence of S1 in all reactions. (C) The ATPase hydrolase activity of 0.25 mg/ml myosin was measured in the presence of 5 uM F-actin or
purified ADPr-actin oligomers. The average activity for triplicate measurements is plotted ± SEM. The ADPr-actin-containing complex activities were significantly different than that in the presence of F-actin (p-values;
ADPr-monomer: 0.003, ADPr-dimer: 0.005, ADPr-trimer: 0.001) ADPr-trimer did not activate myosin ATPase activity beyond that seen with ADPr-monomer (p = 0.679).
113x205mm (300 x 300 DPI)
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Figure 4. Analytical size exclusion chromatography of ADPr-trimer and myosin S1 reactions. (A) A mixture of 2 uM ADPr-trimer and 0.87 uM myosin S1 in G-10 buffer containing 50 mM NaCl or 2 uM ADPr-trimer alone was incubated for 1 hour at room temperature and then developed on a Superdex-200 (10/300) analytical gel filtration column developed in G-10 buffer containing 50 mM NaCl. The bar above the absorbance profile indicates the fractions analyzed in panel (B). (B) Silver-stained SDS-PAGE of 10 uL sample of fractions the chromatographic analysis of the mixture of ADPr-trimer and S1 in (A). The peaks of S1 and ADPr-trimer do
not appear to in the same fractions, suggesting no interaction.
107x102mm (300 x 300 DPI)
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Figure 5. Subfragment-1 binding and myosin ATPase assays in the presence of purified GS:ADPr-trimer. (A) Native gel of reactions containing 0.23 uM of ADPr-monomer, or Gelsolin:ADPr-trimer complex (GS:ADPr-trimer) incubated for 15 min on ice in the presence of 0.20 uM myosin S1 in G-10 buffer containing 50 mM NaCl with 24ug/mL each of antipain, leupeptin, TLCK, and TPCK protease inhibitors. (B) SDS-PAGE of the
samples in A, showing the presence of all components in the reactions. Some contaminating full-length myosin is present in the S1 preparation. (C) The ATPase hydrolysis activity of 0.25 mg/ml myosin was
measured in the presence of 1.5 uM F-actin, purified ADPr-monomer, GS:ADPr-trimer. The average activity for triplicate measurements is plotted ± SEM. GS:ADPr-trimer did not activate myosin ATPase activity
beyond that seen with ADPr-monomer.
168x133mm (300 x 300 DPI)
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Figure 6. Analytical size exclusion chromatography analysis of GS:ADPr-trimer-S1 binding reactions. (A) Chromatograms of a GS:ADPr-trimer-S1 binding reaction containing 0.9 umol of GS:ADPr-trimer and 0.8 umol S1 (dotted line) and 0.9 umol GS:ADPr-trimer alone (solid line) run on a Superdex-200 (10/300)
column. Both samples also contained 150 ug/mL antipain, 150 ug/mL leupeptin, 60 ug/mL TLCK, and 60 ug/mL TPCK. A major peak at an elution volume of about 11.8 mL is observed, along with a minor peak
eluting at just after 15 mL. (B) SDS-PAGE of fractions from the GS:ADPr-trimer-S1 reaction in (A). Fractions B4 to C2 are across the major peak indicated by the top bar in (A) while fraction D9 is from the minor peak. While S1 is present in the loaded sample (Load), S1 is not present as a major band in the peak fractions of the elution profile. (C) Silver stained native gel of major peak fractions indicated by the bottom bar in (A).
Note that S1 is not a major band in the peak fractions.
136x113mm (300 x 300 DPI)
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Figure 7. Purification of DNase I-bound PBM-trimer (D:trimer). (A) Chromatogram of a sample of crosslinked F-actin incubated with DNase-I in a 1:1 molecular ratio developed on a Superdex S-200
(10/300) column as described in Methods and Materials. The identity of DNase-I bound actin oligomers is indicated on the chromatogram. The range of elution fractions analyzed by SDS-PAGE (B) or Native PAGE
(C) is indicated by the box above the chromatogram.
127x196mm (300 x 300 DPI)
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Figure 8: DNase-I bound crosslinked actin trimer (D:trimer) interactions with Myosin S1. (A) Silver stained native PAGE of binding reactions including 0.42 uM DNase-I capped actin oligomers in the presence or
absence of 0.37 uM S1 and 33 ug/mL anitpain, leupeptin, TLCK and TPCK protease inhibitors. DNase-I was also run alone to identify that band. No new bands were seen suggesting S1 bound to the DNase-I bound
actin oligomers. (B) Myosin ATPase activity assays including 0.25 mg/mL myosin in the presence of 1.5 uM F-actin or DNase-I bound crosslinked actin oligomers. The average activity for triplicate measurements is
plotted ± SEM. No significant activity was observed for DNase-I bound actin trimers.
110x115mm (300 x 300 DPI)
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