TheRoleofMeH73inActinPolymerizationand ATP Hydrolysisactin/JMB_02.pdfTheRoleofMeH73inActinPolymerizationand ATP Hydrolysis TomasNyman1,HerwigSchu ¨ler1,ElenaKorenbaum1 ClarenceE.Schutt2,RogerKarlsson1andUnoLindberg1*

doi:10.1006/jmbi.2001.5436 available online at http://www.idealibrary.com on J. Mol. Biol. (2002) 317, 577±589

The Role of MeH73 in Actin Polymerization andATP Hydrolysis

Tomas Nyman1, Herwig SchuÈ ler1, Elena Korenbaum1

Clarence E. Schutt2, Roger Karlsson1 and Uno Lindberg1*

1Department of Cell BiologyThe Wenner-Gren InstituteStockholm University, S-106 91Stockholm, Sweden2Department of ChemistryHenry H. Hoyt LaboratoryPrinceton UniversityPrinceton, NJ 08544, USA

Present addresses: H. SchuÈ ler, DeBiochemistry, NYU Medical CenterYork, NY 10016, USA; E. KorenbauBiochemie I, Medizinische FakultaÈ tD-50931 KoÈ ln, Germany.

Abbreviations used: eATP, 1,N6-e50-triphosphate; EGTA, ethylene glyether-N,N0-tetra acetic acid; F-actin,G-actin, globular actin; Pi, inorganiccritical concentration for polymeriza

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0022-2836/02/040577±13 $35.00/0

In actin from many species H73 is methylated, but the function of thisrare post-translational modi®cation is unknown. Although not withinbonding distance, it is located close to the g-phosphate of the actin-bound ATP. In most crystal structures of actin, the d1-nitrogen of themethylated H73 forms a hydrogen bond with the carbonyl of G158. Thishydrogen bond spans the gap separating subdomains 2 and 4, therebycontributing to the forces that close the interdomain cleft around the ATPpolyphosphate tail. A second hydrogen bond stabilizing interdomain clo-sure exists between R183 and Y69. In the closed-to-open transition in b-actin, both of these hydrogen bonds are broken as the phosphate tail isexposed to solvent.

Here we describe the isolation and characterization of a mutant b-actin(H73A) expressed in the yeast Saccharomyces cerevisiae. The properties ofthe mutant are compared to those of wild-type b-actin, also expressed inyeast. Yeast does not have the methyl transferase necessary to methylaterecombinant b-actin. Thus, the polymerization properties of yeast-expressed wild-type b-actin can be compared with normally methylatedb-actin isolated from calf thymus. Since earlier studies of the actinATPase almost invariably employed rabbit skeletal a-actin, this isoformwas included in these comparative studies on the polymerization, ATPhydrolysis, and phosphate release of actin.

It was found that H73A-actin exchanged ATP at an increased rate, andwas less stable than yeast-expressed wild-type actin, indicating that themutation affects the spatial relationship between the two domains ofactin which embrace the nucleotide. At physiological concentrations ofMg2�, the kinetics of ATP hydrolysis of the mutant actin were unaffected,but polymer formation was delayed. The comparison of methylated andunmethylated b-actin revealed that in the absence of a methyl group onH73, ATP hydrolysis and phosphate release occurred prior to, and see-mingly independently of, ®lament formation. The comparison of b and a-actin revealed differences in the timing and relative rates of ATPhydrolysis and Pi-release.

# 2002 Elsevier Science Ltd.

Keywords: b-actin mutants; actin isoforms; methylhistidine-73;polymerization; ATP hydrolysis
*Corresponding author
partment of, 550 First Ave., Newm, Institut fuÈ rder UniversitaÈ t KoÈ ln,

thenoadenosinecol bisaminoethyl®lamentous actin;phosphate; Acc,tion.

ing author:

Introduction

Actin and myosin use free energy provided byhydrolysis of ATP to generate movements inmuscle and non-muscle cells. In this process, myo-sin is believed to be the central force-producingcomponent, whereas actin is seen as a relativelypassive element, whose role is to activate the myo-sin ATPase and bear the load during force gener-ation.1,2 However, actin also possesses an ATPase

# 2002 Elsevier Science Ltd.

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578 Analysis of Methylhistidine in Actin

activity thought to be important in controlling ®la-ment turnover in the cell (reviewed by Chen et al.3).At low salt concentrations, where the protein existsas monomers in solution, the nucleotide stabilizesthe protein. Furthermore, nucleotide hydrolysisand Pi-release during polymerization have beenimplied to in¯uence the ®nal structure of the ®la-ment formed.4 ± 6 Hydrolysis of ATP on actin andproduct release might also be intrinsic to force gen-eration during muscle contraction and non-musclecell motility.7,8 Therefore, it is of great interest toexplore the mechanism by which actin cleavesATP. We have undertaken a series of investigationsexamining the role of the amino acid residues inand around the nucleotide-binding site duringATP hydrolysis and Pi-release.9 ± 11

Actins from most eukaryotes carry a methylatedhistidine at position 73.12 ± 14 This residue is of inter-est in connection with ATP hydrolysis in actinbecause its d1-nitrogen can form a hydrogen bondwith the carbonyl group of G158, thereby contact-ing the G156-V159 loop that chelates the phosphatetail of ATP.15 This hydrogen-bond bridges the cleftseparating the two major domains of actin. Thepresence of a methyl group on MeH73 confers apositive charge on the side-chain. This enables H73

Figure 1. Comparison of the tight and open states of actinoutlines the area shown in stereo to the right. The barrier rbinding loop-residues S14, D157 and G158; and the hydrogthe open state (bottom row) the b and g-phosphates (yellowhairpin loops chelating the ATP phosphate tail are coloredhelix is colored yellow. All molecular graphics were prepared

to participate in a network of electrostatic inter-actions comprising charged residues E72, MeH73,R177, D179, and R183 that constitute a barrierbetween the terminal phosphate and the solvent(Figure 1). In the crystal structures, the imidazoleof H73 does not make direct contact with the term-inal phosphate, but it is in a position to mediateconformational changes in the hairpin loops associ-ated with nucleotide binding thereby governingnucleotide hydrolysis and polymerization.

The methyl group of S-adenosyl methionine istransferred to H73 by a methyl transferase thatspeci®cally recognizes a peptide in vivo corre-sponding to residues 69-77 of actin.16 The modi®-cation is highly conserved among actins fromdifferent species.17,18 For a long time the onlyknown exception was actin isolated from Naegleriagruberi which has an unmethylated histidine 73.19

However, it was reported recently that the yeastsCandida albicans and Saccharomyces cerevisiae alsolack this modi®cation, suggesting that methylhistidine may be present only in multicellulareukaryotes.20

Two highly conserved b-hairpin loops chelatethe phosphate tail of ATP between the two majordomains of actin. Residues within these loops,

in the pro®lin:b-actin crystals. The overview to the leftesidues E72, H73, R177, D179 and R183; the phosphateen-bond forming residues Y69 and R183 are labeled. In) of the ATP are exposed. In the left overviews, the b-

red, the site of H73 is colored green, and the Q137-S145with RasMol.66

Table 1. Dissociation rate constants for the binding ofDNase I and ATP to actin

Property/cation b-Actinyeast H73A

Kd (DNase I)/Ca2� (nM) 0.28 � 0.04 6.84 � 0.38Kd (DNase I)/Mg2� (nM) 0.66 � 0.09 5.24 � 0.47(kÿATP)/Ca2�(sÿ1) 1.4 � 0.2 � 10ÿ3 1.2 � 0.4 � 10ÿ2

(kÿATP)/Mg2� (sÿ1) 1.6 � 0.3 � 10ÿ2 1.5 � 0.3 � 10ÿ1

The Kd for DNase I:actin interaction was determined usingthe DNase I inhibition assay. The dissociation rate constant forATP (kÿATP) was determined from the rate of eATP-incorpora-tion.

Figure 2. Thermal stability of G-actin. The stability ofb-actinyeast, b-actincow, and H73A-actin was determinedusing the DNase I-inhibition assay. The DNase I-inhi-bition activity at 25 �C was normalized to 100 for eachactin. Circles, b-actinyeast; squares, b-actincow; triangles,H73A-actin. Filled symbols, Ca-actin; open symbols,Mg-actin.

Analysis of Methylhistidine in Actin 579

S14,9 D157,9,21 and V159,22 and residues in thevicinity of the nucleotide binding site, e.g. R1779,23

and H73,24 have been found to be crucial for main-taining the interdomain geometry in actin. How-ever, residues directly involved in the ATPhydrolysis reaction have not yet been identi®ed.

Earlier work on the H73 of actin involves copo-lymerization of actin mutated at position 73 withwild-type actin,25,26 and an extensive study of actinwhere H73 was replaced by several differentamino acid residues.24 However, there have beenno reports on the effects of mutating MeH73 onATP hydrolysis and phosphate release; nor has thesigni®cance of methylation for ATPases in generalbeen explored.

Here, chicken b-actin with histidine 73 replacedby either alanine (H73A) or aspartic acid (H73D)was expressed in S. cerevisiae. Both mutant actinswere puri®ed by DNase I-af®nity chromatography,but only the H73A-actin could be isolated fromendogenous yeast actin. The characterization ofmutated actin (H73A) with respect to DNase I-binding, nucleotide exchange, thermostability,ATPase activity, polymerizability and phalloidin-binding in comparison with non-methylated wild-type actin expressed in S. cerevisiae is describedhere. In addition, non-methylated yeast-expressedwild-type b-actin is compared with methylatedmammalian b and a-actin. The results con®rm thatH73 is important in stabilizing actin in monomericas well as polymeric form, and that in the absenceof a methyl group on the e2 nitrogen of H73, ATPhydrolysis and phosphate release occurs inadvance of polymerization.

The present work also demonstrates that there isa signi®cant difference between the a and b-actinisoforms in the timing of ATP hydrolysis andphosphate release during polymerization. Themechanism of Pi-release and the role of MeH73 inthe interdomain relationship in actin are discussed.

Results

Isolation of mutant actins

Chicken b-actin expressed in S. cerevisiae waspuri®ed from cell extracts using DNase I-af®nitychromatography, and separated from endogenousyeast actin by chromatography on hydro-xylapatite.27,28 The H73A mutant actin was elutedfrom hydroxylapatite at the same ionic strength aswild-type b-actin, well separated from the yeastactin. It was recently shown that yeast actin is notmethylated.20 Also, unlike mammalian actin, yeast-expressed actin retains its N-terminal methionine.29

To distinguish actins expressed in yeast from wild-type mammalian actin, the following nomenclaturewill be used: wild-type mammalian b-actin puri-®ed from calf thymus is referred to as b-actincow;yeast-expressed wild-type b-actin as b-actinyeast

and the H73A mutant actin as H73A-actin. Itshould be noted that the mammalian b-actin isidentical in sequence to chicken b-actin. One actin

mutant, H73D actin, bound to DNase I, but con-ditions for its separation from yeast actin have yetto be found, and it will not be discussed further.

Actin:DNase I-interaction

The binding site on actin for DNase I involvesparts of both subdomains 2 and 4.15 Thus, DNase Ispans the cleft between the two domains of actin atthe (ÿ)-end (so called pointed, or slow growing,end) of the molecule. Earlier work showed that theDNase I-inhibiting activity of actin can be used toprobe interdomain relationships in the molecule.30

It is shown here that the af®nity of b-actinyeast forDNase I is closely similar to that of mammalian band g-actins, whereas the af®nity of H73A-actin forDNase I was greatly decreased (Table 1). Thus,exchanging Ala for His at position 73 causes a sig-ni®cant alteration at the DNase I-binding site, eventhough H73 is 10 AÊ away.

Thermostability

Since the alteration in the DNase I-inhibitingactivity caused by the H73A-mutation suggests


relative changes in the orientation of the two majordomains of actin, it was of interest to investigatethe thermal stability of the different actins. Forthis, the DNase I-inhibition assay was employed asdescribed earlier.30 As shown in Figure 2, the Mgand Ca-forms of b-actinyeast lost their DNase I-inhi-biting activity in a single transition with a t1/2 of55 �C and 58 �C, respectively. The H73A-actin wasless stable, with t1/2-values of 50 �C and 55 �C forthe Ca and Mg-form, respectively. Comparison ofthe thermal stability of b-actinyeast and b-actincow

showed that the non-methylated, incompletelyN-terminally processed b-actinyeast was somewhatmore stable than the methylated, N-terminally pro-cessed counterpart in both the Mg and Ca-states.

Nucleotide exchange

To investigate whether the H73A-mutation in¯u-enced the binding of ATP to actin, measurementsof nucleotide exchange rate were performed. Forthis, ATP-actin was incubated with eATP in theabsence of excess ATP, and the increase in ¯uor-escence resulting from eATP binding to actin wasmonitored. Under the conditions used, including alarge excess of eATP, rebinding of ATP is negli-gible and the rate of incorporation of the ¯uor-escent nucleotide therefore represents the off-ratefor ATP (kÿATP).31 Table 1 shows that for both theMg and the Ca-form, the kÿATP for H73A-actin wastenfold higher than for b-actinyeast.

G-actin ATPase

To evaluate the effect of mutating His73 on theintrinsic ATPase activity of actin, the activities ofboth the Ca and Mg-forms of b-actinyeast andH73A-actin were examined. Figure 3 shows that b-actinyeast in the Ca-form under non-polymerizingconditions hydrolyzed ATP at a rate of 0.05/hour,

Figure 3. ATPase activity of G-actin. The amount ofinorganic phosphate generated by 12 mM Ca- and Mg-actin in buffer G was monitored using the phosphomo-lybdate precipitation assay. Filled symbols, b-actinyeast;open symbols, H73A-actin; circles Mg-actin, triangles,Ca-actin.

whereas the H73A Ca-actin apparently was unableto hydrolyze ATP. In the Mg-form, b-actinyeast

exhibited a fourfold higher ATPase activity (0.2/hour) than in the Ca-form, while the H73A-actin inthe Mg-form expressed only a weak activity (0.02/hour). Thus, under non-polymerizing conditions, itseems that a histidine residue at position 73 isnecessary for actin to hydrolyze ATP.

Polymerization, ATP hydrolysis and Pi-release

In 0.1 mM CaCl2 and 100 mM KCl, theb-actinyeast polymerized to steady state in abouttwo hours (Figure 4(a)). The ATP hydrolysis andphosphate release preceded polymerization duringthe elongation phase. The rate of hydrolysis contin-ued undiminished after steady state of polymeriz-ation had been reached. The overall rate ofhydrolysis during polymerization was 0.5 hÿ1. TheH73A mutant did not form ®laments nor did ithydrolyze ATP under these conditions(Figure 4(d)).

With Mg2� at the high-af®nity cation bindingsite (50 mM MgCl2), and with 100 mM KCl addedto the solution (Figure 4(b) and (e)), the b-actinyeast

polymerized to steady state in 15 minutes and ATPhydrolysis occurred simultaneously with, orslightly ahead of ®lament formation as probed bythe pyrenyl-assay. The H73A-actin was able toform ®laments under these conditions althoughpolymerization did not reach steady state untilafter 25 minutes. As in the case of b-actinyeast ATPhydrolysis preceded ®lament formation.

With Mg2� at the high-af®nity binding site, andwith 1 mM MgCl2 and 100 mM KCl in the solution(Figure 4(c) and (f)), polymerization was faster forboth b-actinyeast and the H73A mutant, reachingsteady state in 12 and 20 minutes, respectively. Theeffect of the additional Mg2� on ATP hydrolysiswas remarkable in that both actins hydrolyzedATP well ahead of ®lament formation, and theamount of ATP consumed at steady state washigher than that expected from the amount of actinadded. In these experiments as well as in those ofFigure 4(b) and (e), ATP hydrolysis proceededwith the same kinetics for both b-actinyeast as wellas H73A-actin, independently of the polymeriz-ation rate.

It is interesting that the gain in polymerizabilityof the H73A-actin (Figure 4(d) and (e)) and theincreased rate of ®lament formation of theb-actinyeast (Figure 4(a) and (b)) apparently are notsolely effects of adding Mg2�, since the mereremoval of Ca2� by EGTA restored polymerizabil-ity to the H73A mutant actin (Figure 5).

S. cerevisiae does not methylate histidine 73 inactin.24 The availability of methylated b-actin iso-lated from calf thymus offered the possibility ofinvestigating the speci®c effect of the methyl groupon the imidazole on ATP hydrolysis and phos-phate release. Figure 6 shows that with Mg-b-actincow supplemented with 1 mM MgCl2 and100 mM KCl, ATP hydrolysis and phosphate

Figure 4. Comparison of b-actinyeast ((a)-(c)) and H73A-actin ((d)-(f)). Filament formation was measured by the pyr-enyl-assay and the concomitant formation of inorganic phosphate was determined by TLC. All measurements wereperformed with 8 mM actin. (a) and (d) Ca-actin � 100 mM KCl. (b) and (e) Mg-actin (50 mM MgCl2) � 100 mM KCl.(c) and (f) Mg-actin � 100 mM KCl and 1 mM MgCl2. Circles, polymerization; ®lled triangles, ATP hydrolysis; opentriangles, Pi-release.


release followed closely the progress of ®lamentformation, whereas b-actinyeast under the same con-ditions hydrolyzed ATP and released Pi prior to®lament formation.

In the polymerization/ATPase experimentsdescribed above, Pi-release appeared to occur sim-ultaneously with ATP hydrolysis. Earlier reportswith a-actin have described a delay betweenhydrolysis and Pi-release.32,33 Thus, it was of inter-est to use the ATPase and Pi-release assaysemployed here in a direct comparison between band a-actins. As seen in Figure 7, a-actin displays aclear delay between hydrolysis and Pi-release, con-®rming previous observations and demonstratinga clear difference between the muscle and non-muscle actins.

Viscometry

To obtain information about the quality of thepolymers formed from the H73A-mutant, ®lamentformation was studied by high shear viscometry(Figure 8). Addition of 1 mM MgCl2 and 100 mMKCl to H73A-actin monomers in the Mg-form ledto a viscosity increase after a much longer lagphase than in the case of b-actinyeast. Furthermore,both the maximal rate of viscosity increase of themutant protein and the steady state level of vis-cosity was signi®cantly lower. Since the Acc ofH73A was only slightly increased in comparisonwith b-actinyeast (Table 2), this suggests that theH73A-mutation affected not only the assemblyprocess, but also the stability of the ®laments.

Figure 5. The effect of EGTA on the polymerization ofCa-actin. Filament formation was induced by theaddition of 100 mM KCl to 8 mM Ca-actins, and moni-tored using the pyrenyl assay. After 270 minutes, EGTAwas added to 0.2 mM. Filled and open circles denote b-actinyeast and H73A-actin, respectively.

Figure 6. Comparison of b-actincow and b-actinyeast

with respect to polymerization, ATP hydrolysis andphosphate release. Polymerization of Mg-actin (8 mM)was induced by 100 mM KCl and 1 mM MgCl2 and fol-lowed using the pyrenyl-assay. Production of inorganicphosphate was monitored by TLC. (a) b-Actincow. (b) b-Actinyeast. Circles, polymerization; ®lled triangles, ATPhydrolysis; open triangles, Pi-release.


Decoration with myosin subfragment 1

The morphology of the ®laments formed fromH73A-actin could not be distinguished from thoseof b-actinyeast as analyzed by electron microscopyafter negative staining, and the mutant ®lamentsbound myosin-S1 fragments, giving rise to the clas-sical arrowhead structure (not shown).

Binding of phalloidin

Histidine 73 is close to residues G158, R177, andD179, which have been implicated in the bindingof phalloidin to actin in the ®lamentous form.21

Figure 9 shows the binding of phalloidin toa-actin, b-actinyeast and H73A-actin, as a functionof F-actin concentration. The slopes of the bindingcurves are approximately equal, indicating that theH73A mutation did not severely affect phalloidinbinding. The rise of the H73A binding curveoccurred at a higher actin concentration, againillustrating the increased Acc of the actin mutant.

Discussion

Conformationally different states of actin

There are biochemical, spectroscopic and elec-tron microscopic observations indicating that the

Table 2. Critical concentration for polymerization for b-actin

Cation Salts added

Ca2� KClMg2� KClMg2� KCl�MgCl2

The Acc values were determined by the polymerization of a seriesaf®nity cation bound to actin prior to polymerization is given in th100 and 1 mM, respectively.

actin monomer has different conformationsdepending on the status of the actin-bound ATP,the nature of the divalent cation (Ca2� or Mg2�) atthe high af®nity site or at additional sites, and tothe degree of oligomerization.34,35 Crystallographicinvestigations on pro®lin:b-actin have directlydemonstrated that actin can exist in a tight and anopen state, which differ signi®cantly in con-formation.36,37 The most dramatic differencebetween the two structures is the opening of theinterdomain cleft. It comes about by a 10 � rotationof subdomain 1, with the Q137-S145 helix andR335 as pivot points38,39 (Figure 1), which resultsin an outward shift of the N12-C17 loop, exposingthe phosphate tail of ATP to solution.37

yeast and H73A-actin

b-Actinyeast (mM) H73A (mM)

2.1 >120.2 0.650.2 0.45

of actin concentrations using the pyrenyl-actin assay. The high-e ®rst column. In the second column, KCl and MgCl2 refers to

Figure 7. Polymerization and ATPase activity of a-actin. Experiments were performed as in Figures 4 and6, using 8 mM Mg-actin. (a) Polymerization induced byaddition of 100 mM KCl. (b) Polymerization induced byaddition of 1 mM MgCl2 and 100 mM KCl. Circles, pol-ymerization; ®lled triangles, ATP hydrolysis; open tri-angles, Pi-release. In (a), the graph describing hydrolysisof ATP levels off before an amount of ATP stoichio-metric to the amount of actin added has been hydro-lyzed, and in both experiments the ®nal amount of Pi

released appears to be slightly higher than the totalamount of ATP hydrolyzed. These observations werereproducible (n � 3), and might be explained by some ofthe radioactive Pi being trapped in the protein precipi-tate on the TLC plate. This anomaly, however, does notaffect the interpretation of the results.

Figure 8. Filament formation analyzed by viscometry.Polymerization of Mg-actin (7 mM) was induced by theaddition of salt to 1 mM MgCl2 and 100 mM KCl. Filledand open circles denote b-actinyeast and H73A-actin,respectively.

Figure 9. Binding of phalloidin to ®lamentous actin.Actin at increasing concentrations was polymerized inthe presence of 0.35 mM rhodamine phalloidin and2 mM MgCl2. After the polymerization had reachedsteady state (two hours) the rhodamine ¯uorescencewas measured. Open circles, H73A-actin; ®lled circles,b-actinyeast; triangles, a-actin.


In the tight state of actin, the terminal phos-phates of the nucleotide are bound by the b-hairpinloops, N12-C17 and D156-V159, which protrudeinto the interdomain cleft from subdomains 1 and3, respectively. The b-phosphate is hydrogen-bonded to the amide nitrogen atoms of S14, G15,M16, and D157, and the g-phosphate is bound toamide nitrogen atoms of S14, D157, G158 andV159. In the open state, the hydrogen bond withthe amide nitrogen of G15 shifts from the O1 oxy-gen to the O2 oxygen of the b-phosphate, while thehydrogen bonds of G158 and V159 to the g-phos-phate are broken.37 In the tight state, there are twohydrogen bonds that span the nucleotide-bindingcleft. These two bridging hydrogen bonds, betweenMeH73 and the carbonyl oxygen of G158 andbetween the guanido group of R183 and the p-elec-trons of the ring of Y69,40 stabilize the closed ATP-

form of the actin molecule. In the open state, thesehydrogen bonds are broken.

In addition to the loop-phosphate-loop links andthe bridging hydrogen bonds, the two majordomains of actin are connected through a chargenetwork involving mostly long-chained residues:E72 and MeH73 from one side of the cleft andD157, R177, D179 and R183 from the other. Thelong-chained residues shield the ATP phosphatesfrom the solvent on one side of the molecule. Onthe opposite side, there is another set of large resi-dues (M16, K18, K336, and Y337) separating thepolyphosphate tail from solvent in both the openand tight states. The transition from the tight toopen state does not result in any major changes inthis barrier region.


The structures of different actins determinedfrom crystals of DNase I:a-actin,15 gelsolin subfrag-ment I:a-actin complexes,41 gelsolin subfragmentI:yeast actin,42 and gelsolin subfragment I:Dictyoste-lium-actin42,43 all correspond to the tight statefound for b-actin in the pro®lin:b-actin crystals. Ithas been noted that the conformation of actinappears relatively unchanged regardless ofwhether the nucleotide is ATP or ADP, or whetherthe tightly bound divalent cation is Ca2� or Mg2�.This may seem contradictory in the light of manyobservations indicating that the actin can attaindifferent conformations depending on the natureof bound ligands.35,44 The invariability in thea-actin crystal structures may be explained byclamping of the two domains by DNase I-bindingacross the cleft between subdomains 2 and 4 ofactin in the DNase I:actin crystals, or by packinginteractions in the case of the gelsolin subfragment1:a-actin crystals. In the pro®lin:b-actin crystals,the actin is similarly bridged across subdomain 1and 2 by a neighboring actin molecule that mimicsthe contact that is made by DNase I to actin. How-ever, in this case the cleft can open and close inresponse to changes in ionic conditions made poss-ible through intramolecular hinging and shearingmovements that are coordinated between thebound actin molecules.37,45 It is also knownthat exchange of ADP and AMP for ATP in thepro®lin:actin crystals signi®cantly in¯uences theirdiffraction.45

Interdomain relationships in actin

Exchanging ATP with ADP in actin results in asigni®cant reduction in the stability of the protein,and removal of the nucleotide leads to a ratherrapid loss in polymerizability.46 As shown pre-viously, the effects of mutations in the nucleotide-binding cleft on the spatial relationship betweenthe major domains of actin can be evaluated bydetermining the DNase I-inhibiting activity, ther-mal stability and nucleotide exchange rate of themutated protein.30 The H73A mutation, as well asmutations in the loops binding the phosphatecause signi®cant decreases in the DNase I-inhibit-ing activity of the actin, whereas the previouslydescribed R177D mutation did not. All fourmutations (H73A, R177D,10 S14C,9 and the doublemutation S14C/D157A9) made the actin less stableat increased temperature, increased the nucleotideexchange rate, and reduced the rate of polymeris-ation. This further emphasizes the importance ofthe non-covalent bonds between the two majordomains to the stability and polymerizability ofactin. The ATPase activity of the actins, however,was not altered much by the mutations, whenassayed in the presence of Mg2�.

The results obtained here with b-actin mutatedat histidine 73 are in close agreement with thosereported by Yao and co-workers.24 These investi-gators reported that replacing H73 with positivelycharged residues, arginine or lysine, stabilized

actin, whereas the introduction of glutamic acidwas destabilizing. This is explainable in terms ofthe coordinating position of H73 in the charge net-work, illustrated in Figure 1.

Recently, a crystal structure of uncomplexedactin in the ADP state was reported.47 Actin waskept in a monomeric state by a covalent binding oftetramethylrhodamine maleimide (TMR) to the Cterminus. The position of subdomain 2 is differentin this structure as compared to previously deter-mined a-actin structures. It has moved via arotation around the same hinge region that isinvolved in the movement of subdomain 2 in thetight-to-open state transition of pro®lin:b-actin.37

However, the rotation of subdomain 1 with respectto subdomains 3 and 4, intrinsic to the opening ofthe nucleotide binding cleft, is not seen in TMR-actin. The changes of subdomain 2 in TMR-ADP-actin might be due not just to the transition fromATP to ADP state but also to crystal contacts, or tothe tight binding of TMR to the base of subdomain1. Evidence for allosteric coupling between the Cterminus and subdomain 2 has been reported ear-lier.48,49

ATPase activity and polymerization

It is commonly held that ATP hydrolysis islinked to the polymerization of actin and that Pi-release takes place only after ®laments haveformed. The results shown in Figure 7 con®rm thatthis is true for skeletal muscle a-actin. However,the non-muscle b-actincow is different in thisrespect, hydrolyzing ATP slightly ahead of theappearance of actin polymers with Pi-release clo-sely following ®lament formation. This implies thatthe use of a-actin might not be appropriate in stu-dies of the interactions of non-muscle actin-bindingproteins with actin.

In both b-actinyeast and the H73A mutant actin,nucleotide hydrolysis and Pi-release in 1 mM Mg2�

occurred well ahead of ®lament formation and fol-lowed closely similar time courses. A comparisonof the results shown in Figures 4 and 6 indicatesthat the main disturbance in the behavior of the b-actinyeast and H73A mutant actin is not so much inthe timing of nucleotide hydrolysis itself, as in thedelayed appearance of actin polymers. It is wellknown that adenosine trisphosphate is importantin holding actin in a polymerization-competentform, which is illustrated by the three to ®vefoldslower polymerization of ADP-actin.50 It is possiblethat after hydrolysis of ATP, the methylated H73stabilizes ADP-Pi actin in an ATP-conformationlong enough for rapid ®lament nucleation andelongation to occur. In the cases of b-actinyeast andH73A mutant actin, where H73 is not methylated,hydrolysis of ATP and Pi-release might rapidlyconvert actin from an ATP-state to the ADP-state.Thus, what is observed in these cases would be thepolymerization of ADP-actins, explaining thedecreased polymerization rates. This does notexclude the possibility of a coupling between Pi-


release and ®lament formation in the case of wild-type actins with a methylated H73.

Nucleotide hydrolysis was slowed down in bothb-actinyeast and H73A mutant actin under certainconditions, but was largely restored by theaddition of Mg2�, whereas polymer formation wasmuch retarded. While it is true that the N terminusof actin is not identical between bovine and yeast-expressed b-actin (the N-terminal methionine ofactin is retained in yeast29), the reason for the pro-nounced difference in ATP hydrolysis versus poly-mer formation is more likely due to the lack of themethyl group on H73, or in the case of the H73Amutant to the lack of the whole histidine side-chain.

In the absence of Mg2� (Figure 4(d)), the H73A-actin did not form ®laments and ATP hydrolysiswas very slow. Adding EGTA under these con-ditions (Figure 5) restored the polymerizability ofthe mutant actin even without the addition ofMg2�. Since Ca2� should still be bound at the highaf®nity site under these conditions, the stimulatoryeffect of EGTA on the polymerization appears tobe due to the withdrawal of Ca2� bound to second-ary divalent cation-binding sites, but this effectcannot yet be explained in structural terms. Adirect effect of EGTA on the protein cannot beexcluded.

Figure 10. The localization of His73 is similar in allstructures of ATP-actin in the tight state. (a) Stereo viewshowing the hydrogen bonds connecting MeH73, G158,V159, and the g-phosphate of ATP as shown in theclosed state of the pro®lin:b-actin structure. Thesehydrogen bonds are all broken in the open state. Thenumbers denote bond lengths in AÊ . (b) Schematic draw-ing showing the electrostatic and hydrogen-bondinginteractions of the methylated His73.

The differences between the a-actin and b-actinisoforms seen here might be a re¯ection of thenecessity for a more rapid turnover of actin®laments in motile processes in non-muscle cells,compared with the more static organization ofactin ®laments in sarcomeres that produce largeforces. It is also possible that actin isoform differ-ences in the timing of phosphate release re¯ectsubtle variation in free energy coupling mechan-isms underlying force generation in the cytoplasmcompared with muscle ®bers.

The importance of methylation

The signi®cance of methylation of H73 in actinmay be understood from its positioning (Figure 10)and biochemical properties. When the MeHis73side-chain is protonated, it is fully substituted andcarries one positive charge, shared between thetwo nitrogen atoms. The charge lends salt-bridgecharacter to the MeH73-G158 hydrogen bond, i.e.making it a strong hydrogen bond. Due to p-elec-tron cooperativity, the G158-V159 peptide bond ispolarized through the electron-pull from MeH73,increasing the positive nature of the V159 back-bone nitrogen. This polarization is furtherenhanced by the charge on MeH73. This chain ofelectrostatic interactions strengthens the V159-g-phosphate hydrogen bond. The positive charge ofthe protonated MeH73 is also vital for the inter-actions with its neighbors E72 and D179. Thus, thecharged MeH73 side-chain makes contact withthree residues, two of them across the base of thenucleotide-binding cleft.

Histidine 12 in bovine pancreatic ribonuclease Ais positioned as H73 in actin, with the d1-nitrogeninvolved in a hydrogen bond to a backbone oxy-gen, and the e2-nitrogen exposed to bulk solution.In this particular histidine, the pKa of the d1-nitro-gen has been determined to be higher than 8,whilst that of the e2-nitrogen is around 6.2.51 Thisimplies that in an unmethylated form of actin, theprobability for the H73 e2-nitrogen to be proto-nated is low. As a consequence, the interactionsbetween H73 and its neighbors in the charge net-work are affected, weakening the strength of boththe H73-G158 as well as the V159-g-phosphatehydrogen bond.

It is clear from this analysis that a positivelycharged residue at position 73 plays a central rolein stabilizing a network of electrostatic interactionsthat bind the polyphosphate tail of ATP in actin.The methylated His in this position in the tightstate of actin is hydrogen-bonded via its d1 nitro-gen. Therefore, its pKa is likely to be elevated tomore than 8.51 In the open state, the H73-G158hydrogen bond is broken and the d1-nitrogen isexposed to solvent causing a dramatic drop in thepKa of the d1-hydrogen. The effect is that the d1-hydrogen is easily lost into solution and theMeH73 side-chain loses its positive charge, abolish-ing the possibilities for electrostatic interactions. Inconclusion, MeH73 may act as a switch in the cleft


opening mechanism, where cleft opening leads todeprotonization, causing further weakening of theinteractions between the two halves of actin.

On the ``front-door'' mechanism of phosphaterelease in actin

In the presence of Mg2�, the actin ATPaseactivity is greatly stimulated. Under these con-ditions, the region near Y69 (R62 - K68) is pro-tected from proteolysis52 suggesting that theinterdomain cleft is closed. Thus, it is possible thatthe binding of Mg2� to actin stabilizes the tightstate of actin allowing nucleotide hydrolysis totake place. Following ATP hydrolysis, breakage ofthe interdomain hydrogen-bond bridges mightallow the opening of the cleft, facilitating release ofthe g-phosphate directly into the solvent. This isclearly seen in the tight-to-open transition, inwhich actin attains a state where the cleft opens upto expose the phosphates (Figure 1).

The phosphate release mechanism envisionedhere is quite different from the ``back-door'' mech-anism proposed earlier,53 which involves a solventchannel underneath H73, rather than through thefront-door suggested by crystallographic analysis(Figure 1). It was proposed, on the basis of molecu-lar dynamics simulations on yeast actin, that for-mation of a salt-bridge between H73 and D184creates a state facilitating Pi-release. This wasthought to be part of a series of transfers, involvingalso R177, that ushers the phosphate ion through asmall solvent channel (the back door) connectingthe interior of the protein to the bulk solvent. Itshould be mentioned that the sequence around thenucleotide-binding site in yeast actin is highly con-served, except that yeast actin lacks the methylgroup on H73.

As shown in Figure 1, MeH73 and D184 in thecrystallographically determined open state of theb-actin, have actually moved further apart, allow-ing D184 to form a salt-bridge with R183 ratherthan with H73. Also, in the tight-to-open state tran-sition, R177 of b-actin moves from hydrogen bond-ing with the backbone atoms of MeH73 to a salt-bridge interaction with the D179. These consider-ations suggest that changes in actin conformationcontributing to an opening of the cleft allow theinorganic phosphate to escape directly into the sol-ution via the front-door. Actin-binding proteinscould trigger these events.

Materials and Methods

Protein preparation

Site-directed mutagenesis of the chicken b-actingene, its subcloning into the S. cerevisiae expressionvector, fermentation of the yeast and isolation of wild-type and mutant b-actins were performed asdescribed.9,28 It should be noted that chicken b-actin isidentical in sequence to mammalian b-actin. Bovine b-actin was isolated from calf thymus pro®lin:actin,54

and rabbit skeletal muscle a-actin was isolated as

described earlier.55 The intactness of the actin C termi-ni was veri®ed by the increase in absorbance uponbinding of Cu2� to actin56 as detailed.9 This is import-ant, since actin that has lost its C-terminal amino-acidresidues behaves abnormally.52,57 Actin with Mg2� atthe high af®nity cation-binding site was obtained byincubating Ca-actin in buffer G (0.5 mM ATP, 0.1 mMCaCl2, 0.5 mM DTT, 5 mM Tris, pH 7.6) sup-plemented with 50 mM MgCl2 and 0.2 mM EGTA forten minutes at room temperature.58 Pyrene-labelingwas done as described.59 Actin concentration wasdetermined from the absorbance at 290 nm using anabsorbance coef®cient of 0.63 ml mgÿ1 cmÿ1.60

DNase I-affinity and monomer stability

The af®nity of the actin:DNase I-interaction was deter-mined from double-reciprocal plots of the DNase I-inhi-bition as a function of actin concentration. The DNase I-inhibition was measured at 25 �C using the DNase I-inhi-bition assay.61 To determine the thermostability of theH73A-actin monomer, the DNase I-inhibition assay wasused as described.30 Brie¯y, Ca and Mg-actin as de®nedabove (7.2 mM) in G-buffer were incubated at 40 �C forten minutes. The temperature was then raised by twodegrees every three minutes. Samples of the incubationmixtures were withdrawn and their DNase I-inhibitingactivity measured at the end of each three-minuteperiod. The temperature at which the DNase I-inhibitingactivity was reduced to 50 % of the activity measured at25 �C was de®ned as t1/2.

Nucleotide exchange

G-actin (in either the Mg or Ca-form) was freed fromexcess nucleotide by gel ®ltration over a Sephadex-G25column (PD-10, Pharmacia, Sweden) equilibrated withATP-free G-buffer (with and without 0.2 mM EGTA and50 mM MgCl2, respectively). Thereafter the protein con-centration was adjusted to 6 mM. After addition of300 mM eATP (Molecular Probes, Eugene, OR), the ¯uor-escence increase at >408 nm (lex � 360 nm) was moni-tored using a Sigma ZWS II spectro¯uorimeter (BiochemWissenschaftliche GeraÈ te GmbH, Puchheim, Germany).Under these conditions, rebinding of ATP is negligible,and the rate of incorporation of the ¯uorescent nucleo-tide represents the off-rate for ATP (kÿATP), e.g. Kinosianet al.31 Estimates of kÿATP were made by ®rst-order curve®tting of the experimental data using Origin (MicrocalSoftware, Northampton, MA).

Polymerization assays

Polymerization of actin was measured with eitherCa2� or Mg2� at the high-af®nity binding site (Ca andMg-actin, respectively) at 25 �C and pH 7.6 using thepyrenyl-assay.59 Polymerization was initiated byaddition of either KCl to 0.1 M ®nal concentration, orKCl and MgCl2 to 0.1 M and 1 mM, respectively. Fila-ment formation was monitored by following the increasein ¯uorescence due to the presence of 2 % pyrene-labeledbovine b/g-actin (lex � 365 nm; lem � 410 nm). Fluor-escence measurements were performed using a micro-plate reader (Fluoroskan II, Labsystems, Finland). Thecritical concentrations for polymerization of b-actinyeast

and H73A-actin in different salt conditions were deter-mined essentially as described.32,62 Actin was mixed with2 % pyrene-labeled actin and diluted to concentrations


ranging between 0.02 and 2.0 mM. After addition of poly-merizing salts the reaction mixtures were incubated for14 hours at 20 �C. The resulting steady-state ¯uorescencewas recorded as above.

ATP hydrolysis and phosphate release

The inorganic phosphate formed by the intrinsicATPase activity of G-actin was measured by the phos-phomolybdate procedure of Sugino and Miyoshi63 asmodi®ed by Spudich.64 All other ATPase experimentswere carried out as follows. Actin at 8 mM was equili-brated with [g-32P]ATP (0.1 Ci/mmol ATP, ®nalactivity), MgCl2 and EGTA prior to the onset of ®lamentformation. Polymerization was induced by the additionof 1.0 mM MgCl2 or 100 mM KCl, or both, aliquots of0.5 ml were withdrawn at different time points andloaded onto TLC plates cut to 4 cm � 8 cm (PEI celluloseF, plastic sheets, Merck, Darmstadt, Germany). Theabsorption of actin to the cellulose quenched furtherATP hydrolysis. The TLC plates were dried thoroughly,developed in 0.2 M NH4HCO3 (pH 8), dried and placedin a storage phosphor screen cassette (BAS cassette 2325,Fuji Photo Film Co., ltd) for 20-30 minutes. The phosphorscreen was read using a Fuji®lm FCA-3000 (Fuji PhotoFilm Co., ltd), and the relative amounts of 32Pi and[g-32P]ATP were quanti®ed using the program ImageGauge (Fuji Photo Film Co., ltd). The amount of hydro-lyzed ATP was calculated as follows: in each sample, theradioactivity of the generated 32Pi relates to the totalradioactivity (32Pi � [g-32P]ATP) as the Pi relates to thetotal amount of ATP added to the buffer, which was0.5 mM. Thus, the amount of hydrolyzed ATP,expressed in mM is (32Pi/total) � 500-to, where to is theamount of Pi in the reaction mixture immediately priorto the addition of polymerizing salt.

For measurement of phosphate release from polymer-izing actin, the reaction mixture was prepared asdescribed for the TLC assay above. At each time point a30 ml aliquot was withdrawn and placed in a 200 ml ®l-tration ®lter unit with a molecular cutoff of 30 kDa(Ultrafree-MC, Millipore Corporation, Bedford, MA,USA). The sample was centrifuged for ®ve seconds, toallow for a few ml to pass through the ®lter. Sub-sequently 0.5 ml from the ®ltrate was placed on a TLCplate as above. All release assays was performed simul-taneously with ATPase measurements and using thesame reaction mixture.

Viscometry

Viscometry was performed at 25 �C in a capillary visc-ometer (Cannon-Manning 100) with a buffer ¯ow-timeof 56 seconds. Measurements were done using 0.7 mlMg-actin at 7.1 mM. After determination of the ¯ow timeof G-actin, polymerization was induced by addition ofsalt to 100 mM KCl and 1 mM MgCl2. The viscosity wasrecorded at two-minute intervals.

Phalloidin-binding

Binding of rhodamine-phalloidin to ®lamentous actinwas monitored spectro¯uorimetrically (lex � 544 nm;lem � 590 nm) using a microplate reader (Fluoroskan II,Labsystems, Finland). This takes advantage of the 20-fold increase in ¯uorescence of rhodamine-phalloidinupon binding to F-actin.65

Acknowledgments

We are grateful for valuable comments given by thereviewers. We acknowledge ®nancial support from theSwedish National Science Research Council (NFR) toU.L. and R.K., from the Swedish Foundation for Inter-national Cooperation in Research and Higher Education(STINT) to U.L., and to C.E.S. from the NIH (GM44038).

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Edited by R. Huber

(Received 1 November 2001; received in revised form 10 January 2002; accepted 14 January 2002)

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TheRoleofMeH73inActinPolymerizationand ATP Hydrolysisactin/JMB_02.pdfTheRoleofMeH73inActinPolymerizationand ATP Hydrolysis TomasNyman1,HerwigSchu ¨ler1,ElenaKorenbaum1 ClarenceE.Schutt2,RogerKarlsson1andUnoLindberg1*