Effect of A22 on the Conformation of Bacterial Actin Mreb

International Journal of Molecular Sciences

Article Effect of A22 on the Conformation of Bacterial Actin MreB

Elvis Awuni 1 and Yuguang Mu 2,*

1 Department of Biochemistry, School of Biological Sciences, CANS, University of Cape Coast, Cape Coast 00233, Ghana; [email protected] 2 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore * Correspondence: [email protected]; Tel.: +65-63162885

Received: 23 January 2019; Accepted: 25 February 2019; Published: 15 March 2019

Abstract: The mechanism of the antibiotic molecule A22 is yet to be clearly understood. In a previous study, we carried out molecular dynamics simulations of a monomer of the bacterial actin-like MreB in complex with different nucleotides and A22, and suggested that A22 impedes the release of Pi from the active site of MreB after the hydrolysis of ATP, resulting in ﬁlament instability. On the basis of the suggestion that Pi release occurs on a similar timescale to polymerization and that polymerization can occur in the absence of nucleotides, we sought in this study to investigate a hypothesis that A22 impedes the conformational change in MreB that is required for polymerization through molecular dynamics simulations of the MreB protoﬁlament in the apo, ATP+, and ATP-A22+ states. We suggest that A22 inhibits MreB in part by antagonizing the ATP-induced structural changes required for polymerization. Our data give further insight into the polymerization/depolymerization dynamics of MreB and the mechanism of A22.

Keywords: molecular dynamics; simulations; actin-like MreB; conformational change; polymerization; depolymerization

1. Introduction The bacterial actin-like MreB forms the cytoskeleton network, and is involved in several life processes of rod-shaped bacteria [1–10]. MreB polymerizes into filaments which are made of two straight and antiparallel strands, unlike the two twisted and parallel strands of eukaryotic actin [11]. Figure1a–c shows the crystal structures of the monomeric (PDB ID: 4CZK [ 11]), single protofilament (PDB ID: 4CZI [11]), and double protofilament (PDB ID: 4CZJ [11]), respectively, of Caulobacter (C) cresentus MreB (CcMreB). The four subdomains (IA, IB, IIA, and IIB) of MreB, A22, and ATP binding sites are indicated in Figure1a. To form a single filament, adjacent monomeric chains of MreB interact longitudinally at the intraprotofilament interfaces (Figure1b). Opposite chains interact laterally at the interprotofilament interfaces to form a double protofilament as illustrated in Figure1c. Thus, the polymerization of MreB involves both single filament and double filament formation. The antibiotic molecule A22 (Figure1d) has been shown to affect bacteria by targeting MreB [8,12,13], but its mechanism is yet to be clearly and fully understood. In a previous study [14], we carried out molecular dynamics (MD) simulations of monomeric bacterial actin-like MreB in complex with different nucleotides (NTs) and A22, and suggested that A22 impedes the release of Pi from the active site of MreB after the hydrolysis of ATP thus resulting in filament instability. On the basis of the observations we made [14], the fact that Pi release occurs on a similar timescale to polymerization [15–17], and that polymerization can occur in the absence of NTs [18], we proposed a hypothesis that A22 interferes with the conformational change in MreB that is required

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Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 14 for polymerization. In this study, MD simulations of the MreB protofilament in the empty (apo), wereATP+, carried and ATP-A22+out to test statesthis hypothesis. were carried We obse out torved test that this (i) hypothesis. ATP induces We a conformational observed that change (i) ATP ininduces MreB athat conformational could favor changethe formation in MreB of that stable could single favor and the formationdouble protofilaments, of stable single and and (ii) double A22 interferesprotofilaments, with the and generation (ii) A22 interferes of this favorable with the generation conformation of this and favorable induces conformationa structure that and may induces not supporta structure polymerization that may not of support MreB into polymerization stable filaments. of MreB into stable filaments.

Figure 1. Forms of MreB and structure of A22. (a) Monomeric structure of MreB. Subdomains IA, IB, Figure 1. Forms of MreB and structure of A22. (a) Monomeric structure of MreB. Subdomains IA, IB, IIA, and IIB as well as the ATP and A22 binding sites are indicated. The α-helix and β-sheet secondary IIA, and IIB as well as the ATP and A22 binding sites are indicated. The α-helix and β-sheet secondary structural elements are labeled. (b) Structure of a single protofilament of MreB composing of chains A, structural elements are labeled. (b) Structure of a single protofilament of MreB composing of chains B, and C. The intraprotofilament interfaces are indicated. (c) Structure of double protofilament of MreB. A, B, and C. The intraprotofilament interfaces are indicated. (c) Structure of double protofilament of The antiparallel strands and the interprotofilament interface are shown. (d) Structure of A22. MreB. The antiparallel strands and the interprotofilament interface are shown. (d) Structure of A22. 2. Results and Discussion 2. Results and Discussion 2.1. A22 Impedes ATP-Induced Backbone Conformational Change in MreB 2.1. A22To Impedes determine ATP-Induced any conformational Backbone Conformational change in the Change apo, ATP+, in MreB and ATP-A22+ MreB, root mean squareTo deviationdetermine (RMSD)any conformational analysis was change carried in out the on apo, the ATP+, backbone and atomsATP-A22+ of chain MreB, B inroot all mean three squaresimulations deviation of each (RMSD) state. Theanalysis RMSDs was were carried calculated out on by the using backbone the corresponding atoms of chain equilibrated B in all initialthree simulationsstructure of of each each state state. of MreBThe RMSDs as the reference.were calculat Theed RMSD by using values the of corresponding the last 50 ns simulationsequilibrated ofinitial each structurestate were of usedeach state to generate of MreB RMSD as the distributionreference. The curves. RMSD The values results, of the as last reported 50 ns simulations in Figure2, of reveal each statethat therewere isused a relatively to generate large RMSD conformational distribution change curves. in The the results, ATP+ state as reported (broad redin Figure distribution 2, reveal curves) that thereof MreB. is a relatively In the apo large and ATP-A22+conformational forms change (narrow in cyanthe ATP+ and greenstate (broad distribution red distribution curves, respectively), curves) of MreB.however, In the the apo backbone and ATP-A22+ atoms undergo forms relatively (narrow smallcyan conformationaland green distribution changes curves, as compared respectively), with the however,ATP+ form. the The backbone closeness atoms of the undergo backbone relatively atom distributions small conformational of the apo changes and ATP-A22+ as compared MreB formswith thesuggests ATP+ thatform. A22 The impedes closeness ATP-induced of the backbone conformational atom distributions change. of the apo and ATP-A22+ MreB forms suggests that A22 impedes ATP-induced conformational change.

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Figure 2. Figure 2. Backbone-atom root mean square deviation (RMSD) distribution curves of apo, ATP+,ATP+, and ATP-A22+ MreB. The solid, dashed, and dotted cyan lines represent backbone RMSD distributions of ATP-A22+ MreB. The solid, dashed, and dotted cyan lines represent backbone RMSD distributions of chain B from simulations 1, 2, and 3, respectively, of the apo filament. The solid, dashed, and dotted red chain B from simulations 1, 2, and 3, respectively, of the apo filament. The solid, dashed, and dotted lines represent backbone RMSD distributions of chain B from simulations 1, 2, and 3, respectively, of red lines represent backbone RMSD distributions of chain B from simulations 1, 2, and 3, respectively, the ATP+ filament. The solid, dashed, and dotted green lines represent backbone RMSD distributions of the ATP+ filament. The solid, dashed, and dotted green lines represent backbone RMSD of chain B from simulations 1, 2, and 3, respectively, of the ATP-A22+ filament. distributions of chain B from simulations 1, 2, and 3, respectively, of the ATP-A22+ filament. 2.2. MreB Adopts One Main Low-Energy Structure in the Apo, ATP+, and ATP-A22+ States 2.2. MreB Adopts One Main Low-Energy Structure in the Apo, ATP+, and ATP-A22+ States To visualize the most essential dynamics and structural variations in the apo, ATP+, and ATP-A22+ statesTo of visualize MreB, principal the most component essential dynamics analyses (PCA) and st wereructural performed variations on thein the backbone apo, ATP+, atoms and of chainATP- BA22+ of each states state of MreB, using principal the last 50component ns trajectories analyses of the(PCA) simulations. were performed The gmxcovar on the backbone tool in Gromacsatoms of 5.4.1chain was B of usedeach tostate generate using the eigenvectors. last 50 ns trajectori For thees three of the simulations simulations. of eachThe gmxcovar MreB state, tool the in trajectory Gromacs with5.4.1 thewas lowest used to cosine generate content eigenvec wastors. selected For forthe thethree generation simulations of aof 2D each projection MreB state, and the a free trajectory energy landscapewith the lowest (FEL) cosine plot using content gmxanaeig was selected and gmxfor the sham generation utilities inof Gromacs,a 2D projection respectively. and a free An energy earlier studylandscape suggested (FEL) thatplot ausing lower gmxanaeig cosine content and valuegmx sham (preferably, utilities <0.2 in Gromacs, on the scale respectively. of 0 to 1) is An indicative earlier ofstudy efficient suggested sampling that fora lower FEL analysiscosine content [19]. value (preferably, <0.2 on the scale of 0 to 1) is indicative of efficientIn each sampling case, the for first FEL and analysis the second [19]. principal components (PC1 and PC2) were projected to generateIn each the case, 2D plot the forfirst the and FEL the diagrams. second principal Figure3 componentsshows the FEL (PC1 plots and andPC2) the were corresponding projected to representativegenerate the 2D structures plot for extractedthe FEL fromdiagrams. each localFigure minimum. 3 shows Figure the FEL3a–c plots represent and the the corresponding FEL diagrams forrepresentative the apo, ATP+, structures and ATP-A22+ extracted systems, from each respectively. local minimum. The FEL plotsFigures show 3a–c one represent main low-energy the FEL regiondiagrams (local for minimum) the apo, ATP+, for the and apo, ATP-A22+ ATP+, and systems, ATP-A22+ respectively. states and The suggest FEL plots that oneshow main one low-energy main low- structureenergy region was adopted(local minimum) by the backbone for the apo, atoms ATP+, of chain and BATP-A22+ in each system. states and This suggest observation that one indicates main thatlow-energy any stuctural structure variation was adopted in the apo by the and, backbone especially, atoms the ATP+of chain and B in ATP-A22+ each system. MreB This forms observation could be restrictedindicates that to some any stuctural local structural variation elements, in the apo but and, not global. especially, the ATP+ and ATP-A22+ MreB forms could be restricted to some local structural elements, but not global.

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FigureFigure 3.3. FreeFree energyenergy landscapelandscape (FEL)(FEL) diagramsdiagrams ofof thethe apo,apo, ATP+ATP+ andand ATP-A22+ATP-A22+ MreB.MreB. ((aa)) FELFEL diagramdiagram of of thethe apoapo MreB.MreB. ((bb)) FELFEL diagramdiagram ofof thethe ATP+ATP+ MreB.MreB. ((cc)) FELFEL diagramdiagram ofof thethe ATP-A22+ATP-A22+ MreB.MreB. TheThe representativerepresentative structures structures were were extracted extracted from from the the low low energy energy regions regions colored colored in in deep deep blue. blue. 2.3. The ATP-A22+ MreB Structure Differs from the ATP+ Form 2.3. The ATP-A22+ MreB Structure Differs from the ATP+ Form To ﬁnd out the main structural variations in chain B of the apo, ATP+, and ATP-A22+ states To find out the main structural variations in chain B of the apo, ATP+, and ATP-A22+ states of of MreB, the backbone atoms of the representative structure of chain B from the local minimum of MreB, the backbone atoms of the representative structure of chain B from the local minimum of the the FEL diagram (Figure3) of each state was superimposed on the backbone atoms of the crystal FEL diagram (Figure 3) of each state was superimposed on the backbone atoms of the crystal structure. The crystal, apo, ATP+, and ATP-A22+ structures are colored in gray, cyan, red, and green, structure. The crystal, apo, ATP+, and ATP-A22+ structures are colored in gray, cyan, red, and green, respectively. Figure4 shows a pair-wise alignment and the corresponding RMSDs between the apo, respectively. Figure 4 shows a pair-wise alignment and the corresponding RMSDs between the apo, ATP+, ATP-A22+, and the crystal structure without ATP and A22. As shown in Figure4, the following ATP+, ATP-A22+, and the crystal structure without ATP and A22. As shown in Figure 4, the following RMSD values were obtained from the alignments: apo/crystal = 1.69 Å (Figure4a), ATP+/crystal RMSD values were obtained from the alignments: apo/crystal = 1.69 Å (Figure 4a), ATP+/crystal = = 2.53 Å (Figure4b), ATP-A22+/crystal = 1.46 Å (Figure4c), apo/ATP+ = 2.47 Å (Figure4d), 2.53 Å (Figure 4b), ATP-A22+/crystal = 1.46 Å (Figure 4c), apo/ATP+ = 2.47 Å (Figure 4d), apo/ATP- apo/ATP-A22+ = 1.66 Å (Figure4e), and ATP+/ATP-A22+ = 2.39 Å (Figure4f). From these RMSD A22+ = 1.66 Å (Figure 4e), and ATP+/ATP-A22+ = 2.39 Å (Figure 4f). From these RMSD values, it can values, it can be observed that the pair-wise alignments of the crystal, apo, and ATP-A22+ structures be observed that the pair-wise alignments of the crystal, apo, and ATP-A22+ structures produced produced RMSDs < 2 Å, which indicates that these structures are close. The ATP+ conformation, on the RMSDs < 2 Å, which indicates that these structures are close. The ATP+ conformation, on the other hand, aligns with each of these structures with a RMSD > 2 Å, indicating that the ATP+ structure

Int. J. Mol. Sci. 2019, 20, 1304 5 of 14 otherInt. J. Mol. hand, Sci. 2019 aligns, 20, with x FOR each PEER of REVIEW these structures with a RMSD > 2 Å, indicating that the ATP+ structure5 of 14 undergoes a large conformational change. The results are consistent with the RMSD distribution curve undergoes a large conformational change. The results are consistent with the RMSD distribution in Figure2, suggesting that A22 blocks ATP-induced conformation change in MreB. curve in Figure 2, suggesting that A22 blocks ATP-induced conformation change in MreB.

Figure 4. Pair-wise alignment of the crystal, apo, ATP+ ATP+,, and ATP-A22+ structures with corresponding RMSDs. ( aa)) Alignment ofof apo/crystalapo/crystal structures.structures. ( b) AlignmentAlignment ofof ATP+/crystalATP+/crystal structures.structures. ( c) d e Alignment ofof ATP-A22+/crystalATP-A22+/crystal structures. (d) AlignmentAlignment ofof apo/ATP+apo/ATP+ structures. ( e) Alignment ofof apo/ATP-A22+ structures. (f) Alignment of ATP+/ATP-A22+ structures. apo/ATP-A22+ structures. (f) Alignment of ATP+/ATP-A22+ structures. To determine the compactness of the apo, ATP+, and ATP-A22+ structures, the radius of gyration To determine the compactness of the apo, ATP+, and ATP-A22+ structures, the radius of gyration (RG) on the backbone atoms of chain B in each state was calculated. The results, as shown in Figure5, (RG) on the backbone atoms of chain B in each state was calculated. The results, as shown in Figure suggest that the backbone atoms of chain B relaxes in the ATP+ form of MreB compared with the 5, suggest that the backbone atoms of chain B relaxes in the ATP+ form of MreB compared with the apo and ATP-A22+ forms. This is indicated by the relatively high RG values of the ATP+ form (red apo and ATP-A22+ forms. This is indicated by the relatively high RG values of the ATP+ form (red curve) as against the relatively low RG values of the apo and ATP-A22+ forms (cyan and green curves, curve) as against the relatively low RG values of the apo and ATP-A22+ forms (cyan and green curves, respectively). Video S1 explains why MreB adopts a relaxed conformation in the ATP+ state and respectively). Video S1 explains why MreB adopts a relaxed conformation in the ATP+ state and compact conformations in the ATP-A22+ and apo states. It can be observed from Video S1 that in the compact conformations in the ATP-A22+ and apo states. It can be observed from Video S1 that in the ATP+ state, helix 4 (H4) in subdomain IA is destabilized during the simulations. This leads to the ATP+ state, helix 4 (H4) in subdomain IA is destabilized during the simulations. This leads to the disordering of the H4-S9 (Sheet 9) loop which links subdomains IA and IIA (Figure1a). The H4-S9 disordering of the H4-S9 (Sheet 9) loop which links subdomains IA and IIA (Figure 1a). The H4-S9 loop then extends freely to allow the subdomain IIA to move outwards leading to a relaxed structure. loop then extends freely to allow the subdomain IIA to move outwards leading to a relaxed structure. On the contrary, H4 is ordered and rigid in the ATP-A22+ and apo states and thus makes the structure On the contrary, H4 is ordered and rigid in the ATP-A22+ and apo states and thus makes the structure compact by keeping subdomains IA and IIA closer. compact by keeping subdomains IA and IIA closer.

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Figure 5. Radius of gyration (Rg) of backbone atoms of chain B in the apo, ATP+, and ATP-A22+ Figure 5. Radius of gyration (Rg) of backbone atoms of chain B in the apo, ATP+, and ATP-A22+ protofilaments. The cyan, red, and green curves represent the radius of gyration of chain B of the apo, protofilaments. The cyan, red, and green curves represent the radius of gyration of chain B of the apo, ATP+, and ATP-A22+ filaments, respectively. ATP+, and ATP-A22+ filaments, respectively. Figures S1 and S2 show the alignment of the backbone atoms on the interprotofilament interface Figures S1 and S2 show the alignment of the backbone atoms on the interprotofilament interface (the face where MreB chains interact to form a double protofilament) and the opposite face, respectively, (the face where MreB chains interact to form a double protofilament) and the opposite face, of the crystal, apo, ATP+, and ATP-A22+ structures. It appears that the atoms on the interprotofilament respectively, of the crystal, apo, ATP+, and ATP-A22+ structures. It appears that the atoms on the interface do not produce a good alignment in contrast to atoms on the opposite face, which is indicative interprotofilament interface do not produce a good alignment in contrast to atoms on the opposite of conformational change on the interprotofilament interface and not the opposite face. To confirm face, which is indicative of conformational change on the interprotofilament interface and not the this observation, the atoms of the main secondary structural elements on the interprotofilament opposite face. To confirm this observation, the atoms of the main secondary structural elements on interfaces and the opposite faces of the crystal, apo, ATP+, and ATP-A22+ structures were grouped the interprotofilament interfaces and the opposite faces of the crystal, apo, ATP+, and ATP-A22+ separately, and, in each case, the RMSD of each group in the apo, ATP+, and ATP-A22+ structures structures were grouped separately, and, in each case, the RMSD of each group in the apo, ATP+, and was calculated by superimposing the group on the corresponding reference group in the crystal ATP-A22+ structures was calculated by superimposing the group on the corresponding reference structure to determine the extent of structural variation. The main secondary structural elements group in the crystal structure to determine the extent of structural variation. The main secondary on the interprotofilament interface selected as a group (Group 1) include H2, H3, H5, H8, S6, and structural elements on the interprotofilament interface selected as a group (Group 1) include H2, H3, S11. Those on the opposite face selected as a group (Group 2) include H1, H6, H12, S3, S12, and H5, H8, S6, and S11. Those on the opposite face selected as a group (Group 2) include H1, H6, H12, S13. Group 1 from the apo/crystal, ATP+/crystal, and ATP-A22+/crystal structures aligned with S3, S12, and S13. Group 1 from the apo/crystal, ATP+/crystal, and ATP-A22+/crystal structures RMSDs of 1.16 Å, 3.51 Å, and 1.12 Å, respectively. On the other hand, RMSDs of 1.47 Å, 1.25 Å, aligned with RMSDs of 1.16 Å, 3.51 Å, and 1.12 Å, respectively. On the other hand, RMSDs of 1.47 Å, and 1.39 Å were obtained from the alignments of group 2 from the apo/crystal, ATP+/crystal, and 1.25 Å, and 1.39 Å were obtained from the alignments of group 2 from the apo/crystal, ATP+/crystal, ATP-A22+/crystal structures, respectively. Relatively, the data suggest that the Group 1 in the ATP+ and ATP-A22+/crystal structures, respectively. Relatively, the data suggest that the Group 1 in the structure undergo significant conformational change. On the basis of these observations, we suggest ATP+ structure undergo significant conformational change. On the basis of these observations, we that the dynamics of some secondary structural elements on the interprotofilament interface could suggest that the dynamics of some secondary structural elements on the interprotofilament interface influence the polymerization/depolymerization dynamics of MreB and that A22 possibly interferes could influence the polymerization/depolymerization dynamics of MreB and that A22 possibly with the dynamics of these secondary structural elements. interferes with the dynamics of these secondary structural elements. 2.4. A22 Affects the Conformational Change of the Ile55-Ile219 and Ser232-Val316 MreB Segments 2.4. A22 Affects the Conformational Change of the Ile55-Ile219 and Ser232-Val316 MreB Segments To find out the specific structural elements responsible for the difference between the apo, ATP+To and, find ATP-A22+ out the specific MreB structural conformations, elements root resp meanonsible square for the fluctuation difference (RMSF) between calculations the apo, ATP+ on theand, backbone ATP-A22+ atoms MreB (N, conformations, CA, and C) of chainroot Bmean of each square state fluctuation were carried (RMSF) out to quantifycalculations the atomicon the fluctuations.backbone atoms The RMSF(N, CA, calculations and C) of in chain each systemB of each were state carried were out carried by using out the tolast quantify 50 nssimulations the atomic andfluctuations. the corresponding The RMSF equilibrated calculations initial in each structure system asreference. were carried The cyan,out by red, using and greenthe last curves 50 inns Figuresimulations6 represent and the the corresponding RMSF of the backboneequilibrated atoms initial of thestructure apo, ATP+, as reference. and ATP-A22+ The cyan, structures, red, and respectively.green curves in As Figure illustrated 6 represent in Figure the RMSF6, the of fluctuations the backbone of atoms the atoms of the of apo, a 165-residue ATP+, and ATP-A22+ segment (Ile55-Ile219)structures, respectively. and an 85-residue As illust segmentrated (Ser232-Val316)in Figure 6, the are fluctuations much affected. of the The atoms regions of correspondinga 165-residue segment (Ile55-Ile219) and an 85-residue segment (Ser232-Val316) are much affected. The regions corresponding to the Ile55-Ile219 and Ser232-Val316 are indicated by the orange- and blue-dashed

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Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 14 to the Ile55-Ile219 and Ser232-Val316 are indicated by the orange- and blue-dashed rectangles, rectangles,respectively. respectively. Relatively, theRelatively, backbone the atoms backbone of these atoms two of segments these two recorded segments high recorded ﬂuctuations high in fluctuationsthe ATP+ structure in the ATP+ than instructure the apo andthan ATP-A22+in the apo structures.and ATP-A22+ The data structures. suggest The that data the ATP-inducedsuggest that theconformational ATP-induced change conformational is restricted change to segments is restricted Ile55-Ile219 to segments and Ser232-Val316, Ile55-Ile219 and and Ser232-Val316, that A22 impedes and thatthis A22 conformational impedes this change. conformational change.

Figure 6. Root mean square fluctuations (RMSF) of the backbone atoms (N, CA, and C) of chain B of Figurethe apo, 6. ATP+,Root mean and ATP-A22+ square fluctuations filaments. (RMSF) The cyan, of red,the backbone and green atoms curves (N, represent CA, and RMSFs C) of chain of chain B of B theof the apo, apo, ATP+, ATP+, and and ATP-A22+ ATP-A22+ filaments. filaments, The respectively. cyan, red, and Regions green corresponding curves represent to the RMSFs Ile55-Ile219 of chain and B ofSer232-Val316 the apo, ATP+, are indicatedand ATP-A22+ by the filaments, orange- and respecti blue-dashedvely. Regions rectangles, corresponding respectively. to the Ile55-Ile219 and Ser232-Val316 are indicated by the orange- and blue-dashed rectangles, respectively. Figure7a shows the Ile55-Ile219 and Ser232-Val316 segments colored orange and blue, respectively. The Ile55-Ile219Figure 7a segment,shows the which Ile55-Ile219 consist of and helices Ser232-Val316 (H1, H2, H3, H4,segments H5, and colored H6) and orange B-sheets and (S6, S7,blue, S8, respectively.S9, S10, and S11),The Ile55-Ile219 extends from segment, subdomain which IA consist to IB through of helices IIA (H1, to IIB H2, (Figure H3, H4,7a). H5, The and Ser232-Val316 H6) and B- sheetssegment, (S6, on S7, the S8, other S9, S10, hand, and consists S11), extends of the from helices su H7,bdomain H8, H9, IA andto IB H10 through and B-sheetsIIA to IIB S12, (Figure S13, 7a). S14, Theand Ser232-Val316 S15 and resides segment, in subdomains on the IIAother and hand, IIB (Figure consists7a). of Interestingly,the helices H7, the H8, dimerization H9, and H10 helix and (H3), B- sheetswhich hasS12,been S13, mentionedS14, and S15 in anand earlier resides study in subdom [11] as beingains IIA important and IIB in (Figure the dimerization 7a). Interestingly, of two MreB the dimerizationchains, is found helix within (H3), the which Ile55-Ile219 has been segment. mentioned in an earlier study [11] as being important in the dimerizationFigure7b,c of two show MreB the superimposedchains, is found backbone within the atoms Ile55-Ile219 of the two segment. segments from the apo, ATP+, ATP-A22+,Figure 7b,c and show crystal the structures superimposed colored backbone cyan, red, atoms green, of andthe two gray, segments respectively. from H3the isapo, displaced ATP+, ATP-A22+,outwards inand the crystal ATP+ structures structure colored and inwards cyan, inred, the green, ATP-A22+ and gray, conformation. respectively. This H3 is preferential displaced outwardsdisplacement in the of H3ATP+ in responsestructure to and A22 inwards was also in observed the ATP-A22+ in a previous conformati crystallographicon. This preferential study of displacementCcMreB [11]. Asof H3 shown in response in Figure to7d–f, A22 the was Ile55-Ile219 also observed and Ser232-Val316in a previous segmentscrystallographic extend study into the of CcMreBintra- and [11]. inter-protofilament As shown in Figures interfaces. 7d–f, the Intra- Ile55-Ile219 and inter-protofilament and Ser232-Val316 interactions segments extend promote into single the intra-filament and and inter-protofilament double filament formation, interfaces.respectively, Intra- and inter-protofilament and thus the dynamics interactions of these promote segments single could filamentaffect these and interactions double filament and alter format theion, polymerization/depolymerization respectively, and thus the dynamics dynamics of these of segments MreB. could affect these interactions and alter the polymerization/depolymerization dynamics of MreB.

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Figure 7. PossiblePossible roles roles of of the the Ile55-Ile219 Ile55-Ile219 (orange) (orange) and and Ser232-Val316 Ser232-Val316 (blue) (blue) segments in MreB polymerization. (a)) MonomericMonomeric MreBMreB structure structure showing showing the the secondary secondary structural structural elements elements making making up upthe the Ile55-Ile219 Ile55-Ile219 and Ser232-Val316and Ser232-Val316 segments. segm ATPents. and ATP A22 and are A22 also shown.are also ( bshown.) Superimposed (b) Superimposed structures structuresof the Ile55-Ile219 of the Ile55-Ile219 segments fromsegmen chaints from B of chain the crystal B of the (gray), crystal apo (g (cyan),ray), apo ATP+ (cyan), (red), ATP+ and ATP-A22+(red), and ATP-A22+(green) filaments. (green) ( cfilaments.) Superimposed (c) Superimposed structures of structures the Ser232-Val316 of the Ser232-Val316 segments from segments chain B of from the crystalchain B(gray), of the apo crystal (cyan), (gray), ATP+ (red),apo (cyan), and ATP-A22+ ATP+ (red), (green) and filaments. ATP-A22+ (d )(green) Single protofilamentfilaments. (d) of Single MreB protofilamentillustrating the of roles MreB of theillustrating Ile55-Ile219 the and roles Ser232-Val316 of the Ile55-Ile219 segments and at theSer232-Val316 intraprotofilament segments interfaces. at the intraprotofilament(e–f) Double protofilaments interfaces. of MreB(e–f) Double illustrating protofilaments the roles of theof MreB Ile55-Ile219 illustrating and Ser232-Val316 the roles of the segments Ile55- Ile219at the interprotofilamentand Ser232-Val316 interface.segments at the interprotofilament interface. A focused view of the interprotofilament interface (Figure8a) shows that H3 in one chain interacts A focused view of the interprotofilament interface (Figure 8a) shows that H3 in one chain with H3 of the opposite chain, and H5 in one chain interacts with H8 in the opposite chain. Furthermore, interacts with H3 of the opposite chain, and H5 in one chain interacts with H8 in the opposite chain. as shown in Figure8b, at the intraprotofilament interface, H9 interacts with S12 and S13 in an adjoining Furthermore, as shown in Figure 8b, at the intraprotofilament interface, H9 interacts with S12 and chain, and H4-S9 and S10-S11 loops interact with the H1-S6 loop of the adjacent chain. Thus on the S13 in an adjoining chain, and H4-S9 and S10-S11 loops interact with the H1-S6 loop of the adjacent basis of our simulation results, we suggest that in the ATP+ structure, (i) the displacement of the chain. Thus on the basis of our simulation results, we suggest that in the ATP+ structure, (i) the H3s outwards could maximize the H3–H3 interactions at the interprotofilament interface between displacement of the H3s outwards could maximize the H3–H3 interactions at the interprotofilament opposite MreB chains, (ii) the displacement of H8 outwards could maximize the H8–H5 interactions interface between opposite MreB chains, (ii) the displacement of H8 outwards could maximize the at the interprotofilament interface between opposite chains, (iii) the displacement of H9 outwards H8–H5 interactions at the interprotofilament interface between opposite chains, (iii) the displacement at the intraprotofilament interface could possibly increase its interactions with S12 and S13 in the of H9 outwards at the intraprotofilament interface could possibly increase its interactions with S12 adjoining chain, and (iv) the displacement of S9, S10, and S11 may also optimize intraprotofilament and S13 in the adjoining chain, and (iv) the displacement of S9, S10, and S11 may also optimize interactions between the H4-S9 and S10-S11 loops of one chain with the H1-S6 loop of the adjacent chain. intraprotofilament interactions between the H4-S9 and S10-S11 loops of one chain with the H1-S6 In summary, we suggest that the ATP+ structure and dynamics could facilitate MreB polymerization, loop of the adjacent chain. In summary, we suggest that the ATP+ structure and dynamics could and that A22 directly antagonizes the ATP-induced conformational change and dynamics leading to facilitate MreB polymerization, and that A22 directly antagonizes the ATP-induced conformational the weakening of the intra- and inter-protofilament interactions. change and dynamics leading to the weakening of the intra- and inter-protofilament interactions.

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Figure 8. Interactions of Ile55-Ile219 (orange) and Ser232-Val316 (blue) segments in MreB double and Figure 8. Interactions of Ile55-Ile219 (orange) and Ser232-Val316 (blue) segments in MreB double and single protofilaments. (a) Interactions in double protofilament (filament dimerization). (b) Interactions single protofilaments. (a) Interactions in double protofilament (filament dimerization). (b) in single protofilament. The red dashed-rectangle shows the intraprotofilament interface between Interactions in single protofilament. The red dashed-rectangle shows the intraprotofilament interface chains A and B, and the secondary structural elements that could establish contacts. The arrows signify between chains A and B, and the secondary structural elements that could establish contacts. The the interactions. arrows signify the interactions. 2.5. Water Dynamics in the Active Site of MreB Protofilament 2.5. Water Dynamics in the Active Site of MreB Protofilament In a previous simulation study of monomeric MreB [14], we monitored the dynamics of water moleculesIn a previous in the active simulation sites ofstudy the of ATP+ monomeric and ATP-A22+ MreB [14], states we bymonitored counting the the dynamics water molecules of water moleculesthat entered in the active catalytic sites zone of the (the ATP+ solvent and accessible ATP-A22+ space states near by counting the γ-phosphate the water of molecules ATP and that the enteredcatalytic the Glu140 catalytic residue, zone which(the solvent is defined accessible as the space intersection near the of γ-phosphate two spheres of with ATPthe and radii the catalytic of 3.9 Å Glu140and 3 Å residue, around which the phosphorous is defined as atom the intersection (Pγ) of the γof-phosphate two spheres of with ATP the and radii the carbonylof 3.9 Å and oxygen 3 Å aroundatom (OE1) the phosphorous of Glu140, respectively atom (Pγ) of [ 20the–22 γ-phosphate]) of each state. of ATP We and observed the carbonyl that water oxygen molecules atom (OE1) are ofalways Glu140, present respectively in the catalytic [20–22]) zone of each and arestate. properly We observed oriented that to initiatewater molecules the hydrolysis are always of ATP present in both instates. the catalytic We repeated zone this and process are properly for the oriented simulations to initiate of ATP+ the and hydrolysis ATP-A22+ of ATP filaments in both using states. the lastWe repeated50 ns simulations. this process The for results the simulations for the ATP+ ofand ATP+ ATP-A22+ and ATP-A22+ filaments filaments are presented using inthe Figure last 509a,b, ns simulations.respectively. Interestingly,The results wefor observedthe ATP+ the and presence ATP-A22+ of less filaments but longer are staying presented water moleculesin Figure in9a,b, the ATP+respectively. and ATP-A22+ Interestingly, filaments we comparedobserved the with presence the monomeric of less but states longer studied staying earlier water [14]. molecules We found 93in theand ATP+ 70 water and molecules ATP-A22+ in thefilaments active sitecompared of the ATP+ with andthe ATP-A22+monomeric filaments, states studied respectively, earlier as [14]. against We 196found and 93 165 and in 70 the water active molecules sites of monomeric in the active ATP+ site of and the ATP-A22+ ATP+ and MreB,ATP-A22+ respectively, filaments, in ourrespectively, previous asstudy against [14]. 196 and 165 in the active sites of monomeric ATP+ and ATP-A22+ MreB, respectively, in our previousIn monomeric study [14]. MreB, the cleft between subdomains IB and IIB (Figures1a and7a) could allow the freeIn monomeric movement MreB, of many the water cleft between molecules subdomains in and out ofIB theand active IIB (Figures site. In 1a the and filamentous 7a) could MreB,allow thehowever, free movement the cleft between of many the water subdomains molecules IB in and and IIB out of of chain the Bactive (used site. for In the the analysis) filamentous is closed MreB, by however,the adjacent the chaincleft between A (Figures the1 bsubdomains and7d), which IB and could IIB of lead chain to a B reduction (used for ofthe the analysis) number is ofclosed water by themolecules adjacent that chain move A in(Figures and out 1b of and the active7d), which site.This could could lead explain to a reduction why we observedof the number less but of longerwater moleculesstaying water that moleculesmove in and in theout catalyticof the active zone site. of theThis ATP+ could and explain ATP-A22+ why we filaments observed compared less but longer with stayingthe monomeric water molecules forms. Since in the one catalytic molecule zone of water of the triggers ATP+ and the hydrolysisATP-A22+ offilaments ATP, the compared staying time with of waterthe monomeric molecules forms. in the Since catalytic one zone, molecule but not of water the number triggers of the water hydrolysis molecules of thatATP, enter the staying the catalytic time ofzone, water is criticalmolecules for in ATP the hydrolysis.catalytic zone, The but frequent not the number presence of of water long-staying molecules water that enter molecules the catalytic in the ATP+zone, is filament critical isfor consistent ATP hydrolysis. with the The observation frequent that presence polymerization of long-staying turns onwater ATP molecules hydrolysis in [11the]. Additionally,ATP+ filament the is observationconsistent with of long-staying the observation water that molecules polymerization in the catalytic turns on zone ATP of hydrolysis the ATP-A22+ [11]. Additionally,filament supports the observation our suggestion of long-staying in a previous water study molecules [14] that in A22 the maycatalytic not bezone an of inhibitor the ATP-A22+ of ATP filament supports our suggestion in a previous study [14] that A22 may not be an inhibitor of ATP hydrolysis. The orientations of these long-staying water molecules are similar to those reported in our earlier study [14], and thus are suitable for the hydrolysis of ATP.

Int. J. Mol. Sci. 2019, 20, 1304 10 of 14

hydrolysis. The orientations of these long-staying water molecules are similar to those reported in our Int.earlier J. Mol. study Sci. 2019 [14, 20],, and x FOR thus PEER are REVIEW suitable for the hydrolysis of ATP. 10 of 14

FigureFigure 9. WaterWater dynamic in MreB active site. ( (aa)) Time Time spent by by water molecules molecules in in the the catalytic catalytic zone zone ofof the the ATP+ ATP+ MreB MreB filament. filament. ( (bb)) Time Time spent spent by by water water molecules molecules in in the the catalytic catalytic zone zone of of the the ATP-A22+ ATP-A22+ MreBMreB filament. filament. The The arrows point to long-staying water molecules. 2.6. Proposed Effect of A22 2.6. Proposed Effect of A22 It has been established that as an ATPase, MreB needs ATP to polymerize, and polymerization It has been established that as an ATPase, MreB needs ATP to polymerize, and polymerization turns on the process of ATP hydrolysis [2,17,23]. We constructed the apo, ATP+, and ATP-A22+ turns on the process of ATP hydrolysis [2,17,23]. We constructed the apo, ATP+, and ATP-A22+ protofilaments to test our hypothesis that ATP induces a conformational change that favors protofilaments to test our hypothesis that ATP induces a conformational change that favors polymerization of MreB while A22 impedes this conformational change and directs the MreB structure polymerization of MreB while A22 impedes this conformational change and directs the MreB toward a conformation which does not support the formation of stable protofilaments. These structure toward a conformation which does not support the formation of stable protofilaments. suggestions are supported by the fact that double filamentous structures of the AMPPNP(an ATP These suggestions are supported by the fact that double filamentous structures of the AMPPNP(an analogue)-bound MreB have been solved by X-ray crystallography, but only single filamentous ATP analogue)-bound MreB have been solved by X-ray crystallography, but only single experimental structures of the AMPPNP-A22-bound form are tractable [11]. It might have been filamentous experimental structures of the AMPPNP-A22-bound form are tractable [11]. It might more appropriate to add the crystal structure of monomeric ATP-A22+ form to the simulations for have been more appropriate to add the crystal structure of monomeric ATP-A22+ form to the comparison. However, during the simulations of monomeric CcMreB, the IB and IIB subdomains simulations for comparison. However, during the simulations of monomeric CcMreB, the IB and IIB (Figures1a and7a) could adopt an opened or closed state and thus could adopt certain structural subdomains (Figures 1a and 7a) could adopt an opened or closed state and thus could adopt certain features that are not a reflection of the effects of either ATP or A22. In the single filament form, however, structural features that are not a reflection of the effects of either ATP or A22. In the single filament these domains in chain B are restricted by chain A and chain C (Figures1b and7d), creating similar form, however, these domains in chain B are restricted by chain A and chain C (Figure 1b and 7d), conditions for comparison. Thus, to be able to predict the conformational change that could affect the creating similar conditions for comparison. Thus, to be able to predict the conformational change that polymerization of MreB into filaments, the analysis of simulation data was restricted to the backbone could affect the polymerization of MreB into filaments, the analysis of simulation data was restricted atoms of chain B of the apo, ATP+, and ATP-A22+ filaments. to the backbone atoms of chain B of the apo, ATP+, and ATP-A22+ filaments. Following our observations, we have three suggestions. Firstly, ATP induces MreB conformation Following our observations, we have three suggestions. Firstly, ATP induces MreB conformation that is suitable for polymerization. Secondly, the ATP+ conformation is probably transient and that is suitable for polymerization. Secondly, the ATP+ conformation is probably transient and disappears after ATP hydrolysis which immediately follows polymerization. ATP hydrolysis happens disappears after ATP hydrolysis which immediately follows polymerization. ATP hydrolysis very fast so that the experimental structure of the ATP-bound MreB is not tractable, and thus it is happens very fast so that the experimental structure of the ATP-bound MreB is not tractable, and possible that the ATP-induced conformation disappears at a similar rate and may not be captured thus it is possible that the ATP-induced conformation disappears at a similar rate and may not be in total by an experiment. Interestingly, however, the preferred conformations (displacements) of captured in total by an experiment. Interestingly, however, the preferred conformations the dimerization helix (H3) in the ATP+ and ATP-A22+ states of MreB which were observed in (displacements) of the dimerization helix (H3) in the ATP+ and ATP-A22+ states of MreB which were our simulations are consistent with the experiment in Reference [11]. Thirdly, we suggest that A22 observed in our simulations are consistent with the experiment in Reference [11]. Thirdly, we suggest impedes the ATP+ conformation and induces the ATP-A22+ conformation, which does not support that A22 impedes the ATP+ conformation and induces the ATP-A22+ conformation, which does not the polymerization of MreB into stable double protofilaments. These suggestions are supported by support the polymerization of MreB into stable double protofilaments. These suggestions are an earlier crystallization study of MreB [11] where only the double protofilament structures of the supported by an earlier crystallization study of MreB [11] where only the double protofilament AMPPNP-bound state, and not the AMPPNP-A22-bound state, could be solved. Additionally, a study structures of the AMPPNP-bound state, and not the AMPPNP-A22-bound state, could be solved. involving time-lapse imaging has shown that MreB filaments are dynamic structures in vivo [9,24] and Additionally, a study involving time-lapse imaging has shown that MreB filaments are dynamic probably hydrolyze ATP during movements [25]. We suggest that these movements may be necessary structures in vivo [9,24] and probably hydrolyze ATP during movements [25]. We suggest that these for MreB polymerization and that the mechanism of A22 may partly involve its ability to impede movements may be necessary for MreB polymerization and that the mechanism of A22 may partly these motions. involve its ability to impede these motions.

3. Methods

3.1. Preparation of Structures The NT- and A22-free (apo) crystal structure of the CcMreB single filament, PDB ID 4CZI [11] (shown in Figure 1b), was downloaded from the protein data bank (PDB) and used as the reference

Int. J. Mol. Sci. 2019, 20, 1304 11 of 14

3. Methods

3.1. Preparation of Structures The NT- and A22-free (apo) crystal structure of the CcMreB single filament, PDB ID 4CZI [11] (shown in Figure1b), was downloaded from the protein data bank (PDB) and used as the reference structure for the construction of the ATP+ and ATP-A22+ single filament forms of MreB. All the missing residues were added using the Swiss model package [26]. To add ATP and A22 to 4CZI, a monomeric MreB-ATP-A22 structure that we constructed in an earlier study [14] was superimposed on each chain of 4CZI. Then the ATP or ATP andA22 molecule(s) on the monomeric MreB-ATP-A22 structure was/were transferred to 4CZI. The structures were subjected to several energy optimization steps in Gromacs using the AMBER99SB force field [27] to remove all bad contacts. The low-energy-state structures were used for the MD simulations. Three CcMreB protofilaments including the apo, ATP+, and ATP-A22+ forms were prepared for the simulations. Each of these filaments had three chains (A, B, and C), as shown in Figure1b.

3.2. Molecular Dynamics Simulations The normal MD simulations method was the same as used in our previous study [14] with slight modifications. Gromacs 5.4.1 was used with the AMBER99SB force field [27] and the TIP3P water model [28] to build the topology of the protein in each system. For ATP and A22, the Gaussian09 [29] in the R.E.D.-III.4 tool [30] was used to perform quantum mechanical calculations to determine the atomic partial charges. The topologies of ATP and A22 were then built for the general amber force field (GAFF) after the antechamber utility [31] in the AMBER10 package [32] was used to assign atom types. For each simulation system, the protein was centered in a cubic box and the minimum distance between the edges of the box and the surface of the protein was set to 10 Å. The system was then solvated with an explicit TIP3P water model [28] and neutralized electrostatically with the ionic strength maintained at 0.1 M by adding the appropriate number of Na+ and Cl− ions. The energy of the system was appropriately minimized using the steepest descent algorithm, and the cut-offs for long range electrostatic interactions (treated with the Particle Mesh Ewald algorithm [33]) and Van der Waals interactions were set at 10 Å and 14 Å, respectively. After applying the Berendsen barostat for pressure and the V-rescaling thermostat [34] for temperature coupling, the NPT ensemble was used to equilibrate the system for 10 ns at 300 K. The 10 ns equilibration time was chosen to give ample time for such a huge system to properly equilibrate. A continuation of the simulations to 100 ns was performed for each system, for which three independent simulations were carried out. Table1 shows the summary of the simulation systems.

Table 1. Table summary of the molecular dynamics (MD) simulation systems.

State of MreB System ATP A22 Mg2+ Time (ns) Repeats Protoﬁlament 1 Apo (no ATP and A22) - - + 100 3 2 ATP-bound (ATP+) + - + 100 3 ATP-A22-bound 3 + + + 100 3 (ATP-A22+)

4. Conclusions Experimental techniques such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy have been useful in providing structures that have facilitated the understanding of biomolecular function and biological processes. Unfortunately, these techniques may only capture snapshots and inadequately quantify the ﬂexibility of some proteins and protein-ligand complexes by excluding the several intermediate structures that may be required to fully understand biological phenomena and mechanisms [35]. One possible way of bridging this gap is by applying Int. J. Mol. Sci. 2019, 20, 1304 12 of 14 computational techniques, such as MD simulations, to enhance the sampling of the conformational space of biological structures. Thus, there is a requirement to combine structural information from both experimental and computer-generated structures to provide a better synergy to facilitate the understanding of biological processes. We applied MD simulations to investigate the effect of A22 on the conformation of MreB and the possible consequences on its polymerization/depolymerization dynamics. Single protoﬁlaments of the apo, ATP+, and ATP-A22+ MreB were simulated and the structures compared. It is suggested that A22 impedes the ATP-induced structural changes, which are required for the formation of stable MreB polymers, in two MreB protein segments (Ile55-Ile219 and Ser232-Val316).

5. Recommendation We carried out molecular dynamics simulations of a CcMreB single protoﬁlament in the apo, ATP+, and ATP-A22+ states and, on the basis of our observations, made suggestions that A22 possibly impedes an ATP-induced conformation (ATP+ conformation) that is required for polymerization and induces a conformation (ATP-A22+ conformation) that does not favor polymerization. We recommend that in a further study, these suggestions should be investigated by constructing and simulating double protoﬁlaments of MreB in the apo, ATP+, and ATP-A22+ forms.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/6/ 1304/s1. Author Contributions: Conceptualization, E.A. and Y.M.; methodology, E.A. and Y.M.; software, E.A.; validation, E.A. and Y.M.; formal analysis, E.A.; investigation, E.A.; resources, E.A. and Y.M.; data curation, E.A. and Y.M.; writing—original draft preparation, E.A.; writing—review and editing, E.A. and Y.M.; supervision, E.A. and Y.M.; project administration, E.A. and Y.M.; funding acquisition, E.A. and Y.M. Both authors read and approved the final manuscript. Funding: This research was supported by MOE Tier 1 Grant RG 138/15, RG 146/17 and the computing resources of the National Supercomputing Centre, Singapore. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

PDB Protein Data Bank CcMreB Caulobacter cresentus MreB NT Nucleotide MD Molecular dynamics AMBER Assisted Model Building and Energy Reﬁnement GAFF General Amber Force Field RMSD Root Mean Square Deviation PCA Principal Component Analysis FEL Free Energy Landscape RMSF Root Mean Square Fluctuation AMPPNP Adenylyl imidodiphosphate

References

1. Jones, L.J.F.; Carballido-Lopez, R.; Errington, J. Control of cell shape in bacteria: Helical, actin-like ﬁlaments in Bacillus subtilis. Cell 2001, 104, 913–922. [CrossRef] 2. van den Ent, F.; Amos, L.A.; Lowe, J. Prokaryotic origin of the actin cytoskeleton. Nature 2001, 413, 39–44. [CrossRef][PubMed] 3. Doi, M.; Wachi, M.; Ishino, F.; Tomioka, S.; Ito, M.; Sakagami, Y.; Suzuki, A.; Matsuhashi, M. Determinations of the DNA-sequence of the MreB-gene and of the Gene-products of the Mre-region that function in formation of the rod shape of Escherichia coli cells. J. Bacteriol. 1988, 170, 4619–4624. [CrossRef] 4. Wachi, M.; Matsuhashi, M. Negative control of cell-division by MreB, a gene that functions in determining the rod shape of Escherichia coli cells. J. Bacteriol. 1989, 171, 3123–3127. [CrossRef][PubMed] Int. J. Mol. Sci. 2019, 20, 1304 13 of 14

5. Soufo, H.J.D.; Graumann, P.L. Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins. Curr. Biol. 2003, 13, 1916–1920. [CrossRef][PubMed] 6. Kruse, T.; Gerdes, K. Bacterial DNA segregation by the actin-like MreB protein. Trends Cell Biol. 2005, 15, 343–345. [CrossRef] 7. Kruse, T.; Moller-Jensen, J.; Lobner-Olesen, A.; Gerdes, K. Dysfunctional MreB inhibits chromosome segregation in Escherichia coli. EMBO J. 2003, 22, 5283–5292. [CrossRef] 8. Gital, Z.; Dye, N.A.; Reisenauer, A.; Wachi, M.; Shapiro, L. MreB actin-mediated segregation of a specific region of a bacterial chromosome. Cell 2005, 120, 329–341. 9. Soufo, H.J.D.; Graumann, P.L. Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization. BMC Cell Biol. 2005, 6, 1–11. 10. Gitai, Z.; Dye, N.; Shapiro, L. An actin-like gene can determine cell polarity in bacteria. Proc. Natl. Acad. Sci. USA 2004, 101, 8643–8648. [CrossRef] 11. van den Ent, F.; Izore, T.; Bharat, T.A.M.; Johnson, C.M.; Loewe, J. Bacterial actin MreB forms antiparallel double filaments. Elife 2014, 3, 1–22. [CrossRef] 12. Kruse, T.; Blagoev, B.; Lobner-Olesen, A.; Wachi, M.; Sasaki, K.; Iwai, N.; Mann, M.; Gerdes, K. Actin homolog MreB and RNA polymerase interact and are both required for chromosome segregation in Escherichia coli. Genes Dev. 2006, 20, 113–124. [CrossRef] 13. Iwai, N.; Nagai, K.; Wachi, M. Novel S-benzylisothiourea compound that induces spherical cells in Escherichia coli probably by acting on a rod-shape-determining protein(s) other than penicillin-binding protein 2. Biosci. Biotechnol. Biochem. 2002, 66, 2658–2662. [CrossRef] 14. Awuni, Y.; Jiang, S.M.; Robinson, R.C.; Mu, Y.G. Exploring the A22-bacterial actin MreB interaction through molecular dynamics simulations. J. Phys. Chem. B 2016, 120, 9867–9874. [CrossRef] 15. Esue, O.; Cordero, M.; Wirtz, D.; Tseng, Y. The assembly of MreB, a prokaryotic homolog of actin. J. Biol. Chem. 2005, 280, 2628–2635. [CrossRef] 16. Esue, O.; Wirtz, D.; Tseng, Y. GTPase activity, structure, and mechanical properties of filaments assembled from bacterial cytoskeleton protein MreB. J. Bacteriol. 2006, 188, 968–976. [CrossRef] 17. Bean, G.J.; Amann, K.J. Polymerization properties of the Thermotoga maritima actin MreB: Roles of temperature, nucleotides, and ions. Biochemistry 2008, 47, 826–835. [CrossRef] 18. Mayer, J.A.; Amann, K.J. Assembly properties of the Bacillus subtilis actin, MreB. Cell Motil. Cytoskeleton 2009, 66, 109–118. [CrossRef] 19. Maisuradze, G.G.; Leitner, D.M. Free energy landscape of a biomolecule in dihedral principal component space: Sampling convergence and correspondence between structures and minima. Proteins Struct. Funct. Bioinf. 2007, 67, 569–578. [CrossRef] 20. Parke, C.L.; Wojcik, E.J.; Kim, S.; Worthylake, D.K. ATP hydrolysis in Eg5 kinesin involves a catalytic two-water mechanism. J. Biol. Chem. 2010, 285, 5859–5867. [CrossRef] 21. Grigorenko, B.L.; Nemukhin, A.V.; Shadrina, M.S.; Topol, I.A.; Burt, S.K. Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations. Proteins Struct. Funct. Bioinf. 2007, 66, 456–466. [CrossRef][PubMed] 22. Grigorenko, B.L.; Nemukhin, A.V.; Topol, I.A.; Cachau, R.E.; Burt, S.K. QM/MM modeling the Ras-GAP catalyzed hydrolysis of guanosine triphosphate. Proteins Struct. Funct. Bioinf. 2005, 60, 495–503. [CrossRef] 23. Popp, D.; Narita, A.; Maeda, K.; Fujisawa, T.; Ghoshdastider, U.; Iwasa, M.; Maeda, Y.; Robinson, R.C. Filament structure, organization, and dynamics in MreB sheets. J. Biol. Chem. 2010, 285, 15858–15865. [CrossRef] 24. Kim, S.Y.; Gitai, Z.; Kinkhabwala, A.; Shapiro, L.; Moerner, W.E. Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. Proc. Natl. Acad. Sci. USA 2006, 103, 10929. [CrossRef] 25. Aylett, C.H.S.; Löwe, J.; Amos, L.A. Chapter One—New insights into the mechanisms of cytomotive actin and tubulin filaments. Int. Rev. Cell Mol. Biol. 2011, 292, 1–71. 26. Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling. Bioinformatics 2006, 22, 195–201. [CrossRef] Int. J. Mol. Sci. 2019, 20, 1304 14 of 14

27. Cornell, W.D.; Cieplak, P.; Bayly, C.I.; Gould, I.R.; Merz, K.M., Jr.; Ferguson, D.M.; Spellmeyer, D.C.; Fox, T.; Caldwell, J.W.; Kollman, P.A. A second generation force ﬁeld for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 1995, 5179–5197. [CrossRef] 28. Jorgensen, W.L.; Chandrasekhar, J.; Madura, J.D.; Impey, R.W.; Klein, M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926–935. [CrossRef] 29. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G.A.; et al. Gaussian 09; Gaussian, Inc.: Wallingford, CT, USA, 2009. 30. Dupradeau, F.-Y.; Pigache, A.; Zaffran, T.; Savineau, C.; Lelong, R.; Grivel, N.; Lelong, D.; Rosanski, W.; Cieplak, P. The R.E.D. tools: Advances in RESP and ESP charge derivation and force ﬁeld library building. Phys. Chem. Chem. Phys. 2010, 12, 7821–7839. [CrossRef] 31. Wang, J.; Wang, W.; Kollman, P.A.; Case, D.A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graph. Model. 2006, 25, 247–260. [CrossRef] 32. Case, D.A.; Cheatham, T.E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K.M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R.J. The Amber biomolecular simulation programs. J. Comput. Chem. 2005, 26, 1668–1688. [CrossRef] 33. Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald - An N.log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993, 98, 10089–10092. [CrossRef] 34. Bussi, G.; Donadio, D.; Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 2007, 126, 1–7. [CrossRef][PubMed] 35. Shan, Y.; Arkhipov, A.; Kim, E.T.; Pan, A.C.; Shaw, D.E. Transitions to catalytically inactive conformations in EGFR kinase. Proc. Natl. Acad. Sci. USA 2013, 110, 7270. [CrossRef][PubMed]

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