Architecture and Assembly of a Divergent Member of the Parm

Architecture and Assembly of a Divergent Member of the ParM Family of Bacterial Actin-like Proteins□S Received for publication, November 15, 2010, and in revised form, February 17, 2011 Published, JBC Papers in Press, February 21, 2011, DOI 10.1074/jbc.M110.203828 Christopher R. Rivera‡, Justin M. Kollman§¶, Jessica K. Polka‡, David A. Agard§¶, and R. Dyche Mullins‡1 From the Departments of ‡Cellular and Molecular Pharmacology and §Biochemistry and Biophysics and ¶The Howard Hughes Medical Institute, University of California, San Francisco, California 94158

Eubacteria and archaea contain a variety of actin-like proteins dynamics differ significantly from each other and from conven- (ALPs) that form filaments with surprisingly diverse architec- tional actin in vitro (11, 14–16). A paradigm emerging from this tures, assembly dynamics, and cellular functions. Although work is that, unlike the eukaryotic actin cytoskeleton, whose there is much data supporting differences between ALP fami- architecture and function are determined by accessory factors, lies, there is little data regarding conservation of structure and each bacterial actin appears adapted to a specific function, with function within these families. We asked whether the filament unique properties that reduce its need for accessory factors. architecture and biochemical properties of the best-understood Given the diversity of the ALPs, we asked whether the bio- prokaryotic actin, ParM from plasmid R1, are conserved in a chemical properties we proposed to be important for the cellu- divergent member of the ParM family from plasmid pB171. Pre- lar function of one actin-like protein, ParM from the R1 plas- Downloaded from vious work demonstrated that R1 ParM assembles into fila- mid, are conserved across the entire ParM family. R1 ParM is ments that are structurally distinct from actin and the other the best understood bacterial actin (17), and it drives plasmid characterized ALPs. They also display three biophysical proper- segregation in Gram-negative enteric pathogens by forming a ties thought to be essential for DNA segregation: 1) rapid spon- polymerization-based motor (10, 18) that pushes plasmids to

taneous nucleation, 2) symmetrical elongation, and 3) dynamic opposite poles of rod-shaped cells (19). We previously identi- http://www.jbc.org/ instability. We used microscopic and biophysical techniques fied three properties that appear to be essential to the cellular to compare and contrast the architecture and assembly of function of ParM and reduce its requirement for accessory fac- these related proteins. Despite being only 41% identical, R1 tors. These properties are as follows: 1) a stochastic switch and pB171 ParMs polymerize into nearly identical filaments between growth and shrinking, called dynamic instability, 2) with similar assembly dynamics. Conservation of the core symmetrical filament elongation, and 3) rapid spontaneous assembly properties argues for their importance in ParM-me- nucleation (18, 20). by guest on May 14, 2020 diated DNA segregation and suggests that divergent DNA- Two recently characterized bacterial ALPs assemble into segregating ALPs with different assembly properties operate structures that look very different from both ParM and conven- via different mechanisms. tional actin filaments. The first, an ALP from plasmid pSK41, was identified initially as a potential member of the ParM family (10). Its sequence similarity to R1 ParM (18%), however, is Prokaryotes were long believed to lack cytoskeletons, but below the 20% cut-off proposed by Derman et al. (6) for defin- recent work demonstrates that eubacteria and archaea use ing ALP families, and its atomic structure appears more closely actin-like filaments, tubulin-related polymers, and intermedi- related to that of an archeal actin, Ta0583, from Thermoplasma ate filaments to control cellular shape (1), divide (2), establish acidophilum. Perhaps not surprisingly, pSK41 ALP assembles order in the cytoplasm (3, 4), and move intracellular cargo (5). into filaments with strikingly different architecture and assem- To understand the evolution of these bacterial cytoskeletal sys- bly dynamics than R1 ParM; it forms one-strand helical fila- tems, we must understand both their diversity and the struc- ments, which are very different from the two-stranded R1 ParM tural and functional relationships between them. A recent filaments. Nucleation of these filaments is slower than that of sequence analysis (6) identified 41 families of actin-like pro- R1 ParM and elongation proceeds from a dimeric rather than a teins (ALPs)2 in eubacteria and archaea. Seven are known to trimeric nucleus. Finally, and most interestingly, the pSK41 form filaments, and their functions include controlling cell wall ALP filaments are not dynamically unstable (15). synthesis (MreB) (7), segregating DNA (ParM, AlfA, Alp7A, The second ALP, AlfA, also is a plasmid-segregating actin and pSK41 ParM) (6, 8–10), and aligning organelles (MamK) with little sequence homology to R1 ParM (15% identity). It also (3, 4). Five ALPs, MreB, ParM, Ta0583, AlfA, and Psk41 ALP, forms unique filaments that bundle spontaneously and lack have been studied in vitro (11–15). Their architectures and dynamic instability (14). These findings, especially the differences in polymer assembly dynamics, invite the intriguing con- □ clusion that different ALP families partition plasmid DNA via S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7 and additional information. distinct mechanisms. These results also suggest an important 1 To whom correspondence should be addressed: Department of Cellular and question; how well conserved are the biochemical and biophys- Molecular Pharmacology, University of California, San Francisco, CA 94158. ical properties of more closely related ALPs, especially as indi- Tel.: 415-502-4838; Fax: 415-502-4838; E-mail: [email protected]. 2 The abbreviations used are: ALP, actin-like protein; AMP-PNP, adenosine vidual ALP families can be more diverse than the entire family 5Ј-(␤,␥-imino)triphosphate; TIRF, total internal reflection fluorescence. of eukaryotic actins?

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To address this question, we purified and characterized an High Speed Sedimentation Assays actin-like protein encoded by the StbA gene from the Par1 We combined various concentrations of pB171 ParM with 10 operon of plasmid pB171 from enteropathogenic Escherichia mM nucleotide in buffer F (100 mM KCl, 30 mM Tris-HCl, pH 7.5,

coli (21, 22). R1 ParM and pB171 stbA share 41% identity and 1mM MgCl2,1mM DTT). For experiments with ATP and GTP, an 52% similarity. Because this level of conservation is within the additional 10 mM MgCl2 was added to the reactions. The reactions cut-off proposed by Derman et al. (6) for prokaryotic ALP fam- were then immediately centrifuged for 15 min at 355,000 ϫ g at ilies, we will refer to StbA as pB171 ParM. This level of conser- 25 °C in a Beckman Coulter TLA 120.1 rotor. The supernatants vation is, however, weak compared with that of eukaryotic were resolved on 4–12% precast gradient NuPAGE acrylamide actins and is more characteristic of the conservation between gels (Invitrogen) or on self-cast 13.75% SDS polyacrylamide gels. conventional actin and the eukaryotic actin-related proteins, The gels were stained with SYPRO Red (Invitrogen), scanned with which have different activities and cellular functions (23). Using a Typhoon 9400 variable mode imager (GE Healthcare), and quan- time-resolved light scattering, as well as electron and TIRF tified using ImageQuant TL software (GE Healthcare). The steady microscopy of single filaments, we asked whether the structure state monomer concentration was estimated as the x-intercept of and basic biophysical properties of R1 ParM are conserved in lines fit to a plot of the calculated amount of protein in the pellet pB171 ParM. versus the total initial protein. Etheno-ATP Binding and Nucleotide Competition Assays EXPERIMENTAL PROCEDURES Dissociation Rate Constant—Reaction mixtures containing Cloning, Expression, and Purification equimolar pB171 ParM and 1,N6-etheno-ATP (Invitrogen) in Downloaded from We PCR-amplified the pB171 ParM gene from a mini pB171 buffer Q (100 mM KCl, 30 mM Tris-HCl, pH 7.5, 1 mM MgCl2,1 plasmid with primers that appended a C-terminal GSKCK tag mM DTT, 200 mM acrylamide) were incubated for 15 min at for later use in maleimide labeling reactions and cloned it into a room temperature and then combined with equal volumes of 10 pET-11a vector (New England Biolabs, Ipswich, MA). We mM ATP in buffer Q using an SFA-20 rapid mixer (Hi-tech, transformed E. coli BL21 cells with the construct, grew them at Bradford-on-Avon, UK). We monitored the fluorescence at 420 37 °C to an optical density of 0.7 at 600 nm, and induced with nm (excitation, 315 nm) over time with a K2 fluorimeter (ISS, http://www.jbc.org/ Champagne, IL) and fit exponential decay functions to the data 0.75 mM isopropyl-␤-D-thiogalactopyranoside for 3–5 h. We to estimate the dissociation rate constant. harvested bacterial pellets via centrifugation and flash froze Association Rate Constant—We mixed equal volumes of 1 them in liquid N2. We purified pB171 ParM-GSKCK using the ␮M pB171 ParM in with a range of concentrations of etheno- same protocol as for R1 ParM (20) with the following modifica-

ATP in buffer Q using the rapid mixer and recorded the fluo- by guest on May 14, 2020 tion; a 0–20% ammonium cut was used to precipitate the pB171 rescence over time. The observed rate constants were esti- ParM protein out of the clarified bacterial extract as the initial mated by fitting exponential rise functions to the data and purification step. R1 ParM-GSKCK was expressed and purified plotted versus the etheno-ATP concentrations. The slope of a as described previously (20). line fit to the plot estimated the association rate constant (kon) and the y-intercept provided a second estimate of the disasso- Electron Microscopy and Image Analysis ciation rate constant (koff). ␮ pB171 and R1 ParM were polymerized with 5 mM nucleotide Affinity Constants (Kd)—A 1.6 M ParM-etheno-ATP-buffer for 5 min and then prepared by negative staining as described Q solution was mixed with equal volumes of a range of concen- (24). Samples were imaged on a Tecnai T12 microscope oper- trations of ATP and GTP. Following a 15-min incubation at ating at 120 kV at 62,000ϫ magnification. Images were room temperature, the fluorescence of the individual reaction recorded on a Gatan Ultrascan 4,000 ϫ 4,000 CCD camera, at a mixtures was measured. We fit a four-parameter logistic func- pixel size of 1.72 Å. The defocus of each micrograph was deter- tion to a plot of the percentage of relative binding versus the mined using the CTFFIND program (25), and the entire micro- concentration of competitor nucleotide to estimate the IC50 graph was corrected by phase flipping. and converted the IC50 values to a Ki using the online IC50-to-Ki Three-dimensional reconstructions of both pB171 and R1 converter tool (BotDB Database (28)). ParM were performed by iterative helical real space reconstruc- Bulk Polymerization and Phosphate Release Assays tion, as described (26). A total of 5006 pB171 ParM filament For the bulk polymerization assays, we rapidly mixed a range segments 260 Å in length were used in an initial reconstruction. of concentrations of pB171 ParM with equal volumes of 10 mM Heterogeneity of the helical symmetry within the dataset was MgCl2-ATP or MgCl2-GTP in buffer F and recorded the right sorted by comparison with a series of references with different angle light scattering intensity over time with an excitation helical symmetries, as described for R1 ParM (27). The largest wavelength of 314 nm. Each trace for a particular concentration class from this analysis, corresponding to particles with an azi- is the average of five or more runs performed on the same day. muthal angle of 166.2°, had 1111 helical segments. This class For the assays with varied nucleotide, we rapidly mixed 10 ␮M was used in an independent reconstruction, yielding the final pB171 ParM in buffer F with equal volumes of a dilution series structure at a 19 Å resolution. The R1 ParM reconstruction of ATP or GTP and recorded the right angle light scattering used 4799 helical segments and did not require classification by over time. We measured phosphate release by 5 ␮M ParM helical symmetry. polymerized with ATP or GTP in buffer F using the EnzChek

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FIGURE 1. pB171 ParM forms filaments in AMP-PNP (A), ATP (B), and GTP (C). pB171 ParM (9.5 ␮M) was polymerized with 5 mM nucleotide, stained with 0.75% uranyl formate, and visualized by transmission EM. The conditions used were as follows: 100 mM KCl, 30 mM Tris-HCl, pH 7.5, 1 mM MgCl2,1mM DTT, 25 °C. Scale bar, 50 nm.

phosphate assay kit (Invitrogen) with an Ultrospec 2100 Pro spec- FIGURE 2. EM reconstruction of pB171 ParM filaments. A, the pB171 ParM tophotometer controlled with SWIFT II software (GE Health- filament structure, calculated from a subset of filament segments that con- care). A values were converted to inorganic phosphate concen- verged to helical symmetry of 166.1° rotation and 24.2 Å rise per subunit. The 360 map is filtered to 19 Å, the estimated resolution of the reconstruction. Each tration by using a phosphate standard and parallel right angle light filament strand is rendered in a different color. B, for comparison, a recon- scattering assays were performed on the same day. struction of R1 ParM filaments was calculated, with a refined helical symmetry Downloaded from of 165.4° rotation and 24.5 Å rise per subunit. C, the crystal structure of R1 pB171 ParM Labeling and TIRF Microscopy ParM was manually fit into the EM structure of the pB171 ParM filament, and the fit along a single strand is shown. For labeling reactions, monomeric pB171 ParM was combined with Alexa 488-maleimide (Invitrogen) at a 1:1.6 molar trast, R1 ParM filaments assembled in AMP-PNP yielded a sta-

ratio in buffer F lacking DTT for 30 min at 4 °C. The reactions ble solution after ϳ20 iterations (supplemental Fig. S1A). The http://www.jbc.org/ were quenched by the addition of 10 mM DTT, and the protein helical twist of R1 ParM filaments has been shown to be some- was separated from free dye by gel filtration. The labeling effi- what variable (16, 27), and we interpret the failure of pB171 ciency was 80–100%. ParM images to produce a stable reconstruction as evidence To monitor single filament polymerization dynamics, we that the variation in angles between protomers in these fila- directly applied 2.7 ␮l of 25% Alexa 488-labeled pB171 ParM in ments is even higher. by guest on May 14, 2020

TIRF buffer (100 mM KCl, 15 mM Tris-HCl, 1 mM MgCl2,1mM To deal with these heterogeneities, we performed multiref- DTT, 0.8% methylcellulose, 0.5% BSA) and 0.3 ␮l of 100 mM erence classification of the data set using nine models with dif- ATP or AMP-PNP to ethanol-base washed coverslips and per- ferent helical symmetries. The largest class, which contained formed time-lapse TIRF microscopy using a Nikon Eclipse 20% of the entire data set, corresponded to an azimuthal rota- TE2000-E inverted microscope equipped with an Andor iXonϩ tion of 166.1° between adjacent protomers. In an independent EM digital camera and a 40-milliwatt 488/514 argon ion laser. reconstruction performed using only this class of the data, Data were analyzed with ImageJ software (29). helical symmetry converged from different initial values to the same solution after ϳ10 iterations. Following initial conver- Sequence Alignments and Phylogenetic Analysis gence, however, the azimuthal rotations oscillated between Representative actin and ParM sequence were identified 166.0° and 166.25° in subsequent iterations, suggesting some using BLASTP on the NCBI website. The sequences were degree of twist heterogeneity even within this class (supple- aligned using the MUSCLE global alignment algorithm (30) and mental Fig. S1C). The final structure of pB171 ParM, with an the Jalview alignment editor (31). Phylogenetic analysis was estimated resolution of 19 Å (supplemental Fig. S1D), closely performed with the MEGA4 software (32) using the Neighbor resembles both the present and previously reported structures joining (33) and Bootstrap (34) methods. of AMP-PNP R1 ParM filaments (Fig. 2A) (27). We fit the atomic structure of ADP-bound R1 ParM (12) into RESULTS our pB171 ParM AMP-PNP reconstruction without steric Comparing Structures of R1 and pB171 ParM Filaments— clashes. The inter- and intrastrand contacts between protomers Using electron microscopy, we examined negatively stained are nearly identical to the model of Galkin et al. (27) (Fig. 2C). pB171 ParM filaments polymerized with AMP-PNP, ATP, and Nucleotide Binding and Sedimentation Assays Demonstrate GTP. Under all conditions, we observed well separated, heli- That pB171 ParM Binds More Tightly to ATP, but Is More Sta- cally wound filaments composed of two strands (Fig. 1, A–C). ble in GTP—We determined the distribution of pB171 ParM Filaments formed in AMP-PNP (Fig. 1A) were longer than between monomeric and polymeric states using high speed those formed in either ATP or GTP (Fig. 1, B and C). centrifugation. Similar to other actin-like proteins, assembly of Initial attempts to construct a high resolution model of pB171 ParM into filaments required nucleotide triphosphates

pB171 ParM filaments in AMP-PNP using iterative helical real (either ATP or GTP) and was promoted by MgCl2. ATP-ParM space reconstruction (26) failed to converge to a stable solution, polymerization was reduced in the absence of added MgCl2 and even after 60 refinement cycles (supplemental Fig. S1B). In con- inhibited by 1 mM EDTA. Like other actin-like proteins, pB171

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FIGURE 3. pB171 ParM binds preferentially to ATP over GTP. A, pseudo-first order association kinetics of pB171 ParM binding to etheno-ATP. The slope of the line estimates the second order association rate constant and the y intercept estimates the first order disassociation rate constant. The inset shows ␮ ␮ representative association curves for 75 M and 13.5 M 1,N6-etheno-ATP. Buffer conditions were as follows: 100 mM KCl, 30 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1mM DTT, 200 mM acrylamide. B, competitive binding experiments of 1,N6-etheno-ATP versus ATP and GTP for pB171 ParM. The data were fit to a four-

parameter logistic curve to estimate the IC50. Buffer conditions were the same as above. Downloaded from

ParM polymerized poorly in the presence of CaCl2. A larger ble) polymer, we can convert it into a one-state polymer using fraction of the pB171 protein pelleted in GTP than ATP in all point mutants or nonhydrolyzable nucleotide analogs. Under ␮ conditions except buffer containing CaCl2. In this condition, either condition, R1 ParM has a critical concentration (0.6 M) pelleting was identical in ATP and GTP (supplemental Fig. S2, governed by Equation 1. We will call this the ATP critical con-

ATP http://www.jbc.org/ A and B). centration (mcc ). R1 ParM is unstable in ADP and has an ADP ␮ Actins and the prokaryotic ALPs studied to date bind to and ADP critical concentration (mcc ) greater than 120 M. Wild polymerize in the presence of ATP and GTP with varying effi- type R1 ParM filaments in the presence of ATP can switch from ciencies (13–15, 27, 35–37). Stopped-flow experiments indi- stable elongation to rapid depolymerization, and the measured cate that pB171 ParM binds the fluorescent ATP analog, 1,N6- steady state monomer concentration under these conditions is 3 Ϫ1 Ϫ1 etheno-ATP with a rate constant of 25.8 Ϯ 1.1 ϫ 10 s M , 2.3 ␮M. Because this monomer concentration reflects the by guest on May 14, 2020 and the pB171 ParM-etheno-ATP complex disassociated with a behavior of two filament populations, each with a different crit- Ϯ Ϫ1 rate constant of 0.368 0.142 s , corresponding to a Kd of ical concentration, we will refer to it simply as the steady state Ϯ ␮ 14.2 5.5 M (Fig. 3A). Competition binding experiments monomer concentration (mss) of the polymer. If we assume that ATP between etheno-ATP and either ATP or GTP indicated that the reason mss is greater than mcc is because at steady state ϭ pB171 ParM has a significantly higher affinity for ATP (Kd some fraction of filaments (r) have ATP caps at their ends and Ϯ ␮ ϭ Ϯ ␮ Ϫ 2.7 1.2 M) than GTP (Kd 114.4 33.4 M) (Fig. 3B). are governed by Equation 1, whereas the rest (1 r) have ADP- Assuming intracellular ATP and GTP concentrations of 9.4 and bound protomers at one or both ends and are catastrophically

4.9 mM (38), respectively, and ignoring the presence of other shortening at a rapid rate (ks), then the steady state monomer nucleotide binding proteins, the measured affinities suggest concentration is given by Equation 3. that, in vivo, 98.8% of pB171 ParM is bound to ATP, and 1.2% is kϪ k 1 bound to GTP. ͓ ͔ ϭ ϩ S ͩ Ϫ ͪ mSS 1 (Eq. 3) For quantitative comparison of cytoskeletal polymers, we kϩ kϩ r will define the following three terms: critical concentration, steady state monomer concentration, and instability ratio. We Using parameters measured for R1 ParM we calculate that, at define a critical concentration only for single-state polymers. steady state, 88% of filaments are stable and 12% are shrinking. Briefly, if polymer assembly is governed by, Finally, for polymers whose stability changes upon nucleotide hydrolysis, the ratio of the critical concentration in nucle- dP/dt ϭ kϩ͓m͔͓e͔ Ϫ kϪ͓e͔ (Eq. 1) otide diphosphate (ADP or GDP) over that in nucleotide triphosphate (ATP or GTP) is a convenient measure of dynamic where kϩ and kϪ are rate constants for monomer association instability. We will call this the “instability ratio” of the polymer. and dissociation, and [m] and [e] are concentrations of mono- For example, actin, which is not generally considered dynami- mer and filament ends, then the critical concentration is cally unstable, has an instability ratio of 1.6 (39). AlfA, which defined as the monomer concentration at which polymer nei- segregates DNA in Bacillus cells, has a similar instability ratio of ther grows nor shrinks. 4.2 (14). In contrast, dynamically unstable R1 ParM filaments Ͼ kϪ have an instability ratio 160 (20). ͓m͔ ϭ (Eq. 2) Finally, sedimentation assays indicated that pB171 ParM has kϩ GTP ϭ a lower steady state monomer concentration in GTP (mss Ϯ ␮ ATP Ϯ ␮ Although R1 ParM is normally a two-state (dynamically unsta- 1.1 0.21 M) than in ATP (mss 1.5 0.12 M). We find

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that like R1 ParM, pB171 ParM has an instability ratio Ͼ140 mer, followed by a brief decrease, and then a slower approach to (Table 1), in both adenosine and guanosine nucleotides. steady state (Fig. 4A). pB171 ParM also rapidly assembled in Rapid Nucleation and Nucleotide Hydrolysis by pB171 ParM— GTP (Fig. 4B). However, the traces lacked the middle phase For linear helical polymers such as actin and R1 ParM, filament observed in ATP. assembly is governed by several parameters that include To estimate the nucleus size and nucleation rate, we plot- nucleus size, nucleation rate, elongation rate, stability of the ted the intensity-normalized maximum polymerization rate polymer in subsaturating nucleotide, and rates of nucleotide versus the protein concentration on a log-log plot and fitted hydrolysis and phosphate release. Using right angle light scat- a line to the transformed data (14, 20, 40). Using this method, we tering, pB171 ParM rapidly assembles in the presence of ATP, estimate that the size of the nucleus, the last unstable interme- suggesting that nucleation is fast. The ATP-pB171 ParM diate in the filament assembly pathway, for pB171 ParM fila- assembly curves have three phases: an initial increase in poly- ments is a dimer in ATP and GTP (Fig. 4C). In contrast, assem- TABLE 1 bly of actin and R1 ParM begins with creation of a trimeric pB171 ParM steady state monomer concentrations nucleus (20, 41). Overall, our analysis indicates that pB171 The steady state monomer concentrations were determined by sedimentation assays. ParM filaments assemble spontaneously much more quickly Steady-state monomer than actin filaments. Nucleotide concentration We also used the method of Flyvbjerg et al. (42) to estimate ␮M the nucleus size from early time points of our light scattering ATP 1.5 Ϯ 0.12 ADP Ͼ114 data. We normalized the amplitudes and times of light scatter- AMP-PNP 0.5 Ϯ 0.04 ing curves collected at different concentrations of pB171 ParM GTP 1.1 Ϯ 0.21 Downloaded from GDP Ͼ114 and observed that all of the ATP data collapsed on to one curve. GMP-PNP 0.77 Ϯ 0.1 The GTP data collapsed onto a similar curve, indicating that http://www.jbc.org/ by guest on May 14, 2020

FIGURE 4. Light scattering assays demonstrate that pB171 ParM nucleates and assembles rapidly in ATP and GTP. A and B, rapid assembly of pB171 ParM in 10 mM MgCl2-ATP (A) and 10 mM MgCl2-GTP (B) monitored by light scattering. Buffer conditions for all experiments in this figure were as follows: 100 mM KCl, 30 mM Tris-HCl, 1 mM MgCl2,1mM DTT. C, determination of the nucleus size and relative rates of nucleation in ATP and GTP. The log of the maximal rate of assembly (Vmax) normalized by the maximum light scattering intensity was plotted versus the log concentration of pB171 ParM. The nucleus size (n) is estimated as two times the slope of the linear fit, and the x-intercept is proportional to the square root of the nucleation rate constant times the elongation rate constant.

The error bars are the S.D. of calculated values from three separate experiments. D, normalized intensity (I(t)/Imax) plotted versus normalized time (t/t1⁄2) for the highest two concentrations of pB171 ParM in ATP (dark gray) and GTP (light gray). Inset, earliest time points (triangles, ATP; circles, GTP).

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gest that pB171 ParM filaments are more stable in GTP than ATP, because the rate of decay in 40 ␮M ATP was faster than the rate of decay in 40 ␮M GTP (supplemental Fig. S4G). Similar results were obtained for pB171 ParM polymerized with vari-

ous concentrations of MgCl2-ATP and MgCl2-GTP (supplemental Fig. S4, A–D). Phosphate release assays indicated that phosphate production lagged behind polymerization in low (0.1 mM) and high (1 mM) concentrations of ATP and GTP (Fig. 5, C–F) Surprisingly, we observed similar rates of phosphate production in 1 mM ATP (0.577 Ϯ 0.001 ␮M/s) and 1 mM GTP (0.593 Ϯ 0.006 ␮M/s) at steady state. Consistent with the difference in measured affinities, steady state pB171 ParM phosphate production was greater in ATP (0.513 Ϯ 0.017 ␮M/s) than GTP (0.445 Ϯ 0.09 ␮M/s) at 0.1 mM nucleotide concentrations. TIRF Microscopy of Individual Filaments Confirm That pB171 ParM Elongates Symmetrically, Is Dynamically Unsta- ble, and That Nucleotide Hydrolysis Is Required for Dynamic Instability—Using time-lapse TIRF microscopy to monitor

individual filaments of 25% Alexa 488 labeled ParM polymer- Downloaded from ized in ATP, we observed many short individual filaments that diffused rapidly, binding only transiently to the coverglass before detaching. However, all filaments that remained attached to the coverslip surface for an extended period elon-

gated symmetrically from both ends prior to undergoing cata- http://www.jbc.org/ strophic depolymerization, which appeared to occur primarily from one end. No rescue events were observed; all filaments that we tracked either detached from the slide or completely depolymerized. We measured the rates of filament elongation (10.6 ϩ/4.9 monomers/s) and depolymerization following by guest on May 14, 2020 catastrophe (22.9 Ϯ 9.4 monomers/s) (Fig. 6, A and B), which are similar to those we measured previously for R1 ParM (20). To determine whether nucleotide hydrolysis regulates FIGURE 5. Light scattering and phosphate release assays demonstrate dynamic instability in pB171 ParM, we performed TIRF that pB171 ParM is unstable in limiting concentrations of nucleotide and rapidly hydrolyzes nucleotide and releases phosphate. A and B, assembly microscopy on various concentrations of pB171 ParM poly- of pB171 ParM in various concentrations of ATP (A) and GTP (B). C–F, phos- merized in AMP-PNP. At all concentrations tested, the AMP- ␮ phate release assays. The amount of phosphate released by 5 M pB171 ParM PNP filaments appeared to elongate from both ends and attain polymerized in 1 mM ATP (C),1mM GTP (D), 0.1 mM ATP (E), or 0.1 mM GTP (F) are plotted versus time (closed squares). Parallel assembly reactions were lengths much greater than ATP filaments (Fig. 6, C and D). monitored with light scattering using the same stock protein solutions (closed Although filament fragmentation occurred, we observed no circles). Buffer conditions for all experiments in this figure are the same as those used for the experiment in Fig. 4. examples of catastrophe. We occasionally observed two types of bundling behaviors that appeared to be length dependent: 1) pB171 ParM assembles via the same mechanism in ATP and lateral binding of a smaller filament to the center of a longer GTP (Fig. 4D) but with different rate constants. To estimate the filament and 2) the collision of two long filament ends that lead nucleus size, we plotted the normalized data for the earliest to filament zippering. We measured an elongation rate con- Ϫ1 Ϫ1 time points on log-log plots. The slope for the earliest time stant in AMP-PNP of 2.3 monomers ϫ s ␮M per filament points reflects the number of kinetic steps in nucleation (42). end and a noncatastrophic depolymerization rate constant of This analysis indicated that nucleus formation occurs in one 0.6 monomers per filament end per second (Fig. 6E). Using step in both ATP and GTP, further indicating that the pB171 these parameters, together with the steady state monomer con- ParM nucleus is a dimer (supplemental Fig. S3). centrations in ATP and AMP-PNP, we calculate from Equation To determine the stability of pB171 ParM in limiting concen- 2 that, at steady state, 89% of the pB171 ParM filaments are trations of nucleotide, we polymerized 10 ␮M ParM in the pres- stable and growing, whereas 11% are unstable and shrinking ence of varying concentrations of ATP and GTP. Following an when polymerized in ATP. Comparing this steady state behav- initial rapid polymerization, pB171 ParM filaments depolymer- ior to R1 ParM indicates that the two polymers maintain a sim- ized when assembled in limiting concentrations of ATP and ilar balance between nucleation, growth, and catastrophe. GTP (Fig. 5, A and B). Consistent with our measurements of nucleotide affinity, the initial rates of pB171 ParM polymeriza- DISCUSSION tion were more sensitive to limiting concentrations of GTP Eukaryotic actins are highly conserved, probably due to the than ATP (supplemental Fig. S4, E and F). These data also sug- large number of conserved binding partners that regulate their

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FIGURE 6. Time-lapse TIRF microscopy observing individual filaments demonstrate that pB171 ParM is dynamically unstable when polymerized in ATP and appears to elongate symmetrically. A, montage of an individual pB171 ParM filament in ATP. 25% Alexa 488-labeled 2.8 ␮M B171 ParM was polymerized in the presence of 10 mM ATP and imaged via TIRF microscopy every 2 s. Buffer conditions were as follows: 100 mM KCl, 30 mM KCl, 1 mM MgCl2,1 mM DTT, 0.8% methylcellulose, 0.5% BSA. Scale bar,1␮m. B, length versus time for six representative filaments polymerized in ATP. C and D, montage of individual filaments polymerized in nonhydrolyzable AMP-PNP. 25% Alexa 488-labeled 0.8 ␮M or 1.0 ␮M pB171 ParM was polymerized in the presence of 10 mM AMP-PNP. The time interval is 20 s between each frame. Buffer conditions are the same as in ATP. Scale bar,1␮m. E, rate of elongation of AMP-ParM. The rate by guest on May 14, 2020 of elongation was measured at various concentrations of pB171 ParM in AMP-PNP and plotted versus the ␮M pB171 ParM. The line fit to the data represents the ϭ ␮ ␮ equation: rate of filament elongation kon x( M protein)-koff. Inset shows three representative filaments growth over time from 0.6, 0.8, and 1.0 M pB171 concentrations. assembly and function. Across metazoan species, for example, actin sequences are ϳ98% identical. The primary sequences of protozoan actins are more variable but the level of sequence conservation is still much greater than that observed in bacterial actin families. Bacterial actin families are, in fact, much less well defined than eukaryotic actins. The majority of known bacterial actins have been identified by genome searches and the homology cut-off proposed by Derman et al. for defining families (6) is more or less arbitrary. We were interested in deter- mining whether one particular clade of bacterial actins, the Alp3 or ParM group, represents a bona fide family with conserved structure and activity. As Fig. 7 demonstrates, the ParM family is quite divergent in comparison with the eukaryotic FIGURE 7. Evolutionary relationships demonstrate that the ParM fam- actins. ily is more divergent than eukaryotic actins. The tree represents a boot- The ParM protein, encoded by the R1 plasmid is, to date, the strap consensus tree inferred from 1000 replicate trees generated using most well characterized bacterial ALP (17). It polymerizes into the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next left-handed, double-stranded, helical filaments that nucleate to the branches. The branch lengths are proportional to the relative evo- rapidly, elongate symmetrically, and are dynamically unstable lutionary distance, and the scale bar is in units of the number of amino acid (20). Here, we show that ParM from the Par1 locus of pB171, substitutions per site. The GI numbers for the sequences and the sequence alignments used to generate this phylogram are provided in the supple- although only 41% identical to R1 ParM, polymerizes via a sim- mental material. Evolutionary analyses were conducted in MEGA4. ilar pathway into filaments that have a remarkably similar structure. Using electron microscopy, we found that pB171 (MreB, AlfA, or the pSK41 ALP). Light scattering assays dem- ParM monomers build filaments that are more similar to R1 onstrated that pB171 ParM filaments rapidly and spontane- ParM than to actin or any other characterized actin-like protein ously form filaments in the absence of nucleation factors in

14288 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 16•APRIL 22, 2011 Architecture and Assembly of a Member of the ParM Family

both ATP and GTP. Sedimentation assays demonstrated that that it promotes plasmid partitioning via a very different mech- pB171 ParM does not polymerize in ADP or GDP, and light anism. Our investigation and that of Popp et al. highlight the scattering assays revealed that pB171 ParM filaments rapidly importance of careful structural and functional studies in defin- depolymerize in limiting amounts of ATP and GTP prior to ing families of bacterial actin-like proteins. complete nucleotide hydrolysis. This is consistent with our pre- vious observation that substoichiometric ratios of ADP to ATP REFERENCES (ϳ20%) destabilize R1 ParM filaments, regardless of the total 1. Jones, L. J., Carballido-Lo´pez, R., and Errington, J. (2001) Cell 104, ATP concentration (20). For both R1 and pB171 ParM, we 913–922 attribute this effect to nucleotide exchange on the terminal sub- 2. Erickson, H. P. (1995) Cell 80, 367–370 3. Komeili, A., Li, Z., Newman, D. K., and Jensen, G. J. (2006) Science 311, unit of the filament, a phenomenon initially observed for actin 242–245 filaments (43). Finally, TIRF microscopy of labeled pB171 ParM 4. Pradel, N., Santini, C. L., Bernadac, A., Fukumori, Y., and Wu, L. F. (2006) revealed both symmetrical filament elongation and dynamic Proc. Natl. Acad. Sci. U.S.A. 103, 17485–17489 instability. 5. Møller-Jensen, J., Borch, J., Dam, M., Jensen, R. B., Roepstorff, P., and In addition to the basic similarities, we also note three minor Gerdes, K. (2003) Mol. Cell 12, 1477–1487 differences between the biochemical and biophysical proper- 6. Derman, A. I., Becker, E. C., Truong, B. D., Fujioka, A., Tucey, T. M., Erb, M. L., Patterson, P. C., and Pogliano, J. (2009) Mol. Microbiol. 73, ties of ParM proteins from plasmids R1 and pB171. First, the 534–552 structure of pB171 ParM filaments is more heterogeneous. This 7. Daniel, R. A., and Errington, J. (2003) Cell 113, 767–776 is shown mainly as variability in the degree of helical twist of the 8. Becker, E., Herrera, N. C., Gunderson, F. Q., Derman, A. I., Dance, A. L., strands that compose the filament. We hypothesize that this Sims, J., Larsen, R. A., and Pogliano, J. (2006) EMBO J. 25, 5919–5931

reflects weaker lateral interactions between the strands that 9. Schumacher, M. A. (2008) Biochem. J. 412, 1–18 Downloaded from permit them to either rotate or slip more freely. Second, we find 10. Møller-Jensen, J., Jensen, R. B., Lo¨we, J., and Gerdes, K. (2002) EMBO J. 21, 3119–3127 that pB171 ParM is slightly more stable in GTP than ATP, as 11. van den Ent, F., Amos, L. A., and Lo¨we, J. (2001) Nature 413, 39–44 evidenced by the lower steady state monomer concentration. 12. van den Ent, F., Møller-Jensen, J., Amos, L. A., Gerdes, K., and Lo¨we, J. Increased stability in GTP may reflect an increase in the rate of (2002) EMBO J. 21, 6935–6943

polymerization, a decrease in the rate of depolymerization of 13. Hara, F., Yamashiro, K., Nemoto, N., Ohta, Y., Yokobori, S., Yasunaga, T., http://www.jbc.org/ stable or unstable filament ends or a decrease in the propensity Hisanaga, S., and Yamagishi, A. (2007) J. Bacteriol. 189, 2039–2045 14. Polka, J. K., Kollman, J. M., Agard, D. A., and Mullins, R. D. (2009) J. of GTP filaments to undergo catastrophe due to a reduced rate Bacteriol. 191, 6219–6230 of nucleotide hydrolysis or phosphate release. Although our 15. Popp, D., Xu, W., Narita, A., Brzoska, A. J., Skurray, R. A., Firth, N., Gos- analysis did not identify the mechanism of the increased stabil- hdastider, U., Mae´da, Y., Robinson, R. C., and Schumacher, M. A. (2010) ity in GTP, our phosphate release assays suggests that pB171 J. Biol. Chem. 285, 10130–10140 by guest on May 14, 2020 ParM filaments hydrolyze GTP at similar rates as ATP. Third, 16. Orlova, A., Garner, E. C., Galkin, V. E., Heuser, J., Mullins, R. D., and the size of the pB171 ParM filament nucleus is a dimer in ATP Egelman, E. H. (2007) Nat. Struct. Mol. Biol. 14, 921–926 17. Needleman, D. J. (2008) Curr. Biol. 18, R212–214 and GTP, whereas the apparent size of the R1 ParM nucleus is a 18. Garner, E. C., Campbell, C. S., Weibel, D. B., and Mullins, R. D. (2007) trimer (20, 27). This probably reflects slight differences in one Science 315, 1270–1274 of the monomer-monomer interfaces. Depending on buffer 19. Campbell, C. S., and Mullins, R. D. (2007) J. Cell Biol. 179, 1059–1066 conditions, the nucleus size of conventional actin ranges from 20. Garner, E. C., Campbell, C. S., and Mullins, R. D. (2004) Science 306, two to four subunits (41). Osawa and Kansai (44) predicted that 1021–1025 all linear helical polymers would generally assemble from nuclei 21. Tobe, T., Hayashi, T., Han, C. G., Schoolnik, G. K., Ohtsubo, E., and Sa- sakawa, C. (1999) Infect. Immun. 67, 5455–5462 in this size range. 22. Ebersbach, G., and Gerdes, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, Popp et al. (15) recently characterized the structure and 15078–15083 assembly of a divergent ALP from plasmid pSK41, identified 23. Dion, V., Shimada, K., and Gasser, S. M. (2010) Curr. Opin. Cell Biol. 22, previously by Møller-Jensen et al. (10) as a possible member of 383–391 the ParM family. This ALP is, however, only 18% similar to R1 24. Ohi, M., Li, Y., Cheng, Y., and Walz, T. (2004) Biol. Proced. Online 6, 23–34 25. Mindell, J. A., and Grigorieff, N. (2003) J. Struct. Biol. 142, 334–347 ParM and falls below the proposed cut-off for definition of ALP 26. Egelman, E. H. (2000) Ultramicroscopy 85, 225–234 families. As noted above, however, this sequence similarity cri- 27. Galkin, V. E., Orlova, A., Rivera, C., Mullins, R. D., and Egelman, E. H. terion is fairly arbitrary and a more rigorous definition of fam- (2009) Structure 17, 1253–1264 ilies requires combining sequence similarity with structural and 28. Cer, R., Mudunuri, U., Stephens, R., and Lebeda, F. (2009) Nucleic Acids functional information. Filaments formed by the pSK41 ALP Res. 37, W441–W445 protein lack many of the longitudinal monomer-monomer con- 29. Abramoff, M., and Magelhaes, P. (2004) Biophotonics International 11, 36–42 tacts that define the two long-pitch helices found in actin and 30. Edgar, R. C. (2004) Nucleic Acids Res. 32, 1792–1797 ParM and these filaments are best described as single-stranded. 31. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M., and Barton, In addition, they are not dynamically unstable, and they do not G. J. (2009) Bioinformatics 25, 1189–1191 elongate symmetrically. Finally, and most intriguingly, the 32. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) Mol. Biol. Evol. 24, atomic structure of the pSK41 ALP is more similar to the 1596–1599 archeal actin Ta0583 from T. acidophilum than to R1 ParM. 33. Saitou, N., and Nei, M. (1987) Mol. Biol. Evol. 4, 406–425 34. Felsenstein, J. (1985) Evolution 39, 783–791 Popp et al. argue that the pSK41 protein represents an evolu- 35. Wen, K. K., Yao, X., and Rubenstein, P. A. (2002) J. Biol. Chem. 277, tionary intermediate between a chromosomally encoded ALP 41101–41109 and the plasmid-encoded ParM-family proteins and suggested 36. Esue, O., Wirtz, D., and Tseng, Y. (2006) J. Bacteriol. 188, 968–976

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14290 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 16•APRIL 22, 2011 Architecture and Assembly of a Divergent Member of the ParM Family of Bacterial Actin-like Proteins Christopher R. Rivera, Justin M. Kollman, Jessica K. Polka, David A. Agard and R. Dyche Mullins J. Biol. Chem. 2011, 286:14282-14290. doi: 10.1074/jbc.M110.203828 originally published online February 21, 2011

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by guest on May 14, 2020 Supplementary Information: The phylogenetic analysis in Figure 7 was performed using the following sequences: Sequence name: GI accession number. ParM sequences: V. furnissii CIP 102972: 260768794 P. ingrahamii 37: 119947163 K. pneumoniae MGH 78578: 152973663 E. coli plasmid pB171:10955418 S. dysenteriae Sd197: 82524481 A. nasoniae: 284008122 K. pneumoniae 342: 206581098 E. coli plasmid R1: 9507578 E. coli prophage CP-933T: 91209978 S. enterica plasmid R64: 32470180 M. extorquens AM: 240141815 PsK41 ALP S. aureus Psk41: 284793988 Actin Sequences N. gruberi: 290974733 T. vaginalis: 123444869 P. caudatum: 15212111 T. gondii: 606857 S. cerevisiae: 170986 H. sapiens alpha skeletal: 4501881 C. elegans: 51011295 D. discoideum: 66804711 A. thaliana: 1002533

Note: supplementary Figures 5,6,7 are not mentioned explicitly in the manuscript; however, they are the alignments that were used in the generation of the phylogram in Figure 7.

Supplementary Figure Legends

Supplementary Figure 1: Variability in twist states and filament resolution of pB171 ParM IHRS reconstruction. (A) The global reconstruction of R1 ParM filament in AMP-PNP rapidly converged to a stable solution. (B) The global reconstruction of pB171 ParM AMP-PNP failed to converge to stable solution. (C) A reconstruction based on a single class of pB171 filament particles converged to an oscillating solution. (D) The resolution of the final pB171 ParM AMP- PNP reconstruction is estimated to be 19 Å.

Supplementary Figure 2: pB171 ParM polymerization is enhanced by divalent cations and requires nucleotide. (A) High-speed sedimentation assay with pB171 ParM polymerized in different buffer conditions in the presence or absence of either ATP or GTP. The bands are from the supernatant fraction of the sedimentation reaction. Base buffer: 100 mM KCl, 15 mM Tris- HCl 1 mM DTT ± 1mM DTT, MgCl2, or CaCl2. Nucleotide concentration: 1 mM. (B) Quantification of sedimentation experiment from A. Bars: Black: buffer no nucleotide, Grey: buffer + ATP, White: buffer + GTP. Error bars are S.D. from two experiments. (C and D) Quantification of High-speed sedimentation assays measuring the steady state monomer concentration of pB171 ParM in different nucleotides. (C) 10 mM MgCl2 ATP (circles) or 10 mM MgCl2-GTP (triangles). (D) 10 mM AMP-PNP (circles) or GTP (triangles). Buffer conditions: 100 mM KCl, 15 mM Tris-HCl, 1mM MgCl2 , 1 mM DTT.

Supplementary Figure 3: Log-Log plots of the initial time points from normalized light scattering data of various concentrations of pB171 ParM polymerized in 10 mM MgCl2-ATP or MgCl2-GTP indicate that the nucleus size is two in both conditions. (A&B) The amplitudes and times of light scattering data from ParM polymerized in (A) ATP or (B) GTP were normalized by the maximum intensity and half times respectively. The initial 20 data points of 15 normalized data series were combined into a single data series and plotted on a log-log plot. The slope of the line fit to the combined data estimates the number of kinetic steps in nucleus formation; therefore the nucleus size is estimated as the slope+1. (C&D) Determination of the slope at the earliest time points provides an additional estimation of the number of kinetic steps in nucleus formation. The slopes of lines fit to the first n (5-20) data points from each normalized data series from (A) and (B) were determined using the MATLAB poly-fit function. The slopes of the lines were averaged. The average slope was plotted versus the number of points (i.e. the value at 10 points is the average slope of lines fit to the first 10 data points of each series and the value at 11 points is the average slope of lines fit to the first 11 points of each data series). A line was fit to the plot. The slope value when the number of points is extrapolated to 1 estimates the slope at the earliest time points of the reaction. This initial slope is equal to the number of kinetic steps in nucleus formation. Again, the nucleus size is equal to the number of kinetic steps +1. Error bars represent the standard deviation of the slopes calculated for n points.

Supplementary Figure 4: Plots of light scattering data from pB171 ParM polymerized with a range of nucleotide concentrations. (A and B). 10 uM pB171 ParM was polymerized with a range of concentrations of (A) MgCl2-ATP or (B) MgCl2 -GTP. The light scattering intensity was plotted versus time. Buffer conditions are the same as in Figure 5. (C and D). Fast data acquisition of light scattering data from the first ten seconds of the polymerization time courses of the same samples as in (A) MgCl2-ATP and (B) MgCl2 -GTP. (E and F). Fast data acquisition of light scattering data from the first ten seconds of the polymerization time courses of the same samples as in (A) ATP and (B) GTP from Figure 5A and 5B. (G) Rate of disassembly of pB171 ParM polymerized in 40 µM ATP or GTP. The amplitude of the time series data for 40 µM nucleotide from Figure 5A and 5B were normalized by the maximum amplitude and plotted versus time. Circles: ATP and triangle: GTP.

Supplementary Figure 5: Sequence alignment of diverse eukaryotic actins demonstrates a high level of conservation. Residues that are conserved in 25% of the sequence in the alignment are colored using a scheme based on Clustal X.

Supplementary Figure 6: Sequence alignment of representative proteins of the ParM demonstrates less sequence conservation than the actin family. Residues that are conserved in 25% of the sequence in the alignment are colored using a scheme based on Clustal X.

Supplementary Figure 7: Joint sequence alignment of the actin and ParM proteins from supplementary Figure 5 and 6 used to generate the phylogenetic tree in Figure 7. Residues that are conserved in 25% of the sequence in the alignment are colored using a scheme based on Clustal X.

Supplementary Figure 1

A B 168 168

167 167

166 166

165 165 Twist (degrees) Twist (degrees) 164 164

163 163 0 20 40 60 0 20 40 60 Iterations Iterations C D 168 1

167

166 19 Å 0.5 165 Twist (degrees) 164 Fouirer Shell Correlation

163 0 0 20 40 60 0 0.05 0.1 0.15 Iterations Resolution (A) Supplementary Figure 2

A B

1.2 Bu er ATP GTP Bu er 1

0.8 EDTA

0.6 MgCl 0.4 CaCl Fraction of ParM in Supernatant 0.2

0 Bu er EDTA MgCl2 CaCl2 Bu er Conditions C D 10 10 9 9 8 8

t 7 7 6 6 GTP AMP-PNP 5 x-intercept=0.91 5 x-intercept=0.475 R=0.999 R=0.998 4 4 uM ParM in Pelle uM ParM in Pellet 3 ATP 3 GMP-PNP x-intercept= 1.45 x-intercept=0.855 2 2 R=0.995 R=0.999 1 1 0 0 0 2 4 6 8 10 0 2 4 6 8 10 uM ParM Total uM ParM Total Supplementary Figure 3

A B 0 0

−0.2 −0.2

−0.4 −0.4

−0.6 −0.6

−0.8 −0.8

−1 −1 y=0.91x−0.29 y=1.16x−0.153 −1.2 R=0.972 −1.2 R=0.982 Log (A(t)/Amax) Log (A(t)/Amax) −1.4 −1.4

−1.6 −1.6

−1.8 −1.8

−2 −2 −1.5 −1 −0.5 0 0.5 −1.5 −1 −0.5 0 0.5 log(T/T) log(T/T) C D 1.5 1.5

1.4 1.4

1.3 1.3

1.2 1.2

1.1 1.1

1 1 Slopes Slopes 0.9 0.9 y=−0.00948x+1.18 y=−0.00274x+1.12 0.8 R=0.978 0.8 R=0.754

0.7 0.7

0.6 0.6

0.5 0.5 0 5 10 15 20 0 5 10 15 20 Points Points Supplementary Figure 4

A B C 4 x 10 10 10 2 10 mM 10 mM 8 8 2.5 mM 2.5 mM 1.5 10 mM 1.25 mM 2.5 mM 1.25 mM 6 6 1.25 mM 1 0.156 mM 4 4 0.040 mM

uM Polymer 0.156 mM uM Polymer 0.156 mM 0.5 2 2

0.040 mM 0.040 mM Light Scattering Intensity (AU) 0 0 0 0 25 50 75 100 0 25 50 75 100 0 2 4 6 8 10 Time(s) Time(s) Time (s) D E F 4 x 10 2 6000 6000

10 mM 5000 10 mM 5000 10 mM 2.5 mM 1.5 2.5 mM 2.5 mM 4000 4000 1.25 mM 1.25 mM 1 1.25 mM 3000 3000 0.156 mM 0.156 mM 2000 2000 0.156 mM 0.5 0.040 mM 1000 1000 0.040 mM Light Scattering Intensity (AU) 0.040 mM Light Scattering Intensity (AU) Light Scattering Intensity (AU) 0 0 0 0 2 4 6 8 10 0 5 10 0 5 10 Time (s) Time (s) Time (s) G

0.8

0.6

GTP 0.4

0.2 ATP

0 Light Scattering Intensity Normalized 0 20 40 60 Time (s) Supplementary Figure 5

10 20 30 40 Homo_sapiens_alpha_skeletal/1-377 1 MCDEDETTALVCDNGS GLVKAGFAG DDAPRAV FP S IVGRPRHQ GVMV GMG 50 Caenorhabditis_elegans/1-362 1 MNG ------ERAGFAG DDAPRAV FP S IVGRPRHQ GVMV GMG 35 Drosophila_melanogaster/1-376 1 MCD- EEVAALVVDNGS GMCKAGFAG DDAPRAV FP S IVGRPRHQ GVMV GMG 49 Dictyostelium_discoideum/1-377 1 MDG- EDVQALVIDNGSGMCKAGFAG DDAPRAV FP S IVGRPRHT GVMV GMG 49 Arabidopsis_thaliana/1-377 1 MADGEDIQPLVCDNGT GMVKAGFAG DDAPRAV FP S IVGRPRHT GVMV GMG 50 Saccharomyces_cerevisiae/1-375 1 MD S --EVAALV IDNGS GMCKAGFAG DDAPRAV FP S IVGRPRHQ GIMV GMG 48 Toxoplasma_gondii/1-376 1 MAD- EEVQALVVDNGS GNVKAGVAG DDAPRAV FP S IVGKPKNPGIMV GM E 49 Trichomonas_vaginalis_G3/1-375 1 MA E- EDVQTLVIDNGSGMCKAGFSGDEAPRS VFPSVVGRPKYKQQLV GGN49 Paramecium_caudatum/1-375 1 MSE- -EHPAVVIDNGSGQ CKAG IAG DDAPRCCFPA VVGRPKHQG IMVGMD 48 Naegleria_gruberi/1-345 1 MT E ---NPPLI IDNGSSLMKAGLS DDDAPCAV FPTVTGRQKVS TLMV GMN 47

60 70 80 90 Homo_sapiens_alpha_skeletal/1-377 51 QKD- S YVGDEAQS-KRGILTLKYPIEHGI ITNWDDMEKIW HHT FYNELRV 98 Caenorhabditis_elegans/1-362 36 QKD- S YVGDEAQS-KRGILTLKYPIEHGIVTNWDDMEKIW HHT FYNELRV 83 Drosophila_melanogaster/1-376 50 QKD- S YVGDEAQS-KRGILTLKYPIEHGIVTNWDDMEKIW HHT FYNELRV 97 Dictyostelium_discoideum/1-377 50 QKD- S YVGDEAQS-KRGILTLKYPIEHGIVTNWDDMEKIW HHT FYNELRV 97 Arabidopsis_thaliana/1-377 51 QKD- AYVGDEAQ S -KRGILTLKYPIEHGIVSNWDDMEKIW HHT FYNELRV 98 Saccharomyces_cerevisiae/1-375 49 QKD- S YVGDEAQS-KRGILTLRYPIEHGIVTNWDDMEKIW HHT FYNELRV 96 Toxoplasma_gondii/1-376 50 EKD- CYVGDEAQ S -KRGILTLKYPIEHGIVTNWDDMEKIW HHT FYNELRV 97 Trichomonas_vaginalis_G3/1-375 50 AKD- VFVGDEAC S -KAGVLILKYPIEHGIVNNWDDMEKIW HHT FYNELRV 97 Paramecium_caudatum/1-375 49 S KE-AYVGDEAQA-KRGVLTLKYPIENGIVNNWDDMERIW HHA FFNELRV 96 Naegleria_gruberi/1-345 48 NIDKTFVGDNTFGLRRGINVFYYP IDRGLITDWDQMEK IWDY IFNSELRV97

110 120 130 140 Homo_sapiens_alpha_skeletal/1-377 99 AP EEHPTLLT EAPLNPKANR EKMTQIMFET FNVPAMYVAIQAVLS LYASG 148 Caenorhabditis_elegans/1-362 84 AP EEHPVLLT EAPLNPKANR EKMTQIMFET FNTPAMYVAIQAVLS LYASG 133 Drosophila_melanogaster/1-376 98 AP EEHPVLLT EAPLNPKANR EKMTQIMFET FNTPAMYVAIQAVLS LYASG 147 Dictyostelium_discoideum/1-377 98 AP EEHPVLLT EAPLNPKANR EKMTQIMFET FNTPAMYVAIQAVLS LYASG 147 Arabidopsis_thaliana/1-377 99 AP EEHPVLLT EAPLNPKANR EKMTQIMFET FNTPAMYVAIQAVLS LYASG 148 Saccharomyces_cerevisiae/1-375 97 AP EEHPVLLT EAPMNPKSNR EKMTQIMFET FNVPAFYVS IQAV LSLY SSG 146 Toxoplasma_gondii/1-376 98 AP EEHPVLLT EAPLNPKANR ERMTQIMFET FNVPAMYVAIQAVLS LY SSG 147 Trichomonas_vaginalis_G3/1-375 98 DP TEHPVLLT EAPLNPKANR EKMI S LMFDTFNVPSFYVGIQAV LSLY SSG 147 Paramecium_caudatum/1-375 97 TPEDHPALLT EAPMNPKANR EKMTQILFET FNVPS FYVA IQAV LSLYASG 146 Naegleria_gruberi/1-345 98 AP EYHEV LLLEIPLNPMETKEKMS QSMFET FRVRS LCLANQ CAMSMFASG 147

160 170 180 190 Homo_sapiens_alpha_skeletal/1-377 149 R TTGIVLDSGDGVTHNVPIYEGYALPHAIMR LDLAGRDLTDYLMK ILTER 198 Caenorhabditis_elegans/1-362 134 R TTGVVLDSGDGVTHTVPIYEGYALPHAILR LDLAGRDLTDYLMK ILTER 183 Drosophila_melanogaster/1-376 148 R TTGIVLDSGDGV S HT VP IYEGYALPHA ILRLDLAGRDLTDY LMKILT ER 197 Dictyostelium_discoideum/1-377 148 R TTGIVMD S GDGV S HT VP IYEGYALPHA ILRLDLAGRDLTDYMMKILT ER 197 Arabidopsis_thaliana/1-377 149 R TTGIVLDSGDGV S HT VP IYEGYALPHA ILRLDLAGRDLTDY LMKILT ER 198 Saccharomyces_cerevisiae/1-375 147 R TTGIVLDSGDGVTHVVP IYAGFSLPHA ILRIDLAGRDLTDY LMKILSER 196 Toxoplasma_gondii/1-376 148 R TTGIVLDSGDGV S HT VP IYEGYALPHA IMRLDLAGRDLT EYMMKILHER 197 Trichomonas_vaginalis_G3/1-375 148 R TTGIVFDAGDGVSHT VP IYEGYSLPHA IMRLNLAGRDLTAWMV KLLT ER 197 Paramecium_caudatum/1-375 147 R TTGI VVDSGDGV S HT VP IYEGYALPHAVLRIDLAGRACTQY LVNILNEL196 Naegleria_gruberi/1-345 148 RMTGIVLES GDGVTNS VP IYEGYALSHATIRVELGGKDLTDYLRK ILMER 197

210 220 230 240 Homo_sapiens_alpha_skeletal/1-377 199 GY S FVTTAER EIVRDIKEKLCYVALDFENEMATAASSSSLEKSYELPDGQ 248 Caenorhabditis_elegans/1-362 184 GY SFTTTAER EIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYELPDGQ 233 Drosophila_melanogaster/1-376 198 GY SFTTTAER EIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYELPDGQ 247 Dictyostelium_discoideum/1-377 198 GY SFTTTAER EIVRDIKEKLAYVALDFEAEMQ TAASSSALEK S YELPDGQ 247 Arabidopsis_thaliana/1-377 199 GY S FTTSAEREIVRDVKEK LAYIALDY EQ EMETANTSSSVEKSYELPDGQ 248 Saccharomyces_cerevisiae/1-375 197 GY SFSTTAER EIVRDIKEKLCYVALDFEQEMQ TAAQ SSSIEKSYELPDGQ 246 Toxoplasma_gondii/1-376 198 GYGFTTS AEKEIVRDIKEK LCYIALDFDEEMKAAEDSS DIEK S YELPDGN 247 Trichomonas_vaginalis_G3/1-375 198 GNAFN TTAEK EIVRDIKEKLCYVALDFDAEMEK AATDSS INVNYT LPDGN 247 Paramecium_caudatum/1-375 197 GV S FT SSAEME IVRDMK EK LCYVALDY EEELKKYKES AANNRPYELPDGN246 Naegleria_gruberi/1-345 198 GYNFTTKHETEIVRYMK EKTGCVA ------221

260 270 280 290 Homo_sapiens_alpha_skeletal/1-377 249 VITIGNER FRCP ET LFQP S FIGM ESAG IHE TTYNS IMKCDIDIRKDLYAN 298 Caenorhabditis_elegans/1-362 234 VITVGNER FRCP EALFQP SFLGM ESAG IHET S YNS IMKCDIDIRKDLYAN 283 Drosophila_melanogaster/1-376 248 VITIGNER FRCP EALFQP SFLGM EACG IHE TTYNS IMKCDVDIRKDLYAN 297 Dictyostelium_discoideum/1-377 248 VITIGNER FRCP EALFQP SFLGM ESAG IHE TTYNS IMKCDVDIRKDLYGN 297 Arabidopsis_thaliana/1-377 249 VITIGGER FRCP EV LFQP S LVGM E AAG IHE TTYNS IMKCDVDIRKDLYGN 298 Saccharomyces_cerevisiae/1-375 247 VITIGNER FRAP EALFHP S VLGLESAG IDQ TTYNS IMKCDVDVRKELYGN 296 Toxoplasma_gondii/1-376 248 IITVGNER FRCP EALFQP SFLGK E AAGVHRTTFDS IMKCDVDIRKDLYGN 297 Trichomonas_vaginalis_G3/1-375 248 VITIGNER FRCP EMLFKPYFDGMEYDGIDKTLFDS IMKCDIDVRKDLYAN 297 Paramecium_caudatum/1-375 247 VVV IQNQ RFRCPELLFKPAFIGLEVSGLHELT FK S IMKCDIDVRKDLYGN 296 Naegleria_gruberi/1-345 222 ------KRYKTTEILFQPNLVGM EVGG IHEN IFTSIRKSDLDIRKELYSN 265

310 320 330 340 Homo_sapiens_alpha_skeletal/1-377 299 NVMS GGT TMYPGIADRMQK EITALAPSTMKIKIIAPPERKYS VWIGGS IL348 Caenorhabditis_elegans/1-362 284 TVLSGGT TMYPGIADRMQK EITALAPSTMKIKIIAPPERKYS VWIGGS IL333 Drosophila_melanogaster/1-376 298 TVLSGGT TMYPGIADRMQK EITALAPSTMKIKIIAPPERKYS VWIGGS IL347 Dictyostelium_discoideum/1-377 298 VVLSGGT TMFPGIADRMNK ELTALAPSTMKIKIIAPPERKYS VWIGGS IL347 Arabidopsis_thaliana/1-377 299 IVLSGGT TMFPGIADRMS KEITALAP SSMK IKVVAPPERKYS VWIGGS IL348 Saccharomyces_cerevisiae/1-375 297 IVMSGGT TMFPGIAERMQK EITALAPSS MK VK IIAPPERKYSVWIGGS IL346 Toxoplasma_gondii/1-376 298 VVLSGGT TMYEGIGERLTKELTSLAPSTMKIK VVAPPERKYS VWIGGS IL347 Trichomonas_vaginalis_G3/1-375 298 IVLSGGT TMFSGIAERLDK EITALAPPTMK VK IVAP EERKYAVWVGGS IL347 Paramecium_caudatum/1-375 297 VVMSGGT TMFPGIPERLS KELT S LAPSS MK IKVVAPPERKFS VWIGGS IL346 Naegleria_gruberi/1-345 266 IFLSGGS TLFNGIVERLYNELRELSPSS MN IRITAPPERKYTAW IGGS IF315

360 370 Homo_sapiens_alpha_skeletal/1-377 349 ASLSTFQQMWITKQEYDEAGP S IVHRKCF- 377 Caenorhabditis_elegans/1-362 334 ASLSTFQQMWI S KQ EYDES GP S IVHRKCF- 362 Drosophila_melanogaster/1-376 348 ASLSTFQQMWI S KQ EYDES GP S IVHRKCF- 376 Dictyostelium_discoideum/1-377 348 ASLSTFQQMWI S KEEYDES GP S IVHRKCFF 377 Arabidopsis_thaliana/1-377 349 ASLSTFQQMWIAKAEYDESGP S IVHRKCF- 377 Saccharomyces_cerevisiae/1-375 347 ASLTTFQQMWI S KQ EYDES GP S IVHHKCF- 375 Toxoplasma_gondii/1-376 348 SSLSTFQQMWITKEEYDESGP S IVHRKCF- 376 Trichomonas_vaginalis_G3/1-375 348 ASLATFPQMV ITKEEYDEAGPSIVHRKC-- 375 Paramecium_caudatum/1-375 347 SSLSTFQAMW ITRSEYDES GP S IVHRKCF- 375 Naegleria_gruberi/1-345 316 S KLS TFQSMC ITLEEY EESGYNI IHRKGIF 345 Supplementary Figure 6

10 20 30 40 E.coli_plasmid_R1/1-320 1 ------MLVFI DDGSTNIKLQWQESDGT IKQH ISPNSFK 33 K.pneumoniae_342/1-318 1 ------MR IFI DDGSTNIKMLW-EQDGETFTHISPNSFK 32 S.enterica/1-326 1 ------MK IFI DDGSTNIKLAW-LEDGDVKTLISPNSFK 32 E.coli_prophage_CP-933T/1-319 1 ------MK ICI DDGSTNIKLAW-TENGERRNAISPNSFK 32 S._dysenteriae_Sd197/1-319 1 ------MLKVSCDDGSTNVKLAW-LEDGEVRTSLSGNSFK 33 A._nasoniae/1-327 1 ------MK TIFCDDGSTAIKLAWFDDEGK IESI ITYNSFK 34 K._pneumoniae/1-324 1 ------MMNIYCDDGSTNVKLAW-FEGNELQTRVSANSFR 33 V._furnissii_CIP_102972/1-344 1 ------MK FAV DDGSTNVKVSW-LD GGK IRSLVSPNSFR 32 E._coli_plasmid_pB171/1-323 1 ------MNVYC DDGSTTIKLAW-NDNGK ICKSLSQNSFR 32 P.ingrahamii_37/1-335 1 ------MLTISVDDGSTNVKISW-FDGTK IETIISPNSFR 33 M._extorquens_AM1/1-347 1 MA EAAMKKERGAKEGVQP LVAIDDGYAQTKLYGEGLDGR IVKRVLRSSVR 50

60 70 80 90 E.coli_plasmid_R1/1-320 34 REWA VSF --GDK-KVFNYTLNG-EQY SFDP ISPDA VVTTNIAWQ YSDVNV 79 K.pneumoniae_342/1-318 33 RGWSAT F --GSG-KPFNYTVDD- EKYSFDLITPDA LPTNNIDWQY SP LNS 78 S.enterica/1-326 33 PEWS FSL- L DDA-APANY EIDG-EKFSFDP LSADA VVTTETRYQYSDVNV 79 E.coli_prophage_CP-933T/1-319 33 SEWSAP F --GGT -QPANYMLDG-VRYGFDP VSDR FVQ TTDTQYQYSDVNV 78 S._dysenteriae_Sd197/1-319 34 E GWNPGL- FNAG-KVYNYVVDE- KKYTYDLGSTAVIG TTHVSYQYSTTNL 80 A._nasoniae/1-327 35 KSWNVSL --NGK-ATYNYTVDG-EQY SYDP YSPDAIK TTNIDFQYSTENL 80 K._pneumoniae/1-324 34 H GWK VAE- -FSA-ATFNYQVGT -LKYTWDSVSRDAIP TTNVEYQYGDLNL 79 V._furnissii_CIP_102972/1-344 33 KDWK SAALRKDK-QVYNYTIDG-FKYTYDATSDKALE TTHVDYQYDDLNL 80 E._coli_plasmid_pB171/1-323 33 H GWK VDG- LGIR-QTFNY ELDG- KKYTYDEVSNQ SIL TTHIEYQYTD VNL 79 P.ingrahamii_37/1-335 34 K GWK S AA- LRADK- VISNYLVGE- TKYTYDITSDKALETTHIDYQYSDLN 80 M._extorquens_AM1/1-347 51 IGSHGLGSFAGDG- AIGLWQ TEEGNKYTVSD- EIEAEDPRFTDFHLSP IN 98

110 120 130 140 E.coli_plasmid_R1/1-320 80 VAV HHA LLTSGLPVSEVDIVC TLPLTEYYD-RNNQPNT EN IER KKANFRK 128 K.pneumoniae_342/1-318 79 IAV HHA LLTSGLEPQDVEI VVT LP LT EFYD-EDAQYRLDN IER KKKSLLR 127 S.enterica/1-326 80 VA IQHA LQQTGLKAQPVDVIVT LP ISEY LD-ANNQKNKQN IER KKKNVMR 128 E.coli_prophage_CP-933T/1-319 79 IAI HHA LVKSGITPQEVDVVVT LP LSEY FD-TNAQPDMAN INR KKANVMR 127 S._dysenteriae_Sd197/1-319 81 LA I HHA LLTSGLQPQDVELTVT LPVT EFFD-NDNQPNEER IER KKANV LR 129 A._nasoniae/1-327 81 LA I HHA LLESK IQPQ EIELIVTLPISEFYT- SDMK PNLDNIA KKKKNLLR 129 K._pneumoniae/1-324 80 LAV HHA LLNSGLEPQPVSLTVT LP LSE YYD-GDCQRNEEN I RRKRENLMR 128 V._furnissii_CIP_102972/1-344 81 LAV HHA LLQTGIEPC PVALVVT LP ITE YYRFEDCQKNEKNIARKRHNLMR 130 E._coli_plasmid_pB171/1-323 80 LAV HHA LLNSGLAPQPVSLTVT LP ISEFYT-KECQKNELNIQRK IENLMR 128 P.ingrahamii_37/1-335 81 LLSV HHA LLKTGLKPQK IKIVAT LP ITA YYNA DDCQKNEGN IQR KKDNLM 130 M._extorquens_AM1/1-347 99 RVLVNHALS AAGF GGAKVDIVTGLPVKEFF--KEQRKDEER IQRKRENLQ 146

160 170 180 190 E.coli_plasmid_R1/1-320 129 KITL--NGGDTFTIKDVKVMPESIPAGY EV L- -QELDE----LDSLLI ID 170 K.pneumoniae_342/1-318 128 DVKL--NKGVVFN ITKVTVRPESIPAG ISLC--DELKP----SHSV LI ID 169 S.enterica/1-326 129 EVRV--QGSDAFVIRSVSVLPESIPAGFSV L --AGLEG----DESLLIVD 170 E.coli_prophage_CP-933T/1-319 128 PVAY--QNGKAFTIRNVRVMPESIPAGFKAL --ADM SP----FESLLIVD 169 S._dysenteriae_Sd197/1-319 130 EISL--NKGET FK I KKVNVMPESLPAAFELL KKDKVNK----LERSLI ID 173 A._nasoniae/1-327 130 KIES--DSGMV FT LKKIN VVPESLP S AANSLEQDNVHE----MEVSL VVD 173 K._pneumoniae/1-324 129 ELVL--NKGRAFTVTDVKVMP ESLPAAFSRLAELKPGP ----AETTLIID 172 V._furnissii_CIP_102972/1-344 131 QITL--NKGQT FDIVDV EVMP ESLPAV LSTLVNSNCNE----FTRSLV ID 174 E._coli_plasmid_pB171/1-323 129 PIRL--NKGDV FT IEHVDVMP ESLPAV FSRLVVDKVGQ ----FEKSL VVD 172 P.ingrahamii_37/1-335 131 RT LVL --NKGKVFNI AAV EVMP ESVPAV LST LLESNVNE----FSRSLV I 174 M._extorquens_AM1/1-347 147 RGAFSR SAGV EAPVLADIQVGCQA IAAFVDWA LDDEMK TRND IAKT IAIV 196

210 220 230 240 E.coli_plasmid_R1/1-320 171 L GGT TLDISQVMGK LSGISK IYGDSSLGV SLVT SAVKDA LSLA-RTKGSS 219 K.pneumoniae_342/1-318 170 L GGT TLDISMVAGQMT SV SR IYGDPKLGVSLVTDAVKLALARA- NT DT SS 218 S.enterica/1-326 171 L GGT TLDV SHVRSKMT GITKTWC DP NIGV SLITSGVKEQMAVHANTRVSS 220 E.coli_prophage_CP-933T/1-319 170 L GGT TLDVAKVQGQ LAGISQVFC DP HVGV SLMADAVLSVMATN-GMR TSH 218 S._dysenteriae_Sd197/1-319 174 L GGT TLDCGL ILGA FEGISEIRGY SEIGTSRITHTVMNALTKA- STPCNY 222 A._nasoniae/1-327 174 L GGT TLDCGT IAGKYAHISHVSGDST IGVSLVTNAVYNALDRA- S TTTSY 222 K._pneumoniae/1-324 173 L GGT TLDAGV IVGQ FDDISAVHGNPSVGVSQVTRAAAGALR AA- DSET SA 221 V._furnissii_CIP_102972/1-344 175 L GGT TLDMGIVVGEFDEVSAVYGNNEIGV SMVTNT VRKALAFA-DSDSSY 223 E._coli_plasmid_pB171/1-323 173 I GGT TLDVGI IVGQ FDSV SA IHGNSG IGVSSVTKAAMSALRMA- SSDT SF 221 P.ingrahamii_37/1-335 175 DIGGTTVDSAV IVGQ FDEISSVYGNTEIGVGLVTNTARQALANA- DSDA S 223 M._extorquens_AM1/1-347 197 DIGGRTTDVAVVVGGK SIDHGR SGTADVGV LDVYKGVLDRVKGR FELDDD 246

260 270 280 290 E.coli_plasmid_R1/1-320 220 Y ---LADD IIIHRKDNNY LKQR INDENK ISIVTEAMNEALRKLEQRVLNT 266 K.pneumoniae_342/1-318 219 Y ---NVDQ IIINRHDEEYLTDNINDPDAV SEV KKV INNS IERLTTRVLTA 265 S.enterica/1-326 221 F ---QADN IIVHRNEPDY LSRR IYNA EQRESI INVINERQKLLIKRVNDV 267 E.coli_prophage_CP-933T/1-319 219 H --- IANT IIEHRHDEAWLRQH IHNDAHYDSLIAVIREKEET LKQRVIRA 265 S._dysenteriae_Sd197/1-319 223 F --- IADELIKNRHDNEY LQTLINDVAEIKNISHVIDR EVKSLAESIRQE 269 A._nasoniae/1-327 223 L ---VADQLV KKNDDEELYKTLINDK SK I DDVKNSFNDAVK LLSDRVISH 269 K._pneumoniae/1-324 222 L --- IADT VIRNRNDRQY LQRV INDAGK IDEVRNKITEAITSLGARVT SE 268 V._furnissii_CIP_102972/1-344 224 L ---VANELI RRRNDDEFVSEV INDEAQ IDMV REKMEAKIHELGKQVVT E 270 E._coli_plasmid_pB171/1-323 222 L ---VADELIKRRNDP DFVRQV INDETKTDLVLNTIEGAIASLGEQVVNE 268 P.ingrahamii_37/1-335 224 YL---VANELIKNRHDSEFVKEVINDLSQVDVVLKK IEDR INELGQ TVAE 270 M._extorquens_AM1/1-347 247 SLPLSLMN EAVRTGSMQLFGKPHDVSEIVSD VVGQVQERALR EIQRRLGR 296

310 320 330 340 E.coli_plasmid_R1/1-320 267 LNE- F- SGYTHVMV I GGGA ELICDAV KKHTQ IRDERFFKTNNSQYDLVNG 314 K.pneumoniae_342/1-318 266 IDS- F- KGYSHA IVI GGGAPLVADA IRERMG LR EDRFVVAEEPQ FALVRG 313 S.enterica/1-326 268 ISR- F- TDYTHVMCV GGGA EIVAEAVKNLTKVPDERFYLSSSPQ FDLVMG 315 E.coli_prophage_CP-933T/1-319 266 LAG- F- SGYGRVM VVGGGA EIVAPAIREAC GVN- AT FIADGVPQ FALVNG 312 S._dysenteriae_Sd197/1-319 270 IST- F- SGMNRIYLT GGGA ELIYPHIKQY FPNL--KVNKVDEPQ FALVKA 315 A._nasoniae/1-327 270 IKHEYKKSFNRIYLT GGGA SLIYPAVKSAYPNQ -- IITIMEDSQTALVT S 317 K._pneumoniae/1-324 269 LTA- F- RNVNRVFLV GGGA SLIEEA IRQAWP LAHDRIEV IGDPQMALAR E 316 V._furnissii_CIP_102972/1-344 271 A KK- FAKNPNRIYLV GGGA SLIYPAVCAAYPSLGERVILIENAQ SALAQE 319 E._coli_plasmid_pB171/1-323 269 LGD- F- HHVNRVYVVGGGAPLIYDS IKTAWHHLGQ KVVMMESPQTALVEA 316 P.ingrahamii_37/1-335 271 EMKK- FCKNPNRVYIV GGGAPLVYDAVKAAY SV LGDRVIILENAQ IALAK 319 M._extorquens_AM1/1-347 297 AAT------LQAVVFV GGGSALFKD-IASAYPNG ----VMSDDP EFANAR 335

360 370 E.coli_plasmid_R1/1-320 315 MY LIGN------320 K.pneumoniae_342/1-318 314 LK IIG ------318 S.enterica/1-326 316 MIKMK GGVTNE ------326 E.coli_prophage_CP-933T/1-319 313 LYAMDK E ------319 S._dysenteriae_Sd197/1-319 316 MV HA------319 A._nasoniae/1-327 318 IASMRAVKKQ------327 K._pneumoniae/1-324 317 IALYNK ED------324 V._furnissii_CIP_102972/1-344 320 ICLYHADQ PETEMSDLPLAEEMSDE 344 E._coli_plasmid_pB171/1-323 317 IAAFKEE ------323 P.ingrahamii_37/1-335 320 ENMLYALSDGEEESVV------335 M._extorquens_AM1/1-347 336 GLYKYFASRRRS ------347 Supplementary Figure 7

10 20 30 40 50 60 70 80 90 100 H.sapiens/1-377 1 ------MCDEDETTALVC---DNGSGLVKAGFAGD --DAPRAVFPSIV------GRPRHQGVMVGMGQKD- SYVGDEAQ S- KRGILT LKYP IEHG I 77 C.elegans/1-362 1 ------MNGE---RAGFAGD--DAPRAVFPSIV------GRPRHQGVMVGMGQKD- SYVGDEAQ-SKRGILT LKYP IEHG I 62 D.discoideum/1-377 1 ------MDGEDVQALV I ---DNGSGMCKAGFAGD --DAPRAVFPSIV------GRPRHT GVMV GMGQ KD-SYVGDEAQS-KRGILT LKYP IEHG I 76 A.thaliana/1-377 1 ------MADGEDIQPLVC---DNGT GMVKAGFAGD--DAPRAVFPSIV------GRPRHT GVMV GMGQ KD-AYVGDEAQS-KRGILT LKYP IEHG I 77 S.cerevisiae/1-375 1 ------MD SE-VAALV I ---DNGSGMCKAGFAGD --DAPRAVFPSIV------GRPRHQG IMVGMGQKD- SYVGDEAQ S- KRGILT LRYP IEHG I 75 T.gondii/1-376 1 ------MADEEVQAL VV---DNGSGNVKAGVAGD --DAPRAVFPSIV------GKPKNPGIMVGMEEKD- CYVGDEAQ S- KRGILT LKYP IEHG I 76 T.vaginalis/1-375 1 ------MA EEDVQT LV I ---DNGSGMCKAGFSGD- -EAPRSVFPSVV------GRPKYKQQLV GGNAKD- VFVGDEAC S- KAGV LILKYP IEHG I 76 P.caudatum/1-375 1 ------MSEE-HPAVV I ---DNGSGQCKAGIAGD --DAPRCCFPAVV------GRPKHQG IMVGMD SK E- AYVGDEAQA- KRGV LT LKYP IENG I 75 N.gruberi/1-345 1 ------MT ENPPLI I ---DNGSSLMK AGLSDD--DAPCAVFPTVT------GRQKVST LMVGMNNIDKT FVGDNT FGL RRGINVFYYP IDRGL 76 H.sapiens/1-418 1 ------MAGR LPACVV---DCGT GYT KLGYAGN --TEPQFIIPSCIAIKESAKVGDQAQ RRVMK GVDDLD-FFIGDEA I- -EKPTYAT KWPIRHGI82 S.dysenteriae/1-319 1 ------MLKVSC---DDGSTNVKLAWLED- GEVRT SLSGNSF ------KE GWNPGLF-NAGKVY- NYVVDEKK------55 V.furnissii/1-344 1 ------MK FAV ---DDGSTNVKVSWLDG- GK IRSLVSPNSF------RKDWK SAALRKDKQVY- NYT IDGFK ------55 E.coli/1-319 1 ------MK ICI ---DDGSTNIKLAWT EN-GE RRNAISPNSF ------KSEWSAP F --GGTQPA- NYMLDGVR------53 K.pneumoniae/1-318 1 ------MR IFI ---DDGSTNIKMLWEQD- GET FTHISPNSF ------KRGWSAT F --GSGKPF- NYT VDDEK ------53 S.enterica/1-326 1 ------MK IFI ---DDGSTNIKLAWLED- GDVKT LISPNSF ------KPEWS FSLLDDAAPA --NYEIDGEK ------54 A.nasoniae/1-327 1 ------MK T IFC ---DDGSTAIKLAWFDDEGK IESI ITYNSF------KKSWNVSL --NGKATY- NYT VDGEQ ------55 K.pneumoniae/1-324 1 ------MMNIYC---DDGSTNVKLAWFEG- NELQT RVSANSF ------RH GWK VAEFSA--AT F- NYQVGT LK------54 E.coli/1-320 1 ------MLVFI ---DDGSTNIKLQWQESDGT IKQH ISPNSF------KREWA VSF --GD KKVF- NYT LNGEQ ------54 E.coli/1-323 1 ------MNVYC ---DDGSTT IKLAWNDN-GKICK SLSQNSF ------RH GWK VDGL-GIRQT F- NY ELDGKK------54 P.ingrahamii/1-335 1 ------MLT ISV ---DDGSTNVKISWFDG- T KIET IISPNSF ------RK GWK SAALRADKVIS- NY LVGETK------56 S.aureus/1-355 1 ------XSNVYVXALDFGNGFVKGK INDEKFVIPSRIGRKTN ------ENNQ LKGFVDNK LDVS-EFI ING NN------DEV L 64 M.extorquens/1-347 1 MA EAAMKKERGAKEGVQP LVAI---DDGYAQTKLYGEGLDGR IVKRVLRSS ------VRIGSHGLG-SFAGDGA I ------GLWQT EEGN74

110 120 130 140 150 160 170 180 190 200 H.sapiens/1-377 78 ITNWDDMEKIW HHT FYNELRVAPEEHPT LLT EAPLNPKANR EKMTQIMFET FNVPAMYVAIQAVLSLYASGRT------TGIVLDSGDGVT HNVP IYEGY 171 C.elegans/1-362 63 VTNWDDMEKIW HHT FYNELRVAPEEHPVLLT EAPLNPKANR EKMTQIMFET FNT PAMY VA IQAV LSLYASGRT ------TGVVLDSGDGVT HT VP IYEGY 156 D.discoideum/1-377 77 VTNWDDMEKIW HHT FYNELRVAPEEHPVLLT EAPLNPKANR EKMTQIMFET FNT PAMY VA IQAV LSLYASGRT ------TGIVMDSGDGV SHT VP IYEGY 170 A.thaliana/1-377 78 VSNWDDMEKIW HHT FYNELRVAPEEHPVLLT EAPLNPKANR EKMTQIMFET FNT PAMY VA IQAV LSLYASGRT ------TGIVLDSGDGV SHT VP IYEGY 171 S.cerevisiae/1-375 76 VTNWDDMEKIW HHT FYNELRVAPEEHPVLLT EAPMNPKSNR EKMTQIMFET FNVPAFYVSIQAVLSLYSSGRT------TGIVLDSGDGVTHVVP IYAGF 169 T.gondii/1-376 77 VTNWDDMEKIW HHT FYNELRVAPEEHPVLLT EAPLNPKANR ERMTQIMFET FNVPAMYVAIQAVLSLYSSGRT------TGIVLDSGDGV SHT VP IYEGY 170 T.vaginalis/1-375 77 VNNWDDMEKIW HHT FYNELRVDPT EHPVLLT EAPLNPKANR EKMI SLMFDT FNVPSFYVGIQAVLSLYSSGRT------TGIVFDAGDGV SHT VP IYEGY 170 P.caudatum/1-375 76 VNNWDDMERIW HHA FFNELRVTPEDHPALLT EAPMNPKANR EKMTQILFET FNVPSFYVAIQAVLSLYASGRT------TGIVVDSGDGVSHT VP IYEGY 169 N.gruberi/1-345 77 ITDWDQMEKIWDYIFNSELRVAPEYHEV LLLEIPLNPMETKEKMSQ SMFET FRVRSLCLANQCAMSMFASGRM------TGIVLESGDGVT NSVP IYEGY 170 H.sapiens/1-418 83 VEDWDLMERFMEQV IFKYLRAEPEDHYFLLT EPPLNT PENR EYT AEIMFESFNVPGLY IAVQAV LALAASWT SRQVGERT LTGT VIDSGDGVT HV IPVAEGY 184 S.dysenteriae/1-319 56 -YT YD------LGSTAV IGTTHV SYQY STT NLLA I HHA LLT ------SGLQPQDV E- LTVT LPVT EFF 109 V.furnissii/1-344 56 -YT YD------AT SDKALETTHVDYQYDDLNLLAVHHA LLQ ------TGIEPCPVA-LVVT LP ITEYY 109 E.coli/1-319 54 -YGFD ------PVSDR FVQTT DTQYQY SDVNVIAIHHA LVK ------SG ITPQ EVD- VVVT LP LSEY F 107 K.pneumoniae/1-318 54 -YSFD ------LIT PDALPTNN IDWQ YSPLNS IAV HHA LLT ------SGLEPQDV E- I VVT LP LT EFY 107 S.enterica/1-326 55 -FSFD ------PLSADA VVTT ETRYQY SDVNVVA IQHA LQQ ------TGLKAQPVD-VIVT LP ISEY L 108 A.nasoniae/1-327 56 -YSYD ------PYSPDAIKTT NIDFQY ST ENLLAIHHA LLE------SK IQPQ EIE- LIVT LP ISEFY 109 K.pneumoniae/1-324 55 -YT WD------SV SRDA IPTTNV EYQYGDLNLLAVHHA LLN ------SGLEPQPVS- LTVT LP LSEYY 108 E.coli/1-320 55 -YSFD ------PISPDA VVTT NIAWQY SDVNVVAV HHA LLT ------SGLPVSEVD- IVCT LP LT EYY 108 E.coli/1-323 55 -YT YD------EV SNQSILTTHIEYQYTDVNLLAV HHA LLN ------SGLAPQPVS- LTVT LP ISEFY 108 P.ingrahamii/1-335 57 -YT YD------ITSDKALETTHIDYQY SDLNLLSV HHA LLK ------TGLKPQK IK-IVAT LP ITAYY 110 S.aureus/1-355 65 LFGND ------LDKTT NT GKDT AST NDRYDIKSFKDLVEC ------S ------IGLLAR EVPEE VVNVV IAT GX120 M.extorquens/1-347 75 KYTVS------D-EIEAEDP RFT DFHLSP INRVLVNHALSA------AGFGGAKVD- IVT GLPVKEFF128

210 220 230 240 250 260 270 280 290 300 H.sapiens/1-377 172 ALPHAIMR LDLAGRDLTDY LMKI--LT ER-GYSFVTTAEREIVRDIKEK LCY------VALDFENE ------MAT AASSSSLEK SY E- -LPDGQ VIT - 251 C.elegans/1-362 157 ALPHAILR LDLAGRDLTDY LMKI--LT ER-GYSFTTTAEREIVRDIKEK LCY------VALDFEQ E ------MAT AASSSSLEK SY E- -LPDGQ VIT - 236 D.discoideum/1-377 171 ALPHAILR LDLAGRDLTDYMMKI--LT ER-GYSFTTTAEREIVRDIKEK LAY ------VALDFEAE ------MQ T AASSSALEK SY E- -LPDGQ VIT - 250 A.thaliana/1-377 172 ALPHAILR LDLAGRDLTDY LMKI--LT ER-GYSFTT SAER EIVRDVKEKLAY------IALDYEQE------MET ANT SSSVEKSYE--LPDGQV IT- 251 S.cerevisiae/1-375 170 SLPHAILR IDLAGRDLTDY LMKI--LSER-GYSFSTTAEREIVRDIKEK LCY------VALDFEQ E ------MQ T AAQ SSSIEKSYE--LPDGQV IT- 249 T.gondii/1-376 171 ALPHAIMR LDLAGRDLT EYMMKI--LHER-GYGFTT SAEK EIVRDIKEKLCY------IALDFDEE------MK AAEDSSDIEK SY E- -LPDGN IIT - 250 T.vaginalis/1-375 171 SLPHAIMR LNLAGRDLTAWMV KL--LT ER-GNA FNTTAEKEIVRDIKEK LCY------VALDFDA E ------MEK AATDSSINVNYT --LPDGNV IT- 250 P.caudatum/1-375 170 ALPHAV LR IDLAGRACTQY LVNI--LNEL-GVSFT SSAEME IVRDMK EK LCY------VALDY EEE------L KKYKES AANNRPYE- -LPDGNVVV- 249 N.gruberi/1-345 171 ALSHAT IRVEL GGKDLTDY LRKI--LMER-GYNFTT KHET EIVRYMKEKTGC------VAKRYKTT ------227 H.sapiens/1-418 185 VIGSCIKHIPIAGRDIT YFIQQL--LRDR-EVGIPPEQSLET AKAVKERYSY------VCPDLVKE ------FNKYDT DGSKWIK- -QYTGINA IS263 S.dysenteriae/1-319 110 D- NDNQ PNEERIERKKANV LR EI--SLNK-GET FK I KKVNVMPESLPAAFEL------L KKDKVNK----LERSLI IDL GGT T LDCGLILG--AFEGISEI- 194 V.furnissii/1-344 110 RFEDCQKNEKNIARKRHNLMRQ I --TLNK- GQ T FDIVDV EVMP ESLPAV LST ------L VNSNCNE----FTRSLV IDL GGT T LDMG I VVG--EFDEVSAV- 195 E.coli/1-319 108 D- T NAQPDMAN INR KKANVMRPV--AYQN- GKAFT IRNVRVMP ESIPAGFKA ------LADM SP FE------SLLIVDLGGT T LDVAKVQG--QLAG ISQV- 190 K.pneumoniae/1-318 108 D- EDAQYRLDNIERKKKSLLRDV--KLNK- GVVFNIT KVT VRPESIPAGISL------CDELKPSH ------SV LI IDL GGT T LDISMV AG--QMT SV SR I- 190 S.enterica/1-326 109 D- ANNQ KNKQNIERKKKNVMREV --RVQG- SDAFVIRSVSVLPESIPAGFSV------LAGLEGDE------SLLIVDLGGT T LDVSHVRS--KMT GITKT- 191 A.nasoniae/1-327 110 T -SDMKPNLDN IAKKKKNLLRK I- -ESDS- GMVFT LKKIN VVPESLP S AANS ------LEQDNVHE----MEVSL VVDLGGT T LDCGT IAG --KYAHISHV- 194 K.pneumoniae/1-324 109 D- GDCQRNEENIRRKRENLMR EL--VLNK- GRAFT VTDVKVMP ESLPAAFSR ------LAELKPGP----AETT LI IDL GGT T LDAGVIVG--QFDD ISAV- 193 E.coli/1-320 109 D- RNNQ PNT EN IER KKANFRKKI--TLNG- GDT FT IKDVKVMP ESIPAGYEV ------LQ ELDELD------SLLI IDL GGT T LDISQVMG--KLSGISK I- 191 E.coli/1-323 109 T -KECQKNELNIQRK IENLMR PI--RLNK- GDVFT IEHVDVMP ESLPAV FSR ------L VVDKVGQ ----FEKSL VVDI GGT T LDVGIIVG--QFDSVSAI- 193 P.ingrahamii/1-335 111 NADDCQKNEGN IQR KKDNLMR TL--VLNK- GKVFNIAAV EVMP ESVPAV LST ------LLESNVNE----FSRSLV IDI GGT T VDSAVIVG--QFDE ISSV- 196 S.aureus/1-355 121 PSNE IGT DKQAKFEK LLNK SR LI--EIDG IAKT INVKGVKIVAQPXGT LLDLNX ENGKVFKAFT EG------KYSVLDFGSGT T IIDT YQNXKRVEEESFV- 213 M.extorquens/1-347 129 --KEQRKDEER IQRKRENLQRGA FSRSAG-VEAPVLADIQVGCQA IAAFVDW ------ALDDEMK T RNDIAKT IAIVDIGGRTTDVA VVVG--GKSIDHGR- 218

310 320 330 340 350 360 370 380 390 400 H.sapiens/1-377 252 ------IGNERFRCPET LFQP SFIGMESAG- IHETT YNSIMK CDIDIRKDLYA NNVMS GGT T MY PGIADRMQ KEITALAP STMK I ------329 C.elegans/1-362 237 ------VGNER FRCP EALFQP SFLGMESAG- IHET SYNS IMKCDIDIRKDLYANT VLS GGT T MY PGIADRMQ KEITALAP STMK I ------314 D.discoideum/1-377 251 ------IGNERFRCPEALFQPSFLGM ESAG-IHETTYNSIMK CDVDIRKDLYGNVVLSGGT TMFPGIADRMNKELTALAP STMK I ------328 A.thaliana/1-377 252 ------IGGERFRCPEVLFQPSLVGM E AAG-IHETTYNSIMK CDVDIRKDLYGN IVLSGGT TMFPGIADRMSKEITALAP SSMK I ------329 S.cerevisiae/1-375 250 ------IGNERFRAPEALFHPSVLGLESAG-IDQ TTYNSIMK CDVDVRKELYGN IVMSGGT TMFPGIAERMQ KEITALAP SSMK V ------327 T.gondii/1-376 251 ------VGNER FRCP EALFQP SFLGKEAAG-VHRTTFDSIMK CDVDIRKDLYGNVVLSGGT T MY EG IGER LTKELT SLAP STMK I ------328 T.vaginalis/1-375 251 ------IGNERFRCPEML FKPYFDGM EYDG-IDKT LFDS IMKCDIDVRKDLYANIVLS GGT TMFSG IAER LDKEITALA PPTMK V ------328 P.caudatum/1-375 250 ------IQNQ RFRCPELLFKPAFIGLEVSG- LHELT FK SIMK CDIDVRKDLYGNVVMSGGT TMFPGIPER LSKELT SLAP SSMK I ------327 N.gruberi/1-345 228 ------EILFQPNLVGMEV GG- IHEN IFTSIRKSDLDIRKELY SN IFLSGGST LFNG IVER LYNELR ELSP SSMN I ------296 H.sapiens/1-418 264 KKEFSIDVGY ER FLGP EIFFHP EFANPDFTQP ISE VVDEV IQNCPIDVRRPLYKNIVLSGGSTMFRDFGRRLQRDLKRTVDAR LKLSEELS GGR LKPKPIDV365 S.dysenteriae/1-319 195 ------RGY SEI- GT SR ITHT VMNA LTKAS --TPCNY FIADELIKNRHDNEYLQT LINDVAEI-KNISHVIDR EVKSLAESIRQE------IS- 271 V.furnissii/1-344 196 ------YGNNE I- GV SMVTNT VRKALAFAD- -SDSSY LVANELI RRRNDDEFVSEV INDEAQ I- DMVREKMEAK IHELGKQVVT E ------AK- 272 E.coli/1-319 191 ------FCDP HV-GVSLMADAV LSVMATNG--MR T SHHIANT IIEHRHDEAWLRQH IHNDAHY- DSLIAV IREK EET LKQRVIRA------LA- 267 K.pneumoniae/1-318 191 ------YGDPKL- GV SLVTDAVKLALARAN --TDT SSYNVDQ IIINRHDEEYLT DN INDP DAV- SEV KKV INNS IERLTTRVLTA ------ID- 267 S.enterica/1-326 192 ------WC DP NI-GVSLIT SGVKEQMAVHA- NT RVSSFQADN IIVHRNEPDY LSRR IYNA EQR- ESIINV INERQK LLIKRVNDV------IS- 269 A.nasoniae/1-327 195 ------SGDST I- GV SLVTNAVYNA LDRAS --TTTSY LVADQLV KKNDDEELYKT LINDKSKI- DDVKNSFNDAVK LLSDRVISH------IKH 272 K.pneumoniae/1-324 194 ------HGNPSV- GV SQVTR AAAGALR AAD--SET SALIADT VIRNRNDRQY LQRV INDAGK I- DEVRNK ITEA ITSLGARVT SE------LT- 270 E.coli/1-320 192 ------YGDSSL-GVSLVT SAVKDA LSLAR --TKGSSYLADD IIIHRKDNNY LKQR INDENK I- SIVT EAMN EALRKLEQRVLNT ------LN- 268 E.coli/1-323 194 ------HGNSG I- GV SSVTKAAMSALRMA S- -SDT SFLVADELIKRRNDP DFVRQV INDET KT-DLVLNT IEGA IASLGEQ VVNE------LG- 270 P.ingrahamii/1-335 197 ------YGNTEI-GVGLVT NT ARQALANAD- -SDA SY LVANELIKNRHDSEFVKEVINDLSQV-DVVLKKIEDRINELGQTVAEE------MK - 273 S.aureus/1-355 214 ------INK ----GT IDFYKR IASHVSKKSEGA SIT PRMI EKGLEYKQCK LNQKT VIDFKDEFYKEQDSLIEEVXSNFEITVGNI------288 M.extorquens/1-347 219 ------SGT ADV- GV LDVYKGVLDRV ------KGR FELDDDSLPLSLMN EAVRT GSMQ LFGKP- HDVSEIVSD VVGQVQERALR E ------IQR 292

410 420 430 440 450 460 470 480 H.sapiens/1-377 330 KIIAPPERKYSVWIGGSILASLST F QQMWIT KQ E ----YDEAGPSIVHRKCF ------377 C.elegans/1-362 315 KIIAPPERKYSVWIGGSILASLST F QQMWI SKQE----YDESGPSIVHRKCF ------362 D.discoideum/1-377 329 KIIAPPERKYSVWIGGSILASLST F QQMWI SK EE----YDESGPSIVHRKCFF------377 A.thaliana/1-377 330 K VVAPPERKYSVWIGGSILASLST F QQMWIAKAE----YDESGPSIVHRKCF ------377 S.cerevisiae/1-375 328 KIIAPPERKYSVWIGGSILASLTTF QQMWI SKQE----YDESGPSIVHHKCF------375 T.gondii/1-376 329 K VVAPPERKYSVWIGGSIL SSLST F QQMWIT KEE ----YDESGPSIVHRKCF ------376 T.vaginalis/1-375 329 KIVAPEERKYAVWV GGSILASLAT FPQMVIT KEE ----YDEAGPSIVHRKC------375 P.caudatum/1-375 328 K VVAPPERKFSVWIGGSIL SSLST FQAMWIT RSE ----YDESGPSIVHRKCF ------375 N.gruberi/1-345 297 RIT APPERKYTAW IGGS IFSK LST FQ SMCIT LEE ----YEESGYNI IHRKGIF ------345 H.sapiens/1-418 366 QV ITHHMQ RYAVWFGGSMLASTPEFYQVCHT KKD----YEEIGPSICRHNPVFGVMS------418 S.dysenteriae/1-319 272 T F- SGMNRIYLT --GGGA ELIYPHIKQY FPNL--KVNKVDEPQ FALVKAMV HA------319 V.furnissii/1-344 273 KFAKNPNR IYLV--GGGA SLIYPAVCAAYPSLGERVILIENAQ SALAQEICLYHADQ PET EMSDLP LAEEMSDE- 344 E.coli/1-319 268 GF-SGYGRVMVV--GGGA EIVAPAIREACGVNAT-FIADGVPQFALVNGLYAMDKE------319 K.pneumoniae/1-318 268 SF-KGY SHAIVI--GGGAPLVADA IRERMG LR EDRFVVAEEPQ FALVRGLK IIG ------318 S.enterica/1-326 270 RF-TDYT HVMCV --GGGA EIVAEAVKNLT KVPDER FY L SSSPQ FDLVMGMIKMK GGVT NE------326 A.nasoniae/1-327 273 EYKKSFNR IYLT--GGGA SLIYPAVKSAYPN- -QIIT IMEDSQT ALVT SIASMR AVKKQ ------327 K.pneumoniae/1-324 271 AF-RNVNRVFLV--GGGA SLIEEA IRQAWP LAHDRIEV IGDPQMALAR EIALYNKED ------324 E.coli/1-320 269 EF-SGYT HVMV I --GGGA ELICDAV KKHT QIRDER FFKTNNSQYDLVNGMY LIGN------320 E.coli/1-323 271 DF- HHVNRVYVV--GGGAPLIYDS IKT AWHHLGQKVVMM ESPQT ALVEAIAAFK EE------323 P.ingrahamii/1-335 274 KFCKNPNRVY IV--GGGAPLVYDAVKAAY SV LGDRVIILENAQ IALAKENM LYALSDG EEESVV------335 S.aureus/1-355 289 ---NSIDR IIVT--GGGANIHFDSLSHYYS---DVFEKADDSQFSNVRGY EK LGELLKNKVEQESKRGSHHHHHH 355 M.extorquens/1-347 293 RLGRAAT LQAVVFV GGGSAL- FKDIASAYPNG ----VMSDDP EFANARGLYKYFASRRRS ------347