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Arginine Finger Regulates Sequential Action of Asymmetrical Hexameric

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Arginine Finger Regulates Sequential Action of Asymmetrical Hexameric 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.
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