MCB Accepted Manuscript Posted Online 25 July 2016 Mol. Cell. Biol. doi:10.1128/MCB.00142-16 Copyright © 2016 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
1 Arginine finger regulates sequential action of asymmetrical
2 hexameric ATPase in dsDNA translocation motor
3 Downloaded from
4 Zhengyi Zhao, 1,2 Gian Marco De-Donatis, 2 Chad Schwartz, 2 Huaming Fang, 2 Jingyuan Li,3 and
5 Peixuan Guo1,2 *
6
1 7 College of Pharmacy, Department of Physiology & Cell Biology/College of Medicine, and http://mcb.asm.org/
8 Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH,
9 USA
10 2Nanobiotechnology Center, Department of Pharmaceutical Sciences, College of Pharmacy,
11 Markey Cancer Center, University of Kentucky, Lexington, KY, USA on August 9, 2016 by Ohio State University 12 3CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center
13 for Nanoscience and Technology of China and Institute of High Energy Physics, Beijing, China
14
15 Running Title: Arginine finger primes sequential action of ATPase
16
17 *Address correspondence to:
18 Peixuan Guo, Ph.D.
19 Sylvan G. Frank Endowed Chair in Pharmaceutics and Drug Delivery System, College of
20 Pharmacy, and Department of Physiology and Cell Biology/College of Medicine,
21 Ohio State University, Hamilton Hall, 1645 Neil Ave, Columbus, Ohio, 43210, USA
22 Email: [email protected]
23 Phone: (614)-293-2114 (office)
1 24 ABSTRACT
25 Biological motors are ubiquitous in living systems. Currently, how the motor component
26 coordinates the unidirectional motion is elusive in most cases. Here we report that the Downloaded from 27 sequential action of the ATPase ring in our system is regulated by an arginine finger that
28 extends from one ATPase subunit to the adjacent unit to promote noncovalent dimer
29 formation. Mutation of the arginine finger resulted in the interruption of ATPase
30 oligomerization, ATP binding/hydrolysis, and DNA translocation. Dimer formation was http://mcb.asm.org/ 31 observed when arginine mutants were mixed with others that can offer the arginine to
32 promote their interaction. Ultracentrifugation and virion assembly assays indicated that
33 the ATPase was presenting as monomers and dimer mixtures. The isolated dimers alone
34 were inactive in DNA translocation, but addition of monomer could resume the activity, on August 9, 2016 by Ohio State University 35 suggesting that the hexameric ATPase ring contained both dimer and monomers.
36 Moreover, ATP binding or hydrolysis resulted in conformation and entropy changes of
37 the ATPase with high or low DNA affinity. Taken together, it is concluded that the
38 arginine finger regulates sequential action of the motor ATPase subunit by promoting the
39 formation of the dimer inside the hexamer. The finding of asymmetrical hexameric
40 organization is supported by structural evidences of many other ATPase systems.
41
42
43
44
45
46
2 47 INTRODUCTION
48 The ASCE (Additional Strand Catalytic E) superfamily is a broad class of proteins
49 among which several nano-biological molecular motors, or nanomotors, as listed. Downloaded from 50 Nanomotors facilitate a wide range of functions 1,5,58,59,77; many of which are involved in
51 DNA replication, repair, recombination, chromosome segregation, protein degradation,
52 membrane fusion, microtubule severing, peroxisome biogenesis, gene regulation,
53 DNA/RNA transportation, bacterial division, and many other processes 1,7,16,28,45,60. http://mcb.asm.org/ 54 Despite their functional diversity, ring-shaped P-loop NTPases share a common
55 conserved module of approximately 230 amino acid residues with a Walker A and a
56 Walker B motif 18 to exert their activity through the energy-dependent remodeling for
57 translocation of macromolecules. The Walker A motif is responsible for ATP binding, on August 9, 2016 by Ohio State University 58 while the Walker B is in ATP hydrolysis 67,68. This energy transition process leads to
59 either a gain or loss of affinity for its substrate, thus producing a mechanical force exerted
60 on a macromolecular substrate to create mechanical movement. This motion is used to
61 either make or break the contact with the macromolecules, resulting in local or global
62 protein unfolding; complex assembly or disassembly; translocation of proteins, DNA,
63 RNA or other macromolecules 37.
64 Both the revolution mechanism and the sequential reaction mechanism adapted by
65 biological systems through evolution are efficient methods of translocation of lengthy
66 dsDNA genome unidirectionally, with minimum consumption of energy and without
67 tangling or coiling 8,17,21,51,52,78. However, both the revolution mechanism and/or the
68 sequential reaction mechanism for DNA translocation requires a complex system of
69 communication from one component to another one of the packaging machine.
3 70 It has been reported that ASCE ATPases contain one arginine finger motif along
71 with the Walker A Walker B motif 11,27,34,38. In the active ATPase ring, the arginine
72 residue is located in proximity to the γ-phosphate of the bound ATP in the adjacent
73 ATPase subunit 27,33,36,71. Arginine finger has been confirmed to associate with the Downloaded from
74 formation of the ATP binding pocket 10,33,34,74,75. To understand how the motor
75 component coordinates its motion, necessary for unidirectional DNA translocation
76 activity and sequential action of the ATPase ring, we analyzed the role of Arginine finger http://mcb.asm.org/ 77 motif in the ATPase core of the dsDNA translocation motor. It was found that this motif
78 control the formation of coordinating dimers inside the hexamer of the motor ATPase.
79 The dimer however is not static but varies with time, following a sequential manner, and
80 this sequential reaction mechanism is regulated by the arginine finger.
81 on August 9, 2016 by Ohio State University
82 RESULTS
83 Hypothesis of motor motion mechanism
84 Most biological motor ATPases assemble into hexameric rings with a motion process
85 stimulated by ATP 18. For phi29 dsDNA translocation motor, our hypothesis is that: (1)
86 An arginine finger is present in phi29 motor ATPase gp16. (2) The arginine finger
87 outstretches to the upstream adjacent ATPase subunit to serve as a bridge for the
88 formation of a dimeric sub-complex, and regulates the sequential action of the subunits in
89 the hexameric ATPase ring. (3) Both ATPase dimer and monomers are present in
90 hexameric ring. (4) ATP binding results in the reshaping of the conformation and the
91 change of the entropic landscape of gp16. (5) Due to the DNA dependent ATPase
92 activity 18, binding of DNA to ATP/gp16 complex resulted in ATP hydrolysis, leading to
4 93 a second conformational and further entropy change of the ATPase to a low DNA-
94 affinity configuration that allows the release of dsDNA for its concomitant transfer to the
95 adjacent subunit.
96 The model speculates that the ATPase undergoes a series of conformational Downloaded from
97 changes during DNA binding and ATP hydrolysis that are organized in a sequential
98 manner, and that this sequential mechanism is coordinated by the arginine finger (Fig. 1),
99 with supporting data below. http://mcb.asm.org/ 100
101 Identification of arginine finger motifs in phi29 gp16 ATPase
102 Gp16 shares the common ATP binding domain typical of all ASCE, including AAA+
103 proteins 22,58. This domain contains very well conserved motifs responsible for ATP
104 binding and ATP hydrolysis 67, which have been previously identified as Walker A 18 and on August 9, 2016 by Ohio State University
105 Walker B motifs 51, respectively. However, the detailed information about its arginine
106 finger motif remained elusive. Sequence alignment was subsequently performed with
107 similar ASCE proteins to identify this motif (Fig. 2A). From the alignment, we identified
108 the position of the arginine fingers (residue 146), localized after beta-4 as seen in other
109 ATPases, which correlates well with the known structural information and consensus
110 sequences for this motif found in other proteins 14,33,46,66,69 (Fig. 2A). Single mutant
111 R146A gp16 was produced and examined for its ATPase activity. As expected, the
112 arginine finger mutant was severely impaired in the activity for ATP hydrolysis (Fig. 2B)
113 and also in DNA binding in the presence of γ-s-ATP compared with the wild-type (Fig.
114 2C), possibly due to the impaired affinity for γ-s-ATP similar to the Walker A mutant 51.
115 On the contrary, the Walker B mutants retained their binding affinity for DNA in the
5 116 presence of γ-s-ATP and were also sufficient in binding DNA in the presence of ATP,
117 although they could not hydrolyze ATP 51,52.
118
119 The arginine finger outstretches to the upstream adjacent ATPase subunit to serve Downloaded from
120 as a bridge for the formation of a dimeric sub-complex, and regulates the sequential
121 action of the subunits in the hexameric ATPase ring
122 Arginine finger has been reported to have various functions including the major role in http://mcb.asm.org/ 123 subunit communications by swinging upon ATP hydrolysis to trigger the conformational
124 changes of the subunits of the ATPase 4,12,44,49,57,58,63. The formation of dimeric
125 complexes of gp16 in the absence of ATP were demonstrated by different approaches:
126 glycerol gradient ultracentrifugation (Fig. 3), electrophoresis mobility shift assay
127 (EMSA) (Fig. 4A-C), size exclusion chromatography, and native gel electrophoresis 51. on August 9, 2016 by Ohio State University
128 These assays were based on the previous finding that fusion of the GFP protein to the N
129 terminal of the gp16 did not interfere with activity of the ATPase gp16 in DNA
130 packaging 29,30,41. Mutation of the Arginine finger abolished the dimer formation within
131 the ATPase (Fig. 3). Although the Arginine mutants alone could not form dimers,
132 interactions were observed when the arginine mutants were mixed with either the wild
133 type or the other mutants that contained an intact arginine finger, which can provide
134 arginine for dimer formation (Fig. 3). The disruptive effect of the Arginine finger
135 mutation on the assembly ability was also reflected in the protein activity, since we
136 observed that one single inactive subunit of an arginine finger mutant was able to
137 inactivate the whole ATPase ring in an assembly inhibition assay (Fig. 4D-E); supporting
138 the idea that in the ATPase ring, one adjacent wild type ATPase provided an arginine
6 139 finger to interact with the arginine mutant, and the lack of one arginine in the entire ring
140 completely abolished the activity of the whole ring.
141 To get a better understanding of the structural role of the Arginine finger, we
142 modeled a gp16 hexameric ring using I-TASSER and Phyre2 softwares. The gp16 Downloaded from
143 sequence aligned well with the crystal structure of the hexameric FtsK DNA translocase
144 of E. coli (Fig. 5). Using this model we observed that the position of the Arginine finger
145 of one subunit of gp16 outstretches to the active site of a neighboring subunit. The http://mcb.asm.org/ 146 predicted structure showed that the Arginine finger was part of the ATP binding pocket
147 (Fig. 5). The structural model provides an explanation for the observed cooperativity
148 behavior in the hexameric ring of gp16. Not surprisingly given the importance in the
149 formation of the active site, mutations in Arginine fingers greatly impaired the ability of
150 gp16 to bind to ATP, hydrolyze ATP (Fig. 2B), bind to DNA (Fig. 2C), and consequently on August 9, 2016 by Ohio State University
151 package the DNA (Fig. 4E).
152
153 Both dimer and monomers forms were present in gp16 hexamer
154 As demonstrated in the above sections, Arginine finger serves as a bridge between two
155 independent subunits, thus forming dimers. In wild type g16 we observed that both dimer
156 and monomer forms were present as revealed by glycerol gradient centrifugation
157 experiments. The molecular weight relative to such fractions were confirmed by protein
158 markers calibrating through the same assay (BSA (66 kDa) localized around fraction 23,
159 Alcohol dehydrogenase (140 kDa) around fraction 18, and beta-Amylase (200 kDa)
160 around fraction 15). We thus proceeded to test the packaging activity of the different
161 fractions of gp16 ATPase recovered from the gradient. Interestingly we observed that
7 162 DNA packaging activity was retained with the fractions containing monomers, while
163 fractions containing the only dimer displayed no DNA packaging activity (Fig. 3C).
164 These results are also supported by the finding that the addition of fresh gp16 monomer
165 to the gp16 dimers and ATP are required for re-initiating the DNA packaging Downloaded from
166 intermediates into infection virus 56.
167
168 ATP binding resulted in the change of conformation and e entropic landscape of http://mcb.asm.org/ 169 gp16
170 ASCE proteins undergo a cycle of conformational changes during ATP binding and
171 hydrolysis with basically 2 major states: high or low affinity for the DNA substrate. In
172 recent publications 51-53,76 we proposed a similar model for gp16, in which binding to
173 ATP exerted an effect on the conformational state of the protein that predispose the on August 9, 2016 by Ohio State University
174 binding to DNA (high affinity). Conversely ADP would promote another conformational
175 state, in which DNA binding is not favorable. This notion together with the observation
176 that arginine fingers has a role in regulating both conformational state of gp16 and its
177 interaction with adjacent subunit prompted us to question whether the effect of ATP
178 binding on gp16 was able to modify not only the conformation of the DNA binding
179 portion of the protein, but also the structural characteristics of gp16 altogether. We thus
180 tested if ATP binding was able to alter the shape of gp16 by partial proteolysis treatment
181 and tryptophan intrinsic fluorescence assay (Fig. 6A-B). Interestingly both assays
182 indicated a conformational change in gp16-ATP complex. Moreover, as visible from the
183 partial proteolysis test, the protection from proteolysis is indicative of a larger population
184 of gp16 with constrained conformation before ATP binding.
8 185 An electrophoretic mobility shift assay was also employed to study the interaction
186 between ATPase and dsDNA in the presence of γ-S-ATP, a non-hydrolysable ATP
187 analog. Stronger binding of gp16 to dsDNA was observed when gp16 was incubated with
188 γ-S-ATP (Fig. 6C), suggesting that the gp16/dsDNA complex is stabilized through Downloaded from
189 addition of the non-hydrolysable ATP substrate.
190
191 Hydrolysis of ATP transformed the ATPase into a second conformation with low http://mcb.asm.org/ 192 affinity for dsDNA, thus pushed the dsDNA toward an adjacent ATPase subunit
193 Consequent to the first structural change we also observed that the binding of ATP/gp16
194 complex to the DNA resulted in ATP hydrolysis and also the passage to a second
195 conformational change with a low DNA-affinity configuration 18,32,42. Such state resulted
196 in the release of dsDNA for its concomitant transfer to the adjacent subunit. The on August 9, 2016 by Ohio State University
197 conclusion was also supported by the finding that addition of normal ATP promoted the
198 release of the dsDNA from the gp16-γ-s-ATP-dsDNA complex (Fig. 6D).
199
200 DISCUSSION
201 The viral genomic DNA packaging reaction of phi29 is a complex phenomenon that
202 involves the work of a multi-component packaging system composed of a 12 subunit
203 connector protein, a hexameric RNA molecule called pRNA 15,20, and finally an ASCE
204 ATPase gp16. Great interest has arisen about this packaging system for its intriguing
205 mechanism of action and for its useful applications in nanotechnology 13,18,23-25,54,55,70.
206 Recent publications demonstrated that the pRNA works as a point of connection between
207 the ATPase and the connector, and that the hexameric ATPase 51,52 provides the pushing
9 208 force for the packaging of genomic DNA, acting in coordination with the connector
209 which acts as a valve 35,76.
210 Nanobiomotors have been previously classified into two main categories: linear
211 and rotational motors; which have been clearly documented using single molecule Downloaded from
212 imaging and X-ray crystallography 3,9,26,47,64,65. Recently, it has been discovered that
213 phi29 dsDNA packaging motor uses a revolution mechanism that does not require
214 rotation or coiling of the dsDNA 19,51,52,78. Finding of revolution mechanism establishes a http://mcb.asm.org/ 215 third class of biomotors. This finding resolves many puzzles and debates throughout the
216 history of painstaking studies on the motor 19,21.
217 The ATPase hexameric ring exerts a force, pushing the dsDNA in a sequential
218 manner to advance through the dodecamer channel which acts as a one-way valve 19,21,35.
219 A sequential reaction mechanism of DNA translocation for the motor ATPase has been on August 9, 2016 by Ohio State University
220 reported5,6. The interest in the sequential revolution mechanism relies in the fact that it
221 elegantly integrates all the known functional and structural information about the
222 packaging core (the ATPase, pRNA and connector). Moreover it offers solutions for
223 many questions that arise when investigating the DNA packaging phenomenon (i.e.
224 coordination between energy consumption and DNA packaging, ability to translocate a
225 long strain of dsDNA without twisting or swirling). However, in order to have a
226 sequential mechanism (which has been proposed for many proteins belonging to the
227 family of AAA+/ASCE) 10,39,72 several conditions need to be fulfilled. The most
228 important are: A) Only 1 or 2 subunits of the oligomer are able to bind the substrate with
229 the same affinity exhibited in the entire hexamer; B) Both the ATPase activity and
230 translocation activity need to demonstrate negative cooperativity when one subunit is
10 231 able to bind ATP, it is not able to hydrolyze the nucleotide (as in the case of the Walker B
232 mutation); C) Only the ATP bound state of the protein is the unique state that efficiently
233 binds to DNA.
234 We demonstrated that indeed this is the case for phi29 motor ATPase 52,53. One Downloaded from
235 important question then arises with the demonstration of the sequential mechanism is that
236 how the different subunit of the ATPase can sense the ATP binding/DNA binding state of
237 others? In the present work, we addressed this question by identifying the arginine finger http://mcb.asm.org/ 238 motifs of the ATPase gp16 by sequence alignment and proved that the arginine finger is
239 an essential motif that participates to the formation of the ATP binding pocket by
240 examining the behavior of gp16 mutants with the Arginine finger removed. The gp16
241 mutated in Arginine finger was unable to package DNA, to hydrolyze ATP, or to bind to
242 DNA. The profile of gp16 in ultracentrifugation indicated the presence of a mixture of on August 9, 2016 by Ohio State University
243 monomeric and dimeric form. Mutation of the Arginine finger eliminated the capacity of
244 gp16 to assemble into dimeric forms. Arginine finger motifs were thus shown to link two
245 subunits to each other since the arginine motif of one subunit participates to the formation
246 of ATP binding site of the next subunit. The importance of the dimer is moreover
247 evidenced as shown by DNA packaging assay, in which reconstituted hexamer of gp16
248 can efficiently pack DNA inside the procapsid only when ultracentrifuge fractions
249 containing both dimeric and monomeric gp16 are mixed together (data not shown) 56.
250 In the sequential action of gp16, we proposed that one subunit of the hexamer
251 binds to the DNA, subsequently hydrolyzing ATP to perform a translocation of a certain
252 number of base pairs of DNA 18,48. The DNA is then passed to the subsequent subunit,
253 and the process is repeated. It is intriguing to notice that the position and the function of
11 254 ATPase offers the possibility to carry the information of ATP/DNA binding from one
255 ATPase subunit to another, with a cooperative behavior of gp16 seen in the case of other
256 mutants (Walker B mutations) 52.
257 The sequential action mechanism of phi29 ATPase is essential for optimal Downloaded from
258 translocation efficiency. This mechanism integrates well with our overall model of the
259 revolution motor and the “push through one way valve” model 52,76. Without the
260 coordination during the energy production of gp16, the cycles of binding and release of http://mcb.asm.org/ 261 DNA would create futile cycles of ATP hydrolysis inhibiting the unidirectional
262 translocation process 52,53,78. Arginine fingers acts thus as integrator of information for the
263 entire process of DNA packaging. Years of evolution have created an efficient biomotor,
264 one that can be used in the future for applications in nanotechnology.
265 Furthermore, the conclusion of asymmetrical hexameric coordination was on August 9, 2016 by Ohio State University
266 supported by structural computation, X-ray diffraction and Cryo-EM imaging of other
267 hexameric ATPase systems (Fig. 7) 2,61,62,73. These results could provide some clues for
268 why the asymmetrical hexameric ATPase of gp16 of phi29 and gp17 of T4 was
269 previously interpreted as pentameric configuration by cryo-EM. Since the two adjacent
270 subunits of the ATPase could interact with each other and form a closer dimer
271 configuration, this dimer will appear as a monomeric subunit different from the others
272 and the hexameric ring is asymmetrical (Fig. 7).
273
274 MATERIALS AND METHODS
275 Cloning, mutagenesis and protein purification
12 276 The engineering of eGFP-gp16 and the purification of the gp16 fusion protein have been
277 reported previously 41. The eGFP-gp16 arginine finger mutant (R146A), eGFP-gp16
278 Walker A mutant (G27D), gp16 Walker B mutant (E119A), and mCherry-gp16 mutant
279 R146A were constructed by introducing mutations in the gp16 gene (Keyclone Downloaded from
280 Technologies).
281 Glycerol gradient ultracentrifugation
282 50 μl of eGFP-gp16 (500 μg/ml) were dropped on the top of 5 ml linear 15-35% glycerol http://mcb.asm.org/ 283 gradients in TMS buffer. After centrifuging at 35000 rpm in a SW55 rotor at 4 °C for 22
284 hr, the gradients were collected into 31 fractions from bottom to top and measured using
285 a plate reader under 488 excitation before being applied to in vitro assembly assay.
286 Electrophoretic Mobility Shift Assay (EMSA)
287 Fluorescently tagged protein that facilitates detection and purification was shown to on August 9, 2016 by Ohio State University
288 possess similar assembly and packaging activity as compared to wildtype 41,53. The
289 EMSA method has been described previously 51,52. The gp16 mutants or wild-type were
290 mixed with 33 bp Cy5-dsDNA in the presence or absence of ATP and γ-S-ATP. Samples
291 were incubated at ambient temperature for 20 min and then loaded onto a 1% agarose gel
292 (44.5 mM Tris, 44.5 mM boric acid) and electrophoresed at 4 °C for around 1 hr at 8
293 V/cm. The eGFP-gp16, mCherry-gp16, and Cy5-DNA samples were analyzed by a
294 fluorescent LightTools Whole Body Imager using 488 nm, 540 nm, or 635 nm excitation
295 wavelengths for GFP, mCherry, and Cy5, respectively.
296 Protein structure prediction and analysis:
297 I-TASSER 50 was used to predict the structure of subunit of gp16 through a threading
298 algorithm. The structure prediction processed without restraint allowing the server to
13 299 select template. The N-domain (1-180 aa) of the predicted structure adopts a RecA-like
300 fold, which is the conserved structure for many oligomeric ATPases, including T7 gp4
301 and FtsK. The RMSD between the predicted structure (N-domain of gp16) and FtsK
302 (beta-domain) after the structure alignment is around 3 Å. The predicted structure Downloaded from
303 (monomer) was then used to construct a hexameric structure of gp16 with P. aeruginosa
304 FtsK (pdb ID: 2IUU) as the template 46. VMD was used to render the image of structure
305 31. http://mcb.asm.org/ 306 Proteinase probing assay:
307 3 μl of his-gp16 (2 mg/ml) was mixed with trypsin (0.5 μg) and different amounts of ATP
308 (0 nmol, 16 nmol, 32 nmol, 64 nmol, 128 nmol, 256 nmol, 512 nmol, 1 μmol) in the
309 enzyme reaction buffer: 50 mM NaCl, 25 mM Tris pH8, 0.01% Tween 20, 0.1 mM on August 9, 2016 by Ohio State University 310 MgCl2, 2% glycerol, 1.5% PEG 8000, 0.5% Acetone, and 5 mM DTT. Fresh DTT is
311 added into the buffer right before the reaction. The final volume for this reaction system
312 was 30 μl, the samples were incubated in room temperature for 30 min and applied on 12%
313 SDS-PAGE.
314 Tryptophan intrinsic fluorescent assay:
315 8 μl SUMO-gp16 (1 μg/μl) was incubated with different amount of ATP in the reaction
316 buffer (0.005% Tween 20, 1.5% PEG 8000, 0.5% Acetone, and 2 mM Tris pH 8.0). The
317 fluorescent intensity of the samples were immediately measured through a spectra-
318 fluorimeter under wavelength excitation at 280 nm.
319 ATPase activity assay:
14 320 Enzymatic activity via fluorescent labeling was described previously 42. Briefly, a
321 phosphate binding protein conjugated to a fluorescent probe that senses the binding of
322 phosphate was used to assay ATP hydrolysis.
323 In vitro assembly inhibition assay: Downloaded from
324 Purified in vitro components were mixed and were subjected to the virion assembly assay
325 as previously described 40. Briefly, newly assembled infectious virions were inoculated to
326 Bacillus bacteria and plated. Activity was expressed as the number of plaques formed per http://mcb.asm.org/ 327 volume of sample (pfu/mL).
328 ACKNOWLEDGEMENTS
329 The work was supported by NIH grant R01-EB012135, R01-EB019036, and TR000875
330 to PG. Funding to P.G.'s Endowed Chair in Nanobiotechnology position is by the on August 9, 2016 by Ohio State University 331 William Fairish Endowment Fund. P.G. is a co-founder of Biomotor and RNA
332 Nanotechnology Development Corp. The content is solely the responsibility of the
333 authors and does not necessarily represent the official views of NIH.
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551 FIGURE LEGENDS:
552 Figure 1. The proposed mechanism of ATPase coordination with a series of
553 conformational changes during DNA binding and ATP hydrolysis that is regulated by the
554 arginine finger.
555 Figure 2. Identification and characterization of arginine finger in phi29 gp16
556 ATPase.
557 (A) Sequence alignment among gp16 ATPase with other ATPase in the same family,
558 indicating the location of the Walker A, Walker B, and arginine finger motifs of gp16
559 ATPase which are well aligned with previously established domains 14,33,46,66,69. (B and
560 C) ATP binding and hydrolysis activity assay of gp16 Arginine mutant. After the R146
25 561 residue is mutated, gp16 ATPase loses its ATP hydrolysis activity (B) and DNA binding
562 activity shown by EMSA (C), compared to that of wild-type ATPase.
563 Figure 3. Ultracentrifugation assay showing the presence of both dimers and
564 monomers in the gp16 ATPase rings. Downloaded from
565 One peak of eGFP-gp16 R146A (A) and two peaks of eGFP-gp16 wild-type (B) were
566 shown after parallel ultracentrifugation in 15-35% glycerol gradient, indicating that both
567 monomers and dimers are formed in gp16 wild-type, while dimer formation is interrupted http://mcb.asm.org/ 568 by the mutation of arginine finger. (C) The isolated gp16 dimers did not show any phage
569 assembly activity, supporting the previous finding that addition of fresh gp16 monomer
570 are required for re-initiating the DNA packaging intermediates. (D-F) Ultracentrifugation
571 fractions of protein markers including BSA (66 kDa), Alcohol dehydrogenase (140 kDa),
572 and beta-amylase (200 kDa) are shown, with their peak locations around fractions 23, 18 on August 9, 2016 by Ohio State University
573 and 15, respectively, establishing separation of the monomers and dimers of gp16
574 ATPase.
575 Figure 4. Inter-subunit interaction of gp16 arginine mutant with other gp16s.
576 (A-C) EMSA between gp16 arginine finger mutant titration with (A) gp16 wild-type, (B)
577 gp16 Walker A mutants, and (C) arginine finger mutants. Interactions between gp16
578 arginine finger mutants with gp16 wild-type or gp16 Walker A mutants are shown by the
579 band shift of both ATPase and DNA in the gel, while no obvious band shifts were
580 observed when ATPase arginine finger mutants were mixed together. DNA was labeled
581 with Cy5, and different ATPase were labeled with different fluorescent protein tags for
582 observation in the gel (Green: mCherry channel, Blue: eGFP channel, Red: Cy5 channel).
583 (D-E) Binomial distribution assay to determine the minimum number (y) of defective
26 584 eGFP-gp16 in the hexameric ring to block motor activity. Different rations of buffer (D)
585 or eGFP-gp16 Arginine finger mutants (E) were mixed with gp16 ATPase wild-type for
586 the in vitro phage assembly activity assay. The experimental curve is plotted with
587 theoretical predications made with the equation as seen in the Materials and Methods. Downloaded from
588 Figure 5. Prediction and comparison of gp16 structure.
589 (A) Structural differences of gp16 ATPase domain between top1 model predicted by I-
590 TASSER (cyan) and Phyre2 (pink) Server, RMSD~ 3Å. (B) Structure comparison http://mcb.asm.org/ 591 between crystal structure of FtsK (pdb ID: 2IUU, cyan), and gp16 ATPase model
592 generated by Phyre2 (pink), arginine finger is highlighted as sphere. (C) ATPase hexamer
593 structure is constructed using the predicted structure (monomers) with P. aeruginosa
594 FtsK (pdb ID: 2IUU) as the template 46. VMD was used to render the image of the
595 structure 31. The ATP domains were highlighted in sphere: residue 27 represents Walker on August 9, 2016 by Ohio State University
596 A domain (Green), residue 119 represents Walker B domain (Blue), and arginine finger
597 motif residue (Arg146 Red). The interaction of arginine finger with the upstream adjacent
598 subunit is supported by the relative location of the related domains.
599 Figure 6. ATP binding and ATP hydrolysis reshape the conformation and the
600 entropic landscape of gp16.
601 (A) Trypsin probing showed that the ATPase digested band is decreased with a reduced
602 amount of ATP added into gp16 ATPase samples, supporting that the gp16 ATPase is
603 less constrained after binding to ATP. (B) Intrinsic tryptophan fluorescent assay showing
604 the signal changes of ATPase upon adding different concentrations of ATP. (C) EMSA
605 showing that gp16 ATPase bound to ATP and undergoes a conformational change that
606 has a high affinity for DNA, and ATP hydrolysis triggers a second conformational
27 607 change of gp16 ATPase with a low affinity with DNA. (D) Increasing DNA is released
608 from gp16 ATPase/DNA/ATP complex upon the addition of increased amount of ATP
609 that can be hydrolyzed by the gp16 ATPase.
610 Figure 7. Asymmetrical structure of various ATPase hexamers. Downloaded from
611 Structures of V1-ATPase 2, TRIP13 73, MCM helicase 43, F1-ATPase 62, ClpX 61 are
612 shown as representatives. PDB ID: V1-ATPase, 3VR5; TRIP13, 4XGU; F1-ATPase,
613 1BMF; ClpX, 4I81. http://mcb.asm.org/ on August 9, 2016 by Ohio State University
28 Downloaded from http://mcb.asm.org/ on August 9, 2016 by Ohio State University Downloaded from http://mcb.asm.org/ on August 9, 2016 by Ohio State University Downloaded from http://mcb.asm.org/ on August 9, 2016 by Ohio State University Downloaded from http://mcb.asm.org/ on August 9, 2016 by Ohio State University Downloaded from http://mcb.asm.org/ on August 9, 2016 by Ohio State University Downloaded from http://mcb.asm.org/ on August 9, 2016 by Ohio State University Downloaded from http://mcb.asm.org/ on August 9, 2016 by Ohio State University