The reovirus cell attachment protein possesses two independently active trimerization domains: Basis of dominant negative effects

Cell, Vol. 71, 479-488,

October

30, 1992, Copyright

0 1992 by Cell Press

The Reovirus Cell Attachment Protein Possesses Two Independently Active Trimerization Domains: Basis of Dominant Negative Effects Gustav0 Leone, Lloyd Maybaum, and Patrick W. K. Lee Department of Microbiology and Infectious Diseases University of Calgary Health Sciences Center Calgary, Alberta T2N 4Nl Canada

Summary The reovirus cell attachment protein, al, is a homotrimer with an N-terminal fibrous tail and a C-terminal globular head. By cotranslatlng full-length and various truncated al proteins in vitro, we show that the N- and C-terminal halves of al possess independent trimerization and folding domains. Trimerization of al is initiated at the N-terminus by the formation of a “loose,” protease-sensitive, three-stranded, u-helical coiled coil. This serves to bring the three unfolded Ctermlni into close proximity to one another, facilitating their subsequent trimerization and cooperative folding. Concomitant with, but independent of, this latter process, the N-terminal fiber further matures into a more stable and protease-resistant structure. The coordinated folding of al trimers exemplifies the dominant negative effects of mutant subunits in oligomeric complexes. Introduction The reovirus cell attachment protein, al, is a minor outer capsid protein strategically located at the 12 vertices of the viral icosahedron (Lee et al., 1981; Furlong et al., 1988). This protein possesses a number of biologically important functions that determine how the reovirus interacts with host cells and the host. Of all the reovirus proteins, 01 alone possesses intrinsic cell binding capabilities, and as such, is responsible for the specific high affinity attachment of virions to susceptible cells (Lee et al., 1981; Armstrong et al., 1984) and thus plays a pivotal role in viral infectivity and tissue tropism (Sharpe and Fields, 1985). Protein 01 is also the viral hemagglutinin (Weiner et al., 1978; Paul and Lee, 1987; Yeung et al., 1987) and is responsible for the triggering of serotypespecific host immune responses (Sharpe and Fields, 1985). In view of the multifunctional nature of 01, much effort has been made to probe the structure-function relationships of this protein. The Sl gene segment, which encodes 01, has been sequenced for all three reovirus serotypes (Nagataet al., 1984; Cashdollar et al., 1985; Bassel-Duby et al., 1985; Duncan et al., 1990; Nibert et al., 1990), and the deduced amino acid sequences have been analyzed to predict various structural motifs. It was initially suggested that the N-terminal portion of al is an a-helical coiled coil, while the C-terminal portion exists as a globular structure (BasseCDuby et al., 1985). Such a prediction was subse-

quently confirmed by electron microscopic studies that showed purified al as a “lollipop’‘-shaped structure with a fibrous tail topped by a globular head (Banerjea et al., 1988; Furlong et al., 1988; Fraser et al., 1990). Similar structures have been found to project from the surfaces of virus particles, with the globular heads most distal from the virions (Furlong et al., 1988). That the globular head and the fibrous tail indeed represent the C- and N-terminal portions, respectively, of al has been confirmed by recent biophysical analysis of the two fragments generated by trypsin digestion of intact (~1, the C- and N-terminal fragments (Strong et al., 1991). It has been demonstrated that the C-terminal portion of 01 harbors the conformationdependent receptor-binding domain (Nagata et al., 1987; Yeung et al., 1989; Duncan et al., 1991; Turner et al., 1992) and that the N-terminal portion possesses intrinsic trimerization and virion anchoring function (Mah et al., 1990; Banerjea et al., 1990; Leone et al., 1991a, 1991b). Recent biochemical and biophysical evidence suggests that intact protein 01, as well as the N- and C-terminal tryptic fragments, are all trimeric (Strong et al., 1991), and that trimerization of al is accompanied by extensive conformational changes necessary for its cell attachment function (Leone et al., 1991c). In the present study, we probe the mechanism of al trimerization and folding. We demonstrate that the N-and C-terminal half of 01 each possesses a distinct trimerization domain. Trimerization of al initiates at the N-terminal half with the formation of an “immature” (proteasesensitive) a-helical coiled coil in this region. This is then followed by trimerization (and cooperative folding) of the C-terminal half to generate a globular head, and maturation folding of the N-terminal half to yield a proteaseresistant, fibrous structure. Our studies also clearly demonstrate the dominant negative effect exerted by mutant subunits in an oligomeric protein. Results The N-Terminal Portion of al Is Required for the Generation, but Not the Maintenance, of a Functional Trimeric C-Terminal Globular Head Previously, we demonstrated that the C-terminal half of 01 (amino acids 223-455), when expressed as a truncated protein (i.e., lacking the N-terminal half), manifests a drastically reduced (>90%) cell binding activity relative to the full-length protein (Nagata et al., 1987). This is in contrast to the subsequent finding that a C-terminal tryptic fragment (amino acids 246-455), generated by trypsin digestion of intact trimeric 01, is fully functional (Yeung et al., 1989; Leone et al., 1991~). These observations, summarized in Figure 1, suggest that the N-terminus is required for the generation, but not the maintenance, of a functional C-terminus. More recently, we demonstrated that the N-terminal half, when expressed as a truncated protein (i.e., lacking the C-terminal half), is capable of forming a three-stranded a-helical coiled coil (Leone et al., 1991a),

Cell 480

FL transcript 5’

,

I

, II

, III / IV ,

dN transcript

Figure 1. Schematic Diagram Comparing the Structural and Functional lntearitv of the Two C-Terminal Portions of 01 Generated by Different Methods

3’

w translation 1

T NAC FL protein

N-fragment

dN protein

C-fragment

(Left) Trypsin treatment of full-length (FL) 01 translated from a full-length Sl transcript (T denotes the trypsin cleavage site located immediately after region II [i.e., in region Ill]). (Right) Direct translation of an N-terminal truncated (dN) transcript. The C-terminal tryptic fragment and the dN protein are 210 and 234 amino acids long, respectively

degraded

(functional)

and that both the N- and C-terminal tryptic fragments of intact al are trimeric (Duncan et al., 1991; Strong et al., 1991) with the trimeric N-terminal fragment being significantly more stable than the trimeric C-terminal fragment (Strong et al., 1991). Collectively, these data suggest that trimerization of al might be initiated at the N-terminus. This would then be followed by C-terminal trimerization with the accompanying conformational changes necessaryforcell binding activity(Leoneetal., 1991c).Therequirement for N-terminal trimerization to precede C-terminal trimerization probably accounts for the relative lack of binding activity of the C-terminal half of 01 when it is expressed as a truncated protein. The inability of such a truncated protein (designated dN) to trimerize (and hence, to bind to cells) is illustrated in Figure 2. Under conditions that allow both the N- and C-terminal tryptic fragments to maintain their trimeric state, the expressed truncated protein (with the N-terminal half deleted) migrated as a monomer, despite the fact that it was slightly larger than the monomeric form of the C-terminal tryptic fragment. As expected, only the trimeric C-terminal tryptic fragment, but not the monomeric truncated protein, manifested cell binding activity. The C-Terminal Half of 01 Possesses Intrinsic Trimeriration Function While C-terminal trimerization is clearly dependent on N-terminal trimerization, there is evidence that the C-terminal half possesses its own assembly and folding characteristics. Previously, using C-terminal deletion mutants expressed in vitro, we demonstrated that the deletion of as few as 4 amino acids from the C-terminus abrogates the cell binding function of the 01 protein (Duncan et al., 1991). Loss of cell binding function was also observed when certain conserved amino acids at the C-terminal half of ol were substituted (Turner et al., 1992). In both cases, the N-terminal half of the protein remained intact (trimeric and protease resistant), whereas the C-terminal half was

grossly misfolded (unassembled and protease sensitive). This suggests that the mechanism of assembly and folding of the C-terminal half is global in nature. This is distinct from N-terminal trimerization, where the requirements are less stringent and which likely occurs in a processive manner during 01 synthesis (Leone et al., 1991a, 1991b). If the two halves of 01 possess distinct trimerization domains, then the involvement of the N-terminal half in C-terminal trimerization would most likely be an indirect one. An obvious possibility is that N-terminal trimerization serves to bring the three C-terminal subunits into close proximity to one another, thereby facilitating their trimerization. To test this hypothesis, and in view of the less stringent conditions required for N-terminal trimerization, we examined the ability of an N-terminal deletion mutant (designated dll) lacking residues 123 to 223 (region II) to form functional heterotrimers with full-length al (Figure 3A). Deletion of region II removes the fiber region adjacent to the globular head, including the last 9 of the 19 heptad repeats that are presumably involved in the stabilization of the N-terminal coiled coil. Despite the loss of a substantial portion of the heptad repeat, dll was capable of forming functional homotrimers and heterotrimers with full-length al (Figure 3A), although, as predicted, these were less stable than full-length al homotrimers (data not shown). The observation that dll homotrimers were functional indicates that the region of the fiber adjacent to the globular head is expendable for the folding of a functional head. Moreover, heterotrimers were also found to be functional, despite obvious distortions in the fiber domain (due to the presumed looping out of region II of full-length subunits [Figure 381) as indicated by the degradation of the N-termini of dll-containing trimers when treated with trypsin (Figure 3C). The C-terminal halves of these proteins were nonetheless trypsin resistant, consistent with the functional integrity of the globular heads. These results suggest that it is not absolutely essential that the three 01 subunits be perfectly aligned throughout their entirety in order to

Reovirus 491

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al Trimerization

Domains

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T dN

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C-trimer

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Figure 2. Comparison between the C-Terminal Tryptic Fragment and the dN Protein in Terms of Oligomerization State and Cell Binding Function [“Sjmethionine-labeled full-length (FL) 01 and N-terminal-truncated (dN) 01 proteins were synthesized in vitro. An aliquot of the lysate containing full-length 01 was digested with 5 uglml trypsin (T), followed by the addition of trypsin inhibitors. All samples were then either analyzed directly by SDS-PAGE (Express.) or applied to L cell monolayers, followed by SDS-PAGE analysis of cell-bound proteins (Bind.). The conditions used for SDS-PAGE analysis were such that both the N- and C-terminal tryptic fragments maintained their trimeric state (i.e., preincubation of samples in protein sample buffer at 4%, followed by electrophoresis carried out also at 4°C). The identities of all the bands have been previously determined (Leone et al., 1991a; Strong et al., 1991).

C generate a trimeric head. Rather, formation of a threestranded coiled coil via the N-terminus allows the three C-terminal subunits to be in close apposition to one another, thereby promoting their subsequent trimerization. This experiment therefore illustrates that the C-terminal half of 01 possesses an active trimerization domain. N-Terminal Trimerization Precedes C-Terminal Trimerization To test the notion that N-terminal trimerization precedes C-terminal trimerization, a pulse-chase experiment was carried out. An in vitro translation mixture was pulse labeled with [35S]methionine for 9 min and then chased with excess unlabeled methionine. Aliquots were taken at 10, 15,30, and 60 min thereafter, incubated in SDS-containing protein sample buffer at 4°C for 30 min, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results (Figure 4, left panel) show clearly the gradual al species conversion (ta =: 30 min) of a slower-migrating (trimer [intermediate]) to one that migrated faster (trimer [mature]). The slower-migrating species corresponded to immature al trimers, which are hydra-like structures with an assembled (trimeric) N-terminal half and an unassembled C-terminal half (Strong et al., 1991). Subsequent trimerization of the C-terminal half resulted in a fastermigrating, lollipop-shaped structure. That this latter species indeed represented the mature 01 form was further confirmed by the demonstration that it alone possessed cell binding function (Figure 4, right panel).

FL dll

FL/dll 43 2:1 1:1 1:2 1:4

---

_

_

-,-A

-N-trimer

@OK)

-N-dimer

(54~)

-

Figure 3. Analysis of In Vitro Cotranslation and Truncated (dll) Si mRNAs

chlWlOlll~r

Products

(23K)

of Full-Length

(A) Cell binding function of cotranslation products. The two transcripts were translated in the presence of [%]methionine either individually or in combination at various concentration ratios as shown. Samples were then applied to monolayers of L cells, and cell-bound proteins were analyzed by SDS-PAGE after the samples were incubated in protein sample buffer at 37OC for 30 min. The compositions of the protein bands are indicated on the right, where (A) and (B) represent the full-length (FL) and dll products, respectively. (B) Structural relationships between the four cotranslation products. (C) Trypsin digestion of cotranslation products. Cotranslation products from (A) were treated with 5 ug/ml trypsin. After the addition of trypsin inhibitors and protein sample buffer, samples were incubated at 37OC for 30 min prior to SDS-PAGE (the trimeric C-terminal tryptic fragments were dissociated into monomers under these conditions).

N-Terminal Half Undergoes Posttrimerization Folding (Maturation Folding) Concurrent with, but Independent of, C-Terminal Trimerization The structural integrity of the in vitro synthesized al products at various stages of maturation was then analyzed by

Cell 482

EXPRESSION 10

-

15

30

BINDING

60 fmin)

--

-

-

-

15

30

60

(min)

I3

trimer (mature)

--monomer

Figure 4. Structural 01 Trimers

A

15 3060

(min)

. .-cc

*

84K

*

N 26K

trimer

trimer

fintermsdiate)

--

-

10

and Functional

-

Figure 6. Trypsin Digestion of a C-Terminal-Truncated tide, d204, Synthesized In Vitro Maturation

of In Vitro Synthesized

Full-length Sl transcripts prepared in vitro were translated in rabbit reticulocyte lysates containing (%]methionine. After 10 min of protein synthesis at 37%, unlabeled methionine was added to the reaction mixture to a final concentration of 16 mM. At various times indicated (with the first time point being the time of addition of unlabeled methio nine), aliquots were taken and were either directly analyzed by SDSPAGE or applied to monolayers of L cells for 10 min. followed by SDS-PAGE analysis of cell-bound proteins (all samples were incubated in protein sample buffer at 4’C prior to electrophoresis, which was also carried out at 4%).

01 Polypep-

TranscriptsencodingtheN-terminal251 aminoacidsofol (i.e., lacking the C-terminal 204 amino acids) were translated in vitro and subjected to trypsin treatment as described in the legend to Figure 5. (A) Analysis of untreated samples by SDS-PAGE under nondissociating conditions. (B) Analysis of trypsin-treated samples by SDS-PAGE under dissociating conditions.

at Vari-

previously shown to be a minor 01 cleavage product N-terminal in origin (Duncan et al., 1991; Leone et al., 1991a). Interestingly, trypsin digestion of the immature trimer (15 min sample) resulted in the degradation of both the N- and C-terminal halves. Whereas degradation of the unassembled C-terminal half of al was expected, that of the trimeric N-terminal half was not. The subsequent acquisition of the trypsin-resistant state by the N-terminal half (see 30 and 60 min samples) suggests that it undergoes a posttrimerization maturation process. Since this process appeared to be concomitant with trimerization of the C-terminal half, it was of interest to determine whether C-terminal trimerization is a requisite for maturation of the N-terminal trimer. To thisend, amutant with the C-terminal 204 amino acids deleted and therefore essentially lacking the entire C-terminal half (designated d204) was synthesized in vitro and analyzed as above. Protein trimerization was essentially complete after a 15 min chase period, since the level of trimers remained unchanged upon prolonged incubation (Figure 6A). However, the trimers from the three chase periods differed significantly in terms of structural integrity: whereas the 26 kd N-terminal fragments of newly assembled trimers (15 min samples) were highly sensitive to trypsin, those from trimers taken at later chase periods (30 and 60 min samples) were significantly more trypsin resistant (Figure 66). Considering the nature of the truncation of the d204 mutant, these results clearly indicate that the N-terminal half undergoes posttrimerization maturation (from the trypsin-sensitive to the trypsinresistant state) independent of C-terminal trimerization.

A pulse-chase experiment similar to the one described in the legend to Figure 4 was carried out. At the indicated times, trypsin was added to the reactions to a final concentration of 5 @ml. After further incubation at 37% for 30 min, trypsin inhibitors were added to the reaction mixtures. The samples were then boiled in protein sample buffer and analyzed by SDS-PAGE. (A) Analysis of untreated samples by SDS-PAGE under nondissociating conditions (both preincubation [in protein sample buffer] and electrophoresis of samples were carried out at 4%). (6) Analysisof trypsin-treated samples by SDS-PAGE under dissociating conditions (samples were boiled in protein sample buffer for 5 min, and SDS-PAGE was carried out at room temperature).

Wild-Type-Mutant al Heterotrimers Adopt Mutant al Conformation We have previously observed that cl needs to be in the trimeric state in order to be functional (Leone et al., 1991 c) and that C-terminal-truncated 01 mutant proteins are nonfunctional (Duncan et al., 1991). These observations have led to the interesting question as to whether in the case of wild-type-mutant al heterotrimers, the presence of one or two wild-type 01 subunits may compensate, perhaps partially, for the mutant al subunit(s) in terms of structural

trypsin digestion. To this end, a pulse-chase experiment similar to the one described above was carried out, and aliquots taken at various chase periods were treated with trypsin, followed by SDS-PAGE (Figure 5). As expected, trypsin digestion of the 60 min sample yielded the characteristic 26 kd N-terminal and 23 kd C-terminal fragments, indicativeofthestructural integrity of the mature al protein (monomeric cl was totally degraded under this condition; also see Leone et al., 1991~). The faint 21 kd band was

A

15 30 60 (min) -,. rrbdl C trimer - - u8 c trimer

(intermediate) (mature)

6 a-

Figure 5. Trypsin Digestion ous Stages of Maturation

N 26K LC 23K N 21K of In Vitro Synthesized

01 Trimers

Reovirus 403

Protein

A

al Trimerization

~

I

.

Domains

II

III

IV

I

A

B

--

B

c

FL

A/B

Bind

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.-A,

82 B3

R/d44 4:1

2:1

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d44

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1:1

1:2

1:4

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1:2

1:4

N26K 23K N21K

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Figure 7. Analysis of In Vitro Cotranslation and 3’-Truncated (d44) Sl mRNAs

*--Products

of Full-Length

Transcripts were translated either individually or in combination. The in vitro synthesized products were then subjected to various assays (see below), followed by SDS-PAGE analysis. The compositions of the protein bands are indicated on the right, where A and B represent the full-length (FL) and d44 products, respectively. (A) Aliquots from the reaction mixture (AfB) containing the four cotranslation products were analyzed for cell binding activity (Bind) and for recognition by the G5 antibody (G5) and by the anti-C-terminus serum (C). Prior to SDS-PAGE, the samples were incubated in protein sample buffer at 37% for 30 min (nondissociating conditions). (8) Cotranslation products synthesized using various ratios of the two transcripts were analyzed by SDS-PAGE under nondissociating conditions (C) Reaction mixtures from (B) were digested with 5 ug/ml trypsin, followed by the addition of trypsin inhibitors. The samples were then boiled in protein sample buffer and analyzed by SDS-PAGE.

and functional integrity. Accordingly, full-length, wild-type al and a truncated al with 44 amino acids deleted from the C-terminus (d44) were cosynthesized in vitro. As noted previously (Strong et al., 1991) four trimeric 01 species could be identified (Figure 7A): two homotrimers (A3 and B3) and two heterotrimers (A2B1 and A1B2). When the translation mixture containing these four species was applied to L cell monolayers, only the wild-type homotrimer (A3) was found to bind. lmmunoprecipitations of the reaction mixture were also carried out using an anti-al monoclonal antibody, G5, (Burstin et al., 1982), which recognizes a conformational epitope on the C-terminal half of cl (Duncan et al., 1991) and a polyclonal serum (anti-C) prepared against the C-terminal 90 amino acids of cl and therefore specific for unassembled C-terminus (Duncan et al.,

1991). Only the wild-type homotrimer (&) was precipitated by the G5 antibody, whereas the other three species were recognized by the anti-C serum. To gain further insight into the structural integrity of the heterotrimers, we varied the ratio of wild-type to mutant transcripts in cotranslation reactions, followed by trypsin digestion. The results (Figures 78 and 7C) show that the N-termini of all the trimeric species were resistant to trypsin, which suggests that the fibrous domains of these trimers were folded correctly. In contrast, the C-termini of the two heterotrimers, like that of the mutant trimer, were sensitive to trypsin. Taken together, both antibody and trypsin analyses indicate that the C-termini of heterotrimers are grossly misfolded, and are thus nonfunctional. Essentially the same results were obtained when smaller C-terminal deletions (up to 4 amino acids) were used instead of d44 in the cotranslation experiments (data not shown). Moreover, when nonfunctional C-terminal sitespecific mutants were cotranslated with a functional N-terminal deletion mutant (dll), the two heterotrimeric species generated were also nonfunctional (data not shown). The possibility that the mutant C-terminus steritally hinders the folding of wild-type C-terminal subunits (thereby rendering the wild-type-mutant heterotrimers nonfunctional) was ruled out by the observation that heterotrimers of wild-type and of d204, which lacks the entire C-terminal half, were also nonfunctional and possessed grossly misfolded C-termini (Figure 8). Collectively, these experiments clearly show that wild-type-mutant al heterotrimers adopt the mutant conformation, and they imply that 01 C-terminal folding is a highly cooperative process that requires that all three subunits be competent (i.e., potentially functional). Discussion Sequence analyses of the reovirus Sl gene have previously identified distinct structural domains in the al protein (BasseCDubyet al., 1985; Duncan et al., 1990; Nibert et al., 1990). The N-terminal third of al is highly a-helical and contains a heptapeptide repeat of hydrophobic residues, suggestive of a coiled-coil structure. This is followed by a middle region composed largely of 8 sheets. The C-terminal third of al does not possess any distinct pattern and is therefore predicted to assume a complex globular conformation. These theoretical predictions are compatible with electron microscopic data that reveal protein 01 (purified from virions or from a mammalian expression system) as lollipop-shaped structures with fibrous tails and globular heads (Banerjea et al., 1988; Furlong et al., 1988; Fraser et al., 1990). The structural differences between the two termini of 01 also reflect their distinct functional roles: the globular head contains a conformation-dependent receptor-binding domain, whereas the fibrous tail anchors the 01 protein to the virion and serves as a stable extension, which presumably facilitates access of the globular head to the cellular receptor. Recent evidence suggests that protein 01 is a homotrimer (Strong et al., 1991) and that both the conformation and function of al are intimately linked to its oligomeric

Cell 494

A

I

I

II

Ill

I

IV

I FL (A) d 204 (B)

.-As ‘APBI -

state (Leone et al., 1991 c). Protease and antibody recognition analyses reveal that trimerization of 01 is accompanied by extensive conformational changes necessary for its cell attachment function. We have taken advantage of the fact that a major protease-sensitive site is present in the middle of the protein, essentially dividing 01 into two halves that are similar in molecular size but drastically different in shape, stability, and function (Yeung et al., 1989; Duncan et al., 1991; Leone et al., 1991a). The bestcharacterized proteolytic fragments are those generated by trypsin, which cleaves ol after Arg-245. The two 01 tryptic fragments (26 kd and 23 kd, respectively) maintain their trimeric status, with the N-terminal trimeric fragment being significantly more stable than the C-terminal trimerit fragment that retains its cell binding function. However, when expressed as a truncated protein, the C-terminal half manifests little, if any, cell binding activity (Nagata et al., 1987). This lack of activity has been attributed in the present study to the inability of the expressed truncated protein (C-terminal monomers) to trimerize. This observation, coupled with the previous demonstration that an in vitro synthesized polypeptide containing the N-terminal heptad repeat is capable of forming stable dimers and trimers (Leone et al., 1991 a), suggests that N-terminal trimerization is a prerequisite for C-terminal trimerization and function. A subsequent pulse-chase experiment (Figure 4) indicates that this is indeed the case. Protein 01 trimerization apparently begins with the formation of a “loose,” three-stranded, coiled-coil structure at the N-terminus (Figure 9). Since the C-terminal subunits are still unassembled at this stage, this structure (trimer [intermediate]) presumably assumes the shape of a hydra whose mobility is retarded in polyacrylamide gels. Importantly, both the N-and C-terminal halves of this intermediate form are highly susceptible to trypsin. Further maturation of al involves two independent, yet apparently concurrent,

AI

B2 B3

Figure 8. Analysis of In Vitro Cotranslahon Products of Full-Length and 3’Truncated (d204) Sl mRNA (A) Transcripts were translated either individually or in combination at various ratios. Aliquots of the reaction mixtures were either analyzed directly by SDS-PAGE or applied to L cell monolayers, followed by SDS-PAGE analysis of cell-bound proteins. All samples were incubated in protein sample buffer at 37% for 30 min prior to electrophoresis. The compositions of the protein bands are indicated on the right, where A and B represent the full-length (FL) and d204 products, respectively. The band indicated by an asterisk on the left represents a misfolded form of the full-length 01 trimer previously reported (Strong et al., 1991). (6) Reaction mixtures from (A) were digested with 5 uglml trypsin. followed by the addition of trypsin inhibitors. The samples were then boiled in protein sample buffer and analyzed by SDS-PAGE.

events: trimerization and cooperative folding of the C-terminal subunits, and maturation of the N-terminal coiled coil into a stable fiber conformation. The resulting structure is a lollipop-shaped homotrimer that migrates at a position more representative of its true molecular size and that is relatively resistant to trypsin. Only the fully matured (lollipop) form of 01 is capable of interacting with cellular receptors. The use of protein (~1 as a model for the study of protein oligomerization and folding has led to three interesting revelations. The first is the existence of independently active oligomerization domains in a single protein. In view of the drastic structural and functional differences between the N-and C-terminal domains of 01, and the requirement of each domain to be in the trimeric state for its functional integrity, the evolvement of two separate, trimerization foci is not surprising, and may well be essential. Based on the observation that al heterotrimers are readily formed in cotranslation experiments, it was originally thought that the initial trimerization event, which takes place at the N-terminus, occurs posttranslationally. However, recent evidence suggests that this is not the case. First, in all of our cotranslation experiments, the distribution of the four oligomeric species (A3, A2Bl, A1B2, and B3) was never found to be in the ratio of 1:3:3:1 (assuming equimolar amounts of A and B were synthesized) as would be the case had assortment occurred randomly (i.e., posttranslationally). Rather, we consistently observed a preference for homotrimer over heterotrimer formation. Second, proportionate reduction of the amounts of the two RNA transcripts invariably resulted in drastic bias against heterotrimer formation (unpublished data). Third, ribonuclease treatment of translation samples immediately after the first round of al monomers are synthesized abrogates N-terminal trimerization of 01 (unpublished data). These observations are compatible with the notion that N-terminal

Reovirus

Protein

ol Trimerization

485

Figure Trimer

C-Terminal Trimerization and Folding

N-Terminal Trimerization

N-Terminal Maturation

INTERMEDIATE TRIMER

trimerization occurs cotranslationally. Formation of heterotrimers would be via the interaction between the N-termini of nascent 01 chains from adjacent transcripts, and would therefore represent an artifact generated at high transcript concentrations. Trimerization of the C-terminus, however, does not commence until synthesis of the third subunit is complete, and thus occurs posttranslationally. It is interesting to note that although mechanisticallydifferent, both trimerization events are triggered by the imposition of spatial proximity between subunits. Trimerization of the N-terminus necessarily involves neighboring nascent cl chains from the same transcript, and is likely facilitated by ribosome stacking and elongation arrest in the 01 reading frame, as recently ObSeNed by Doohan and Samuel (1992). The joining of the three subunits at their N-termini in turn provides the required spatial proximity between the C-termini for their subsequent trimerization. It can easily be seen how such an oligomerization scheme could spare individual 01 subunits the need to search for their “partners” in a soluble pool. Recently we found that certain populations of 01 are complexed with additional factors and that 01 trimerization is an ATPdependent process (unpublished data). It thus appears that the formation of the 01 trimer is chaperone mediated. Whether chaperones are involved in one or both of the aforementioned trimerization events remains to be determined. The second revelation from the present study is the existence of an intermediate state for oligomerization of fibrous proteins. Expression of the N-terminal heptad repeat region alone initially yields a trypsin-sensitive trimeric structure (possibly in the form of a loose coiled coil) that eventually matures into a trypsin-resistant form. Newly assembled trimers are also found to be less thermostable than mature trimers (data not shown). Such a transition state is perhaps equivalent to the “molten globule” state described for globular proteins (Semisotnov et al., 1987) and has led us to entertain the possibility that a molten fiber

MATURE TRIMER

9. Model

for the Generation

of the al

Trimerization of 01 involves three discernible steps. Step 1: N-terminal trimerization. Although not depicted in the diagram, this step presumably occurs cotranslationally and involves interactions between the N-termini of nascent al chains initially to yield a loose, protease-sensitive, three-stranded coiled coil in this region. Step 2: C-terminal trimerization. This step does not commence until the synthesis of the last (third) subunit is complete, and thus occurs posttranslationaiiy. Trimerization in this region iS accompanied by cooperative folding between the three termini, with the end result being the generation of a globular head with cell receptor binding function. Step 3: N-terminal maturation folding. This process occurs independently of, but concomitantly with, trimerization of the C-terminus, and transforms the N-terminal half of 01 into a stable, protease-resistant, fibrous structure. Note: the involvement of cellular factors in these processes is not included in this scheme.

state exists in the oligomerization and folding pathway of fibrous proteins. Interestingly, the N-terminal maturation process and C-terminal trimerization occur concomitantly. However, it is clear that they represent independent events. Recent data suggest that the mature fiber is stabilized by both hydrophobic and ionic interactions between the subunits (Leone et al., 1991a). The third revelation pertains to the molecular basis of the so-called dominant negative effect initially proposed by Herskowitz (1987) to explain how the function of a wildtype gene product could be inhibited by a mutant allele of the same gene. In the case of multimeric proteins, it was suggested that the mutant subunit(s) could exert thiseffect by interacting with wild-type subunit(s) to yield an inactive complex. A requisite for such a hypothesis would be that the oligomerization domain of the mutant subunit remains intact and functional. Results from our full-length/d44 01 heterotrimer analysis in the present study clearly concur with this notion. For both heterotrimeric species (A1B2 and AaB1), trimerization at the N-terminus remains unperturbed. However, a defect at the C-terminus in a single subunit is sufficient to inactivate the other two wild-type subunits in the complex and totally abrogate 01 receptor binding function. That the C-termini of these heterotrimers are unassembled and grossly misfolded has been verified using various criteria. The manifestation of such a dominant negative effect exemplifies the cooperative nature of the second (C-terminal) trimerization event. Our present findings have also raised the interesting question of whether Herskowitz’soriginal hypothesisshould be further refined such that it would be pertinent to only those proteins possessing two independent, active oligomerization domains where, in the case of wild-type proteins, the primary oligomerization event facilitates the subsequent secondary oligomerization step to yield a functional complex. Thus, a mutant protein could exert a dominant negative effect only if it possesses a functionally intact primary oligomerization domain and a defective secondary oligo-

Cell 486

merization domain. Such a mechanism would obligatorily involve a highly efficient primary oligomerization step; the formation of a coiled-coil structure such as the one found in 01 would serve precisely this purpose. It is interesting to note in this regard that dominant negative effects have also been observed in certain tumor suppressors such as p53 (Finlay et al., 1989; Eliyahu et al., 1989; Baker et al., 1989; Nigro et al., 1989) and, more recently, adenomatous polyposis coli (Kinzler et al., 1991; Nishisho et al., 1991; Grodenetal., 1991; Joslynetal., 1991). Inthecaseofp53, it was postulated that mutant p53 exerts this effect by interacting with wild-type p53 to form an inactive complex, thereby promoting tumor progression (Eliyahu et al., 1988; Rovinski and Benchimol, 1988; Finlayet al., 1989; Gannon et ,al., 1990; Milner et al., 1991). This notion has been supported by the recent demonstration in vitro that mutant p53 is capable of driving cotranslated wild-type p53 into the mutant conformation via the interaction between nascent polypeptides (Milner and Medcalf, 1991), an observation in absolute accord with our present findings with protein 01. A similar explanation has also been proposed for adenomatous polyposis coli, in which a wild-typemutant heterozygote exhibits a mutant phenotype. Although this hypothesis has yet to be tested, it is interesting to note that the N-terminal quarter of the adenomatous polyposis coli protein, like that of protein 01, is also a coiled coil. That the coiled coil plays a significant role in the manifestation of dominant negative effects by mutant tumor-suppressor genes has also been theorized and strongly advocated by Bourne (1991 a, 1991 b). If our observations on the ol protein could be generalized, then the formation of the coiled coil would represent the primary and obligatory step in this process, whereas the actual disruptive effect would be the result of the perturbation of subsequent secondary oligomerization and cooperative folding events.

nol precipitated

I” the presence of 2.5 M ammomum acetate. The pellets were washed, resuspended in sterile water, and stored in small aliquots at -7O’C until needed for translation. The resulting mRNA was >95% reaction.

Procedures

Transcription

Capped Sl-specific mRNAs were produced by in vitro transcription using Sp6 RNA polymerase (Pharmacia). The plasmids encoding the full-length and the 44 amino acid C-terminally truncated (d44) 01 products were previously described by Duncan et al. (1991). Deletion of region II of the Sl gene to produce the dll mutant was generated by complete digestion of the full-length parent vector (pG4T3) with Bell, followed by religation of the vector. The full-length, dll, and d44 DNA templates were linearized with Hindlll. The d204 transcripts were generated by linearization of the full-length DNA template with Hgal. The 50 ~1 transcription reactions contained 1 x reaction buffer (40 mM Tris-HCI [pH 7.51, 6 mM MgCI,, 2 mM spermidine, 10 mM NaCl [Promega]), 1 mM ATP, CTP, and UTP, 0.1 mM GTP, 0.5 mM cap analog, 6 mM dithiothreitol, IO mglml bovine serum albumin, 1.0 U/WI RNAguard [Pharmacia], 5 pg of linearized DNA template, and 15 U of Sp6 RNA polymerase per pg of DNA. The reactions were incubated for 30 min at 37OC, at which time GTP was added to a final concentration of 1 .O mM and incubated for an additional 45 min. The DNA templates were digested by the addition of 0.15 pg of RNAaaa-free DNAase I (Bethesda Research Laboratories) and 1 .O U/PI RNAguard. followed by a 15 min incubation at 37OC. The reactions were diluted with 10 mM Tris (pH 7.5) and 1 mM EDTA, extracted with phenol, phenol-chloroform, and chloroform, and etha-

were approximately

lo-15

pg per

L Cell Binding Assay The L cell binding assay was essentially the same as previously described (Lee et al., 1981). Confluent L-929 cell monolayers in 60 mm plates (Falcon) were preincubated with cold phosphate-buffered saline containing 10 mglml bovine serum albumin for 10 min at 4’%, before the addition of 200 PI of diluted translation reactions containing 2.5 mM methionine. The plates were incubated at 4% with intermittent rocking for 60 min (unless otherwise indicated), after which the supernatant was removed and the monolayers were washed five times with phosphate-buffered saline. The cells were lysed with 50 ~1 of lysis buffer(phosphate-buffered saline containing 1% NP-40,0.5% sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride) and the nuclei were pelleted for 2 min in a microfuge at 5000 x g. Protein sample buffer was added to the supernatant and the bound protein was analyzed by SDS-PAGE and fluorography.

lmmunoprecipitation The monoclonal anti-o1 antibody G5 has been described previously (Burstin et al., 1982) and shown to interact with a C-terminal conformational epitope of 01 (Yeung et al., 1987; Leone et al., 1991~). C-terminal specific polyclonal anti-al (C) was prepared in rabbits using SDS-PAGE-purified trpE-al fusion proteins expressed in Escherichia coli using a pATH3 vector (Cashdollar et al., 1989) and was previously shown lo interact with denatured al polypeptide (Leone 81 al., 1991~). [%]methionine-labeled in vitro translation lysates were immunoprecipitated with the above antibodies using a modified immunoprecipitation procedure described previously (Leone et al., 199la).

Digestion

Diluted in vitro translation trypsin

In Vitro

and yields

Translation Capped Sl-specific mRNAs were translated in vitro in rabbit reticulocyte lysates according to the manufacturer’s specifications (Promega). The 25 ~1 translation reactions contained 50-l 00 Kg of RNA and 20 VCi of [35S]methionine (1100 Cilmmol; New England Nuclear). Reactions were incubated at 37OC for 45 min (unless otherwise indicated), then diluted to 200 ~1 with phosphate-buffered saline (pH 7.2) and used for L cell binding assays and analysis by SDS-PAGE.

Trypsin Experimental

full length.

(Sigma)

reactions

at a 5 &ml

were digested

final concentration

with TLCK-treated for 30 min at 37%.

Trypsin inhibitors were then added to the reactions (soybean and egg white trypsin inhibitors [Sigma]) and further incubated in protein sample buffer,

as indicated

in the figure

legends,

prior to SDS-PAGE.

SDS-PAGE Discontinuous SDS-PAGE was performed using the protocol of Laemmli (1970). Both 7.5% and 10% polyacrylamide gels were used. Samples were incubated in protein sample buffer (final concentration: 50

mM Tris [PH. 6.81, 1% SDS, 2% B-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue) for 30 min at either 37OC or 4OC or, alternatively, boiled for 5 min prior to SDS-PAGE, which was performed either at room temperature or at 4X. The various conditions used for SDS-PAGE were necessary owing to the difference in stability between the trimeric N-terminal and C-terminal domains. Under dissociating conditions where samples were boiled for 5 min prior to SDS-PAGE (carried out at room temperature), both N-and C-terminal trimers disso-

ciated and migrated as monomers. Under nondissociating conditions, samples were either incubated for 30 min at 37X, at which temperature only the trimeric N-terminal domain is stable, or incubated at 4OC, at which temperature both the N-and C-terminal trimeric domains are stable (SDS-PAGE was carried out at 4% in both cases). In cases where samples contained heterotrimers, the different trimeric species were resolved by allowing electrophoresis lo continue for an additional 5 hr after the dye front had reached the bottom of the gels.

Reovirus 487

Protein

al Trimerization

Domains

Acknowledgments We thank Dr. B. N. Fields (Harvard Medical School) for the G5 monoclonal anti-al antibody and J. Fernandes and D. Kim for assistance with the artwork. This work was supported by the Medical Research Council of Canada (P. W. K. L.). G. L. is a recipient of the Alberta Heritage Foundation for Medical Research Studentship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “edvertisemenr” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

June

12, 1992;

revised

August

7, 1992

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