Cold-Sensitive Mutations Dictyostelium Myosin Heavy Chain ... · DNA using the Sequenase kit (U.S....

Copyright 0 1996 by the Genetics Society of America

Cold-Sensitive Mutations of Dictyostelium Myosin Heavy Chain Highlight Functional Domains of the Myosin Motor

Bruce Patterson and James A. Spudich

Departments of Biochemistly and Developmental Biology, Stanford University, Stanford, California 94305 Manuscript received November 20, 1995

Accepted for publication February 17, 1966

ABSTRACT Dictyostelium provides a powerful environment for characterization of myosin I1 function. It provides

wellestablished biochemical methods for in vitro analysis of myosin’s properties as well as an array of molecular genetic tools. The absence of myosin function results in an array of phenotypes that can be used to genetically manipulate myosin function. We have previously reported methods for the isolation and identification of rapideffect cold-sensitive myosin I1 mutations in Dictyostelium. Here, we report the development and utilization of a rapid method for localizing these point mutations. We have also sequenced 19 mutants. The mutations show distinct clustering with respect to three-dimensional location and biochemically characterized functional domains of the protein. We conclude that these mutants represent powerful tools for understanding the mechanisms driving this protein motor.

C ONVERSION of chemical energy into mechanical work is a unifjmg feature of the molecular motors

that drive essential cellular processes such as cell divi- sion, motility and chromosome segregation. Despite their pivotal role in these processes, our understanding of the fundamental mechanochemical principles at work in these molecular machines is only abstract, largely due to the difficulty in accessing and characteriz- ing intermediate states of the motors in action. One classical system for investigating these energy conversion processes is the motor protein myosin. However, despite 30 years of intensive biochemical characterization of this motor, many central issues remain unre- solved. Indeed, although the basic concepts of the swinging crossbridge model were formulated more than 20 years ago (A. F. HUXLEY 1957; H. E. HUXLEY 1969; HUXLEY and SIMMONS 1971), the mechanism by which myosin drives the motion of actin filaments remains controversial. To further explore this mechanism, we have taken an approach of randomly mutagenizing the genome of Dictyostelium and using the cell’s myosin dependent behaviors to recover conditional mutations in the myosin motor domain (PATTERSON and SPUDICH 1995). Thus, we have generated a bank of cold-sensitive myosin molecules with the goal of slowing down or interrupting the myosin mechanochemical cycle. These mutants should facilitate the identification and characterization of previously inaccessible intermediates and reveal the interactions that drive the conformational transitions.

We generated conditional mutant derivatives of myosin that are blocked or severely impaired in their ability

Cmesponding authvr: Bruce Patterson, Department of Molecular and Cellular Biology, LSS 452, University of Arizona, Tucson, AZ 85721. E-mail: [email protected]

Genetics 143 801-810 [June, 1996)

to perform myosin’s in uiuo roles of development and cortical tension generation. These mutants possess bio- logically detectable myosin function at the permissive temperature (26”) but rapidly and reversibly lose function upon being shifted to 13”. Thus they represent candidates for myosin molecules that are interrupted or significantly impaired in specific points of the biochemical cycle, but that are nonetheless close mimics of the functioning molecule. Here we characterize these mutants by identifying the specific sequence changes that confer the cold-sensitive phenotype.

To facilitate sequence analysis, we have extended a marker rescue technique that we developed to demonstrate that these mutants were in the myosin heavy chain gene (PATTERSON and SPUDICH 1995). By comparing the frequencies with which two overlapping segments of the myosin gene can repair the defects of each mutant, we have been able to infer the location of the mutants with an accuracy of -100 bp, a fraction of the 6.5-kb mhcA gene. Subsequent sequencing of the indicated regions has allowed us to identify the defect in 19 independent myosin mutants, which comprise 17 changes at 15 amino acid positions.

The distribution and nature of the mutations alone provides suggestive information about both the nature of cold-sensitive alleles and their possible functions in myosin-driven motion. While the mutagenesis technique was untargeted, the resulting cold-sensitive mutations are nonrandomly distributed with respect to their positions in the three-dimensional structure as well as the types of amino acid substitutions created. Taken together, these observations suggest that we have indeed identified a subset of those changes that perturb the myosin molecule such that its functioning has be- come susceptible to temperature.

802 B. Patterson and J. A. Spudich

The Dictyostelium system provides powerful method- ologies for a genetically driven approach to understanding the mechanics underlying the myosin motor. Ho- mologous recombination has allowed construction of strains completely lacking wild-type myosin sequences ( MANSTEIN et al. 1989b). Transformation and self-repli- cating plasmid expression systems provide the opportunity to produce mutant myosin derivatives (MANSTEIN

et al. 1989a; RUPPEL et al. 1990; EGELHOFF et al. 1991, 1993). Biochemical techniques for purification and analysis of mutant proteins are well established (RUPPEL et al. 1994), and crystal structures of a Dictyostelium myosin subfragment have recently been published (FISHER et al. 1995a,b). Finally, the array of phenotypes associated with the absence of myosin I1 function provides opportunities for the selection of conditional mutants as well as efficient generation and analysis of intra- genic revertants (PATTERSON and SPUDICH 1995; B. PATTERSON, unpublished results). The isolation of cold- sensitive myosin mutants provides the foundation for in depth analysis of their structural and biochemical defects, and the opportunity to isolate large numbers of revertants offers an avenue toward gaining insights into previously unknown structures akin to recent suc- cesses in analysis of cold-sensitive mutants of functional RNAs (DAMMEL and NOLLER 1993; FORTNER et al. 1994; ZAVANELLI et al. 1994). Here we lay the groundwork for these future analyses by identifylng 19 mutations giving rise to cold sensitivity and demonstrating that these mutations are indeed the causative alteration.

MATERIALS AND METHODS

Strains and media: Mutant isolation and strains are described in (PATTERSON and SPUDICH 1995). Phenotypic analysis of mutants reconstructed on plasmids (see below) was performed by transforming strain HSlO [a cell derived from JHlO (HADWIGER and FIRTEL 1992) in which the myosin heavy chain coding sequence has been completely deleted and replaced with the THYl gene (RUPPEL et al. 1994)] with the appropriate plasmid and selecting transformants on HL-5 containing 8 mg/ml G418.

Cell culture conditions were as described by SUSSMAN (1987). Minimal medium was from FRANKE and KESSIN (1977). MES starvation buffer is 50 mM MES pH 6.8, 2 mM MgClz, 0.2 mM CaClZ. A mixture of 9 volumes starvation buffer with 1 volume HL-5 is “1:9”.

Bacterial lawns were made by spreading 400 pl of an overnight culture of Klebsiella aerogenesonto SM/5 plates (SUSSMAN 1987) and allowing the plates to sit overnight.

PCR: Polymerase chain reaction was performed using Taq Polymerase (U.S. Biochemicals). The oligos employed were as follws: NuXho, 5’-CTCATCCTCGAGAGACGCTCTTGT- CA-3’; XMN2,5‘-AGClTTGAGAAGAGTTTCCAG3’; PCRBy, 5‘-GAACAAGGATCCATTACAACAAG3‘; LAPNV, 5“GGA- ACGTTTGGAGCTAA-3’. Amplifications were performed using an initial denaturation step of 5’ at go”, followed by 25 cycles of denaturation for 1’ at go”, annealing for 1‘ at 52“ followed by extension for 1’ at 72”, with a final extension step of 5’ at 72”.

Sequencing: Plasmids were sequenced as double-stranded DNA using the Sequenase kit (U.S. Biochemicals), following

the protocol suggested by the manufacturer. The following oligonucleotides were used (as well as the ones listed above) to sequence appropriate regions of recovered mutant myosin genes. M13 forward and reverse primers, EVKR: 5”CCGATT-

CT-3’; Hind.30: 5’-TTITCCTCTGGTTGGTCA-3’; NUS: 5‘- CATTGCCAGTCGTGCA-3’

Plasmid constructions: Mapping constructs were created as follows: pTZ-BstBI and pTZ-CluI were created by cutting pDMU (a plasmid containing the wild-type myosin gene and promoter, H. GOO~SON, unpublished data) with ScuI and KpnI (that removes the myosin promoter and part of the head sequences) and cloning into SmaI-Z+nIcut pBluescript. This construct was cut with BstbI or CluI and the ends filled with Klenow. These reactions were stopped by heating, and the mixtures were digested with BamHI. The resulting fragments were purified and ligated into BamHI-SmaIcut pTZ18R. These vectors were cut with BamHI and Sac1 and cloned into BamHI- Sad-cut pBIG to create pMAP.Bstb and pMAP.Cla. pMAF’.Hind was created by taking the 1.4kb Hind111 fragment from pMyDBam and cloning it into the Hind111 site of pTZ18R, followed by the removal of the BumHI-Sac1 fragment encompassing this insert and cloning it into BumHI-Sad-cut pBIG.

Mutation mapping: To adapt the marker rescue strategy, we used four fragments of the myosin coding sequence (see Figure 1). The first, pMAP.BamNco, encompasses the entire myosin gene, and the other three represent different segments of the head. Each mutant strain was transformed with the four head domain mapping plasmids, and four independent transformants of each type were isolated and transferred to 24well plates. After growing to confluence, each trans- formant was spotted twice onto bacterial lawns, creating eight mutant plaques for each cold-sensitive mutant-plasmid combination. After the cells had multiplied sufficiently to clear a 1-cm region of the lawn (usually after 3 days) the cells were irradiated for 40 s 20 cm beneath a germicidal lamp. The irradiation was repeated after 48 hr. The cells were then incubated for 5 days to allow for outgrowth of induced revertant lines. The number of clear reversion events was then scored for each plaque (up to a maximum number of four, higher numbers and completely reverted perimeters also being scored as four). Predicted nucleotide position (PNP) was calculated using the following equation, designed to convert the recombination frequency observed for each construct into a relative position in the overlap region:

1338 + (1087/( 1 + (# pMapBamNco positives/

TCGTCAAACGT-3’; Bg1.30: 5”TTGGTGGTACAACGTIGT-

# pMapHind positives)/l.49)) = PNP.

Because: 1338 = position of the 5’-most nt of myosin homology in

the pMAP.Hind plasmid, thus the 5’ endpoint of potential mutation locations.

1087 = the number of nucleotides in the overlapping region of the myosin homologies in pMAP.BamNco and pMAP.Hind plasmids. Thus the (adjusted) ratio of the repair events caused by each homology will indicate the relative position of the mutation in the overlap region.

1.49 = the ratio of absolute lengths of the myosin homology in pMAP.Hind and pMAP.BamNco plasmids. This ratio is fac- tored out to remove bias introduced by the greater homology (due to length) of pMAP.BamNco.

Quantitative azide assay: Cells were grown in 2 ml HL-5 supplemented with FM medium in six-well tissue culture plates. Cells were grown until, when resuspended in the 2 ml of growth medium, they yielded an OD600 of 0.3-1.2. At this point, cells were resuspended and diluted by adding a further

Dictyostelium Myosin Mutations 803

TAIL FK;I'KI. I.-Fragments uscd to map muta-

pMAP.Hind 1338 "32968

pMAP.UamNco 1 +2425

pMapScaCla - ( . ~ w n ) -)1510

pMapScaBstb -(.lOOO) +lo75

.i ml HI,-.: supplemented with FM medium. The OD600 of 1 ml of this suspcnsion was measured, and the cells were further tlilrltetl to giw a final OD600 of 0.1-0.3. Tllr resulting suspension was placed in 2-nil aliquots into three wells of a six-well plate. Cells were allowcd to settle and were incubated for 1 hr at 22". For the 13" assays, cells were shifted to 13" for I O ' , antl then the growth medium was replaced with 1 :9 buffer at 13" and the cells were incuhated for a further 10'. For 26" assays, cclls were shifted to 26" for 5', and then growth medium M'~S replaced with 1 9 buffer at 26" and cells were incu- hted for ;I further .5'. At this point, the tissue culture plates were placed on a nutator platform and 23 (13") or 1.i (26") pI of 300 m v sodium azide was carefully added to the medium. Cells were nutated 5' at the appropriate temperature, and then 1 ml of the supernatant was harvested and the OD600 of this aliquot was measured. The aliquots were re- turned t o the tissue culture dish, and the entire well was then harvested by trituration antl the OD600 of the resulting suspension was taken. The percent washoff induced by the azide treatment was expressed as the ratio of cells washed off during the 5' nutation period to the total cells as calculated by OD600 of those removed by trituration.

RESULTS

To accurately locate mutations before sequencing them, we adapted the marker rescue strategy that we employed previously (PATTERSON and S r c n ~ r - ~ 199.5) to allow 100-bp resolution in mapping the mutations. Four plasmids capable of replication in Dictyostelium were constructed, each bearing a different fragment encoding part of the myosin head domain (Figure 1). Each mutant was initially transformed with pMAP.Hind and pMap.RamNco, which contain inserts that in combination cover the entire head domain. The myosin homologies on the two plasmids have an 1087-bp overlap corresponding to sequences encoding the C terminal 40% of the head domain. Transformants were spotted onto bacterial lawns and allowed to form plaques. Homologous recombination was stimulated by U V irradiation (WAI.IA(:E and NEMTIL 1982), and plates were incubated for 1 wk to 10 days. In cases where gene conversion events behveen wild-type myosin sequences on the plasmid and mutant chromosomal sequences resulted in repair of the chromosomal mvosin gene, fast growing, fruiting body-filled regions became apparent at the edges o f the plaques. These represent re-

tions within the myosin head. Shown at top is a schematized version of the n h - A gene, indicating which sequences contribute to the head or motor domain (indicated by the o \ a l ) and the tail domain (indicated by the rounded rectangle). Shown below i t are the regions o f the gene cloned into the plasmids u s c d for mutation mapping and the myosin- encoding sequences they contain.

verted cell lines expressing fully functional myosin. The number of revertants observed per eight plaques was recorded. In cases where few or no revertants were o h sewed with the pMAP.Hind construct, the mutant being studied was also transformed with plasmids capable of repairing promoter sequences and coding sequences up to posi t ion 107.5 (pMAP.ScaRst) or 1510 (pMAP.ScaCla), and the procedure was repeated. The results of this analysis are presented in Table 1.

Mutants that were repaired by both the pMAP.Hind and pMAP.RamNco constructs (and therefore resided between base pairs 1338 and 2425) were further studied. The relative rates of recombination observed with the pMAP.Hind and pMAP.RamNco constructs were used to ascertain the approximate location within the overlap region of the mutations that were repaired by both plasmids. We calculated the location of these mutations by making the following assumptions: the ratio of recombination events represents the relative location of the actual mutation to the endpoint of the repairing fragment ( i .~ . , mutations located toward the end of a fragment would be less likely to be repaired by it than a centrally located one) and the frequency of recombination is proportional to the length of the homology ( i . ~ . , a larger fragment is more likely to undergo recombination events than a smaller one; see MATERIALS AND

As an example of the localization of one of the mutants from the recombination data, take the case of HS81 (See Table 1) : 26 repair events were observed with pMAP.RamNco, while the pMAP.Hind plasmid gave rise to four such events. This gives a ratio of 6.5, which reduces to 4.4 when the greater length of the pMAP.BamNco fragment is compensated for. This ratio suggests that the causative mutation in HS81 is fourfold closer to the edge of the pMAP.Hind homology than it is to the edge of the pMAP.BamNco homology, and thus the mutation is predicted to lie at base pair 1541, which proves to be 66 bp 3' of its actual location.

Mutant recovery and sequencing: The information from the mapping experiments was the basis for tar- geting specific regions of the myosin head for recovery by PCR. Mutations predicted to lie between base pairs

METHODS).


TABLE 1

Fine mapping of cs myosin mutants

Mutant ScaBst ScaCla Hind BamNco Predicted Actual ~ ~~~~~~

HS77 HS79 HS81 HS83 HS84 HS86 HS90 HS9 1 HS92 HS93 HS80 HS96 HS97 HS98 HS99 HSlOO HS103 HS104 HS106 HS76 HS85 HS89 HS94 HS102 HS107 HS108 HS109

0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 4 4 4 3 5 4 1 5

0 0 0 0 0 0 0 0 0 1 0

ND ND ND ND ND ND ND ND 17 11 11 8 4

11 15 20

10 1 4 8

15 23 16 3

13 2 4

14 3 4

22 8 5 8

29 0 0 1 1 1 2 0 0

[%’I

26 6

28 13 16

23 [321 22 16 10 29

3 2

27 26 8

28 [321

17 25 25 24

[321

[321

~321

[321

[ 16831 [ 13601 1541 2061 1821 2126 1988

[ 14711 1835

[1431] 1570 1953 1674 1523 2334 2269 1573 1680 2255

1756 1399 1607 2071 1871 2071 2039 1502 1738 1399 1685 1993 1592 1502 2399 2219 1481“ 1685 2336

Recombination data. The first column gives the myosin mutant strain that was transformed with the pMAP plasmids. ScaBst, ScaCla, Hind and BamNco columns show the number of recombination-induced repair events observed for each pMAP plasmid. Brackets are used to indicate that the number of revertants observed with pMAF’.BamNco exceeded the maximum that could be counted accurately, and thus estimates derived from them represent C-terminal limits rather than actual position predictions. The estimated location of the causative mutation, based on the relative number of recombinants observed with the pMAP.Hind and the pMAP.BamNco constructs is shown in the column labeled “Predicted,” while the column labeled “Actual” shows the observed position of mutations in DNA recovered from the strain.

“This mutant actually comprises two changes, one at position 1480 and a second at position 1482.

1338 and 1785 were recovered using primers NuXho and LAPNV that amplify the region from 1230 to 2138. The amplified sequence between base pairs 1338 and 1785 was then cloned into a pBluescript derivative using convenient restriction sites and sequenced. Mutations predicted to lie between 1785 and 2425 were recovered using primers PCRBy and Xm2, which amplify the region between 1760 and 2440. Again the amplified fragments were subcloned into a pBluescript derivative and sequenced. Once candidate changes were identified, a second PCR product was synthesized and sequenced to rule out artifacts caused by the low fidelity of Taq polymerase. In cases where no candidate changes were detected, we recovered the unsequenced portion of the region 1338-2440 and sequenced it. In this way, mutations in all 19 candidates were identified. The nucleotide changes and the amino acid alterations they introduce are shown in Table 2. For convenience in discussing the location of the mutants relative to the published chicken pectoralis myosin structure, we also

include the corresponding change that would occur at the homologous position in the chicken sequence.

Two of the mutants are difficult to analyze because of the changes they introduce: one is the double mutant dE467QdD595Y (references to amino acid positions in Dictyostelium are preceded by a “d”, those in chicken pectoralis myosin are preceded by a “c”). We have chosen to emphasize the single mutation dE467Q because position 467 is found independently in the collection, this position is much more highly conserved than d595, and the E467 mutation alone induces a cs phenotype, although it is less severe than the double mutant. dE580* creates a stop codon, which is clearly translated in vivo as myosin function can be detected. Apparent readthrough of such codons is not without precedent in Dictyostelium (D. KNECHT, personal communication). Since the amino acid(s) substituted is not known, this mutation will not be considered further.

Confirming the location of the myosin mutants: While the mapping experiment demonstrates


TABLE 2

Codon and amino acid changes of cs myosin mutants

Mutant name

~~ ~~

Nucleotide position

Codon change

Amino acid change

Chicken equivalent

HS93

HS79 HS103 HS91 HS98 HS97 HS81 HS80 HS104 HS92 Hs77 HS84 HS96 HS90 HS83 HS86 HSlOO HS106 HS99

~~

1399 1783

1399 1480, 1482 1002 1002 1592 1607 1685 1685 1738 1756 1871 1993 2039 2071 2071 2219 2336 2399

GAA+CAA GAT -+ TAT

GAA+AAA TAT -+ AAA TGG + TTG TGG -+ 'ITG GAA-CAA CCA -+ CGA CGT - CAT CGT -+ C'IT GAG -+ TAG GAt-AAA GGT -+ GAT GCC -+ ACC GGT - GTT GGT -+ GTT GGT - GTT GGT + GAT CGT - CCT CGT + CCT

E467Q D595Y

E467K Y494K W501L W501L E531Q P536R R562H R562L E580* E586K G624D A665T G680V G69 1 C G69 1 C G740D R779P R800P

E476 T606

E476 Y503 w510 W510 E538 P543 K569 K569 N591 E597 G643 G684 G699 G710 G710 G761 R800 R82 1

DNA and protein sequence changes in cs mutants. The first column lists the strains from which the myosin gene was recovered, the second shows the codon(s) altered, and the third shows the change in encoded amino acid resulting from the mutation(s). The final column shows the corresponding amino acid and position in the chicken sequence for comparison with the crystal structure.

that the mutations we sequenced are necessary for the phenotype, they do not demonstrate sufficiency. To determine whether the sequenced mutation was sufficient to cause the cs defect, we cloned the sequenced region into an unmutagenized wild-type myosin gene. By trans- ferring only the sequenced region (see MATERIALS AND

METHODS), we ensured that any resulting phenotype was the consequence of the change(s) identified in Ta- ble 2. We performed this analysis on 13 of the mutants. In every case except one (dA665T; see below), the resulting myosin genes, when introduced into myosin null cells on extrachromosomal plasmids, conferred the same developmental defect as the strain from which they were isolated. We also performed a semiquantita- tive version of the azide selection on mutants at both 13 and 26", as shown in Figure 2. Again we found that the mutants conferred the expected properties, with the exception of dA665T, which seems to impart only a mild loss of function at both temperatures. This mutation may therefore be only one part of a double mutant. We have not attempted to identify further mutations present in this mutant, and it will not be discussed further.

DISCUSSION

Mapping myosin mutations: In our previous work, we developed a marker rescue strategy to determine

which of our bank of cold-sensitive mutants lay in the head domain of the myosin heavy chain gene (PAT- TERSON and SPUDICH 1995). We identified mutants that mapped to this domain by scoring the ability of a fragment of the myosin heavy chain gene to direct gene conversion events that restore function to the mutant myosins. Specifically, we assayed the ability of fragments of the myosin gene to restore efficient growth on bacterial lawns and developmental competence to the mutants. We have now extended this technique to provide more precise information about the location of the myosin mutation present in each strain by comparing the rates at which different fragments of the head direct repair events. We quantitated the ability of overlapping fragments of the mhcA gene to repair the chromosomal lesion in each mutant. In the case of mutants for which the data indicated a position between base pairs 1338 and 2425, we achieved -100-bp resolution by comparing the relative numbers of repair events generated by the overlapping fragments. Thus, by using fragments with a 1087-bp overlap, we were able to localize mutants within -10% of that interval. This technique should be generally applicable for mapping mutations in any Dictyostelium gene where wild type confers a selectable advantage over mutant, and for which cloned genomic DNA exists.

The location of the mutations shows several interesting distributions (see Figure 3). While the myosin heavy

806 B. Patterson and J . A. Spuclich

FIGURE 2.--Azicle-induced washoff of mutants at permissive and nonpermissive temperatures. Percentage of cells that de- tach from the surface in response to azide is shown. Quantita- tive azide assays were performed as described in M,\TEKIAIS AND METHODS, and the proportion of cells detaching at the permissive temperature (26”) and the nonpermissive temperature (13”) were quantitated. The behavior of cells lacking myosin (top, “myosin-”) or containing wild-type myosin (bottom, “MYOSIN+”) are also shown.

chain gene has a 6350-bp coding region, two-thirds of the mutants we recovered lie in a 1100-bp segment encoding the C terminal half of the head domain. To some extent this finding was anticipated, given the pau- city of mutations in Cnmm-halAih ekgans myosin that map to the tail domain (BEJSOVEC and ANDERSON 1990). Clustering within the head itself suggests that the C terminal 40% of the myosin head is in some way a rich target for cold sensitivity. Only eight cs mutants mapped in the first 350 amino acids us. 20 in the last 350, with a 100 amino acid “desert” containing no mutant. separating the two. The Gterminal region in- cludes several stretches of amino acids that show very high conservation among myosins sequenced to date. One interesting possibility is that this domain is criti- cally involved in the conformational changes that take place during the myosin cycle and is thus a particularly good target for cold-sensitive changes.

The distribution of the amino acid substitutions introduced by the mutations is striking. In the most N- terminal region included in this work (d467d624), nine of the subsequent 11 mutations affect charged residues or introduce charge alterations (since the amino acid inserted in dE580* is unknown, we do not score it as a charge alteration). This region encodes three of the four sequences proposed to be involved in actin binding. Of the five residues affected in the region d624-d740, all are glycines in the Dictyostelium structure (position d665 is an alanine in Dictyostelium but a glycine in most other myosins, but recall that this alteration is not sufficient to induce a major cold-sensi-

tive phenotype). Indeed, of the five glycine residues in the Dictyostelium sequence between positions d624 and d740, inclusive, four were recovered as cold-sensitive mutants in this study. Finally, the last two changes (dR779P and dR800P) both represent alterations from arginine to proline i n light chain binding sites. This distinct grouping of amino acid alterations suggests that difyerent properties o r fhctions are altered in three or more regions of the myosin head to give rise to cold sensitivity.

Locations of specific myosin mutants: Within the region we have analyzed, we can group the mutants we have located into four “families” with regard to the sites in the three-dimensional structure at which they occur (Figure 4). These are the actin binding faces, the light chain binding sites, a mobile region that contains chemically reactive cysteine moieties in many myosins, and positions associated with a helix that forms part of a long cleft running along the head domain. We discuss each of these and their implications separately.

Actin In‘ndingsites: The most recent advances in terms of understanding the interface between actin and myosin have come from the myosin crystal structure. Two groups have used high-resolution reconstructions of electron microscopic images of rigor acto-myosin complexes to “dock” the actin and myosin crystal structures (RAWENT et al. 1993; SCHRODER et nl. 1993). Strikingly, both groups derive a similar set of actin-myosin interaction points from their data, despite using different sources of myosin (rabbit and Dictyostelium) for the EM studies. These groups propose interactions at four sites; three between myosin and a “primary” actin protomer and one with a “secondary” (adjacent) actin protomer. The binding of the second protomer s u p ports previous work (MORNET e1 nl. 1981; AMOS el al. 1982; ANDREEV and BORE~DO 1992) that had suggested such an interaction. Biochemical studies demonstrate that the initial binding of myosin to actin is a weak interaction involving the loop separating the proteolyti- cally defined 50 and 20-kD fragments of myosin (SUTOH 1982; CHAUSSEPIED and MORALES 1988; YAMAMOTO 1989-1991; CHEUNG and REISIXR 1992). This is followed by interaction with other contact points, yielding a strongly bound state and potentially driving the conformational changes that underlie the power stroke.

The most N terminal of the proposed actin interaction sites was not marked by a mutation in this study. The Gterminal three actin binding sites each contain one or more mutants. dE531Q(cE538Q) and dP536R(cP543R) lie in a region proposed to be involved in stereospecific interaction with actin (RAWENT et nl. 19934. dG624D(cG643D) alters a glycine residue at the C terminus of the 50-20 K loop, a region known to be involved in charge-charge interactions with actin. Finally, the two mutants dR562H(cK569H) and dR562L(cK569L) alter residues in the region of the myosin head proposed to interact with the secondary


”

ATP) 4

v R821P

ELC

TIN

ELC FIGURE 3.-Positions of cs mutations on chicken ribbon diagram. Two views of the crystal structure of chicken pectoralis

muscle generated by RAYMENT et al. (1993b) are shown. The positions of the amino acids homologous to those recovered as cs mutants in Dictyostelium in this study are shown by black spheres. The numbers shown are the positions of the amino acid residues in the chicken pectoralis myosin structure equivalent to those altered in the Dictyostelium myosin II sequence. Figures 3 and 5 were prepared with the molecular graphics program MOLSCRIPT (KRAULJS 1991).

actin protomer. The dR562H(cK569H) mutant is particularly interesting in that it demonstrates that a change as subtle as histidine substituting for arginine in the proposed interaction between myosin and the secondary protomer can reversibly block myosin function in vivo (SPRINGER et al. 1994), indicating a critical role for this interaction in the motor cycle.

Light chain binding sites: All myosin heavy chains characterized to date contain one or more tandemly repeated binding sites for calmodulin or calmodulinde- rived light chains. The light chains are proposed to perform both structural and regulatory roles. Structur-

ally, they have been proposed to add stiffness to a long a-helix that may act as a “lever arm” to amplify conformational changes occurring within the head domain (RAYMENT et al. 1993b). The so-called essential light chain has also been proposed to be part of the “hinge” about which myosin pivots during the power stroke (FISHER et al. 1995a). The two cold-sensitive mutations isolated in this region each affect one of the two consensus binding sites for calmodulin/light chains. In Dicty- ostelium, there are two matches to the proposed consensus binding site sequence ( I Q m R G m R ) (MOOSEKER and CHENEY 1992), one of which binds the


TKQPR~YFIGVLDIAGFEIFDFNSFEQLCINFTNEKLQQFFNEEMFVLE~

E4afQ,K zG:rDFG W - T M F r K A T U

E531Q P536R

KNKLYDQE~GKSNNFQKPKPAKGKAEAEFSLVEYAGTVDYNISGWLEKNK - ”

t R569H,L ES$O* ES86K

t

DPLNETVIGLYQKSSVKTLALLFATYGGEAEGGGGKKGGKKKGSSFQ~V~

G624D t -

ALFRENLNKLMANLRSTEPEFVRCIIPNETKTPGAMEEELVLEQLRCNGV

G6iFV LEGIRICRKGFPSRVLYADFKQRYRVLANSAIPEGQFMDSKKASEKLLGS

- f

G691C - IDVIETQYRFGETKVFFKAGLLGLLEEMRDDKLAEIITRTQARCRGFLMR

G7tOD R119P

VEYRRMVERRESIFCIQYNVRSFMNVKEWPWMKLFFKIKPLLK v

R800P

FIGURE 4.-Position of cs mutants with respect to secondary structure elements. The sequence of chicken pectoralis myosin I1 is shown. Secondary structure elements are depicted by lines, with a-helices being denoted by lines below the sequence and P-strands denoted by lines above the sequence. Amino acids positions corresponding to those altered in cold- sensitive Dictyostelium myosin mutants are indicated by arrows. The numbers shown are the positions in the Dictyo- stelium sequence.

essential light chain (IQaatRGwia) and the other re- sponsible for binding the regulatory light chain (IQqnl- BAyidfK). The two mutants recovered in this region, dR779P(cR800P) and dR800P(cR821P) change the second and first arginine residues, respectively, to proline (underlined residues in consensus sequences shown above). Clearly, these changes have the potential to disrupt or alter the binding of the light chains.

Reactive thiol region: One region of the myosin head has been extensively implicated in motions associated with the chemomechanical cycle. Biochemical analysis of myosin has included as a focus two chemically con- spicuous residues in the head: reactive cysteines present in many myosins and commonly referred to as the reactive thiols or SH1 (cC707) and SH2 (cC697). In the chicken myosin crystal structure, the acarbons of these two cysteine residues are 18 A apart and the side chains face opposite sides of the molecule (RAYMEW et al. 1993a). However, during the stroking cycle, the two can be directly cross-linked, indicating a separation of only -3 A and a more favorable geometry (HUSTON et al. 1988). Comparison of crystal structures of Dictyostel- ium myosin fragments made in the presence of ADP and either beryllium fluoride (thought to mimic the ATP bound state) or aluminum fluoride (thought to mimic the transition state for hydrolysis of ATP) suggests that the helix containing SHl undergoes a 2.5-A displacement and rotates 10 deg between these two states (FISHER et al. 1995a). This movement, however, is still insufficient to explain the observation of direct cross-linking of the two cysteine residues.

In the chicken mvosin I1 crvstal structure. the two

FIGURE 5.-Close-up view of the region of the chicken pectoralis myosin I1 crystal structure containing the reactive thiols. The positions of the glycines equivalent to those mutated in this study are shown by black spheres, while the positions of SH2 and SHl are shown by yellow spheres.

cysteine residues lie in two alpha helices separated by a glycine residue (Figure 5). This glycine is mutated to a valine dG680V(cG699) in the collection of cold- sensitive mutants. The second helix, which contains the SH1 thiol (cC707) is also bounded on its C terminus by a glycine, which was mutated in dG691C(cG710). We have preliminary evidence that these mutations have different, temperature-dependent effects on myosin’s ATPase activity and differentially affect myosin’s interaction with actin (K. M. RUPPEL and B. PATTERSON, unpublished observations). The discovery of alterations in the glycines in this region raises the exciting possibility that these mutations perturb myosin function by restricting the freedom of motion of the molecule at critical junctions. The restricted mobility could inter- fere with the adoption of certain conformations or block the transition between conformations.

Ckfiasson’ated h l i x ~475~505: The third relevant structural feature of myosin is the “actin-associated” cleft. This cleft runs along the long axis of the myosin head domain, from the actin binding regions at the distal tip of the head toward the ATP binding site (RAY- MENT et al. 1993b). Sliding along and closure of this cleft has been suggested to be coupled to actin translo- cation by the myosin head (&WENT et al. 1993a; FISHER et al. 1995a,b). An a-helix, consisting of residues c475 (d466) -c505 (d496) runs parallel to the cleft,


forming part of its lower face. In a recent survey of 72 sequences of myosins of all types, this region is almost completely invariant at the sequence level (H. GOOD- SON, personal communication). Three of our mutants, dE467K(cE476), dE467Q(cE476), and dY494K(cY503), lie near the endpoints of the helix. Intriguingly, dE467 hydrogen bonds with dS266 in the crystal structure of myosin bound to ADP and AlF; (FISHER et al. 1995b), which is thought to mimic the transition state of ATP hydrolysis. This hydrogen bond is not observed in the prehydrolysis (ADP + BeF,) structure. A fourth mutant, dW501L(cW510), alters an absolutely conserved amino acid in the turn just distal to the C terminus of the helix. Others mutants, while distant in the primary structure, occupy nearby positions in the tertiary structure: near the N terminus of the helix is dE586K(cN597), and in the vicinity of the C terminus of the helix is dG740D(cG761). It is interesting to note that the reactive thiol region mutants also abut the Gterminal portion of the helix, but we have chosen to discuss them separately because of the large body of biochemical data that pertains to them.

Conclusions: The rationale for isolation of these mutants has been to facilitate analysis of the mechanisms driving the myosin motor. Using an azide-based assay for myosin I1 function, we have confirmed that 11 mutations confer rapidly reversible cold-sensitive myosin I1 function in vivo (Figure 2). While the absolute numbers are variable, in all but one case the mutants are significantly more impaired at the nonpermissive temperature than at the permissive temperature when compared with wild type. Finally, it is interesting to note that the three mutants that exhibited near wild-type function in the developmental cycle at the permissive temperature (dE538Q dR562H and dR562L) all proved to be actin binding site mutants.

We are now in a position to determine the biochemical root of the defects conferred by these mutations and to correlate these with their predicted effects on proposed and observed structural states of the myosin motor. We are also embarking on reversion analysis of these mutants in order to further understand their defects and to gain insights into the conformational changes at the heart of the myosindriven motility. These studies should also shed light on the mechanisms of cold sensitivity in proteins and the utility of such mutants for further genetic studies.

We thank T. Q.-P. UYEDA and K. RUPPEL for useful discussions, and K. RUPPEL for generating the structural figures. We also thank I. RAYMENT for generating initial drafts of the structure figures. This work was supported by National Institutes of Health (NIH) grant GM- 40509 to J.A.S. and Damon Runyon-Walter Winchell Fellowship no. DRG1002 and NIH Fellowship ARO823502 to B.P.

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