Antonie van Leeuwenhoek 61: 93-99, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Biophysical studies of recognition sequences for targeting and folding Lila M. Gierasch, Jeffrey D. Jones, Samuel J. Landry & Sarah J. Stradley Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041, USA

Key words: signal sequence, LamB protein, chaperone binding, GroEL protein, DnaK protein

I. Introduction

It has been accepted since the 1960s that the information present in the amino acid sequence of a nascent polypeptide chain determines the final folded structure of the mature protein. However, the mechanism whereby the nascent chain folds and is correctly localized in vivo has only recently begun to receive much attention (see, for example, Fischer & Schmid 1990). These two processes, folding and localization, are now known to be coupled (Eilers & Schatz 1986; Liu et al. 1989; Neupert et al. 1990), and, furthermore, both are facilitated by factors that are collectively known as molecular chaperones (Ellis & van der Vies 1991). We have initiated studies directed at a particularly puzzling aspect of the in vivo folding and localization problems (Landry & Gierasch 1991): how is the nascent chain recognized and bound by chaperones (including species that facilitate targeting)? The recognition takes place when the nascent chain is incompletely folded, and thus must rely on features that are present in polypeptide chains in this (illdefined) state. Also, many different proteins are recognized by the same chaperone, and the regions that are bound lack primary sequence identity. Hence, one must ask how such different sites are recognized. On the other hand, chaperones do not work indiscriminately, so a basis must be found for their specificity in the absence of sequence identity. We and others have studied targeting sequences with a goal of elucidating the features that are required for their binding by proteinaceous export factors and for their roles more generally in the

pathway of protein localization (for reviews, see Gierasch 1989; Jones et al. 1990). We have focused on signal sequences for export from bacteria. Our strategy has been to synthesize peptides corresponding to wild-type and mutant signal sequences and to correlate the physical properties and interactions of the peptides with the phenotypes of the cognate signal sequences in vivo. In section 1I of this review, we describe our recent work on the characterization of signal peptides. Many of the approaches we have used with signal peptides show promise of shedding light on the mode of recognition of nascent polypeptides by chaperones that facilitate folding. Hence, we have made synthetic peptides and have studied their interactions with the E. coli chaperones, GroEL and DnaK, using primarily a nuclear magnetic resonance (nmr) method that can yield a picture of the chaperonebound conformation of the peptide. Preliminary results of these approaches are summarized in section lI1 of this review. Lastly, we discuss in section IV the implications of our results and common themes in recognition events associated with targeting and folding in vivo.

H. Biophysical studies of signal sequences We have carried out biophysical characterization of peptides derived from two families of signal sequences: one set includes the wild-type and several mutants of the Lamb protein (see McKnight et al. 1989), and the other, the wild-type and several mutants of the OmpA protein (Hoyt & Gierasch

94 1992). Both of these proteins are synthesized cytoplasmically in E. coli and exported to the outer membrane. Our results have shown that functional signal sequences have a high propensity to adopt an ~thelical conformation in membrane-mimetic environments such as micelles and vesicles, but are unstructured in aqueous solution. This conformational behavior is a necessary but not sufficient property of a functional signal sequence. In addition, we find that those peptides corresponding to functional signal sequences have a high affinity for lipid membranes and an ability to insert spontaneously well into the hydrocarbon region of a bilayer. We anticipated that signal sequences would be different from transmembrane sequences, since their hydrophobic cores are approximately half as long. Nonetheless, they can stably insert into a membrane. The evidence supporting this conclusion includes the ability of signal peptides to cause substantial surface pressure rises in lipid monolayers (Briggs et al. 1985; McKnight et al. 1989), the large blue shift of tryptophan residues in the signal peptide upon interaction with lipid vesicles (McKnight et al. 1991; Hoyt & Gierasch, 1991), and the efficient quenching of fluorescence of tryptophans in the signal peptide by nitroxide quenchers within the acyl chains of the phospholipids in vesicles (McKnight et al. 1991). The shorter length of the hydrophobic core of signal sequences raises the question of how they interact with bilayers. On the one hand, one might expect a transmembrane orientation to be of marginal stability, since the free energy gain from burying the hydrophobic residues may not compensate for the free energy loss from conformationally restricting the peptide and from desolvating it. On the other hand, we have data from nmr (Bruch & Gierasch 1989; Bruch et al. 1990) and circular dichroism (McKnight et al. 1989; Hoyt & Gierasch 1992) arguing that the signal peptides are largely a-helical in bilayers or micelles, and we know that these helices must penetrate beneath the bilayer/aqueous interface. Given the charged termini of these peptides, the only reasonable modes of insertion would be transbilayer or kinked so as to re-emerge on the same side of the membrane as the insertion. In our current

model, the signal peptides are in equilibrium between the transmembrane conformation and the kinked form that is anchored in one leaflet only. We find stronger evidence of the transbilayer orientation in the case of the OmpA peptides than for the LamB peptides (Hoyt 1991; Sankaram & Jones, unpubl, results), arguing that the two forms are in equilibrium. In both modes of interaction, the positively charged N-terminus remains strongly associated with the headgroup region on the side of insertion. We are now testing this model by attempting to tip the equilibrium towards the transbilayer conformation and/or to label the peptide from the opposite side of the bilayer. Because we have gathered data on several related signal peptides, we can correlate their ability to insert into a bilayer with characteristics of the sequences (Hoyt & Gierasch 1992). As shown in Table 1, assuming a hydrophobic core of minimum length seven residues, the core must have a mean residue hydrophobicity higher than 2.4 (Kyte-Doolittle, 1982, scale) for the signal peptide to insert into a membrane and for the corresponding signal sequence to be export competent in vivo. This hydrophobicity threshold falls roughly midway between the hydrophobicities of Ala and Leu on this scale. Another question pertinent to the roles of signal sequences in vivo is how they interact with the adjacent region of polypeptide chain. We have analyzed the conformational and membrane-interaction properties of a 53-residue peptide corresponding to the LamB signal sequence and the first 28 residues of the mature LamB protein (McKnight et al. 1991). As shown in Fig. 1, the conformationally diagnostic circular dichroism spectrum of the 53-residue peptide in lipid vesicles is essentially the sum of the spectra of the signal peptide and the 28-residue peptide derived from the mature LamB sequence. Similarly, the membrane affinity and mode of interaction of the longer peptide with bilayers are similar to those of the signal peptide alone. While the mature peptide alone has little tendency to associate with a lipid bilayer, it is tethered to the surface region of the membrane by its attachment to the signal peptide. We conclude from these observations that the first part of the

95

mature domain of the L a m B protein, and perhaps most secreted proteins, is selected to be a passive spacer that does not c o m p e t e with the signal sequence for interactions with m e m b r a n e or proteins and, f u r t h e r m o r e , does not perturb the properties of the signal peptide. The implications of the biophysical data for the mechanism of signal sequences must be cautiously extracted, since these data are gathered for isolated molecular species in model systems. Nonetheless, we posit that the signal s e q u e n c e / m e m b r a n e interactions are a central part of the export mechanism. After release of the signal sequence from SecA (or signal recognition particle, SRP, in eukaryotes), the spontaneous insertion of the signal sequence into the m e m b r a n e would tether the nascent chain and lead to two-dimensional diffusion in the plane of the m e m b r a n e . Subsequent association with a

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190 Table 1. Core mean residue hydrophobicity of Lamb and OmpA signal sequences and export activity. Signal sequence" Mean residue hydrophobicity of coreu

Export activityc

LamB WT A78 A78rl A78r2 G17R A13D

2.4 2.0 2.4 2.7 2.4 1.8

++++ 0 ++ ++++ ++ 0

OmpA WT L6L8 A9 A8 ISN A6--9

2.9 2.7 3.0 2.7 2.0 1.8

++++ ++++ ++++ ++ 0 0

"LamB WT: MMITLRKLPLAVAVAAGVMSAQAMA;OmpA WT: MKKTAIAIAVALAGFATVAQA. A 7 8 represents a mutant with residues 10-14 deleted from the LamB WT; A78rl has this deletion plus G17C; A78r2 has the same deletion plus P9L. All other mutants are named to indicate residue changes or deletions using numbering from the N-terminal residue. Calculated using Kyte-Doolittle (1982) parameters for the hydrophobic core, as delimited by polar residues or, for mutants, the seven-residue stretch of highest hydrophobicity which leaves at least seven residues before the cleavage site. CExport activities are derived from data reported in Emr & Silhavy (1983), Stader et al. (1987), Lehnhardt et al. (1987), or Goldstein et al. (1990, 1991).

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Wavelength (nm) Fig. l. Circular dichroism spectrum of the 53-residue peptide corresponding to the LamB signal sequence with the first 28 residues of the mature Lamb protein ( ) compared to the normalized sum of the circular dichroism spectra of the 25residue signal peptide and the 28-residue mature domain (----), in lipid vesicles. From McKnight et al. 1991, reproduced with permission.

membrane-resident translocation apparatus would thus be more probable. The translocation event itself may require direct contact between the signal sequence and lipid (Simons & Blobel 1991). The next stage of our efforts to understand the roles of signal sequences and how varying sequences with c o m m o n patterns of residue type can participate in a c o m m o n export pathway will involve study of protein recognition and binding of signal peptides. We have begun with SecA, which has been shown both genetically (Fikes & Bassford 1989) and biochemically (Akita et al. 1990) to interact with the signal sequence.

IlL Binding of peptide fragments by chaperones The in vitro refolding of several proteins has been shown to be facilitated by addition of the E. coli chaperonin G r o E L / E S and ATP (Goloubinoff et

96 al. 1989; Laminet et al. 1990; Buchner et al. 1991; Mendoza et al. 1991; Martin et al. 1991). Similar results have been obtained on a more limited set of proteins using the E. coli Hsp70 homologue, DnaK (Skowyra et al. 1990). We reasoned that the recognition of a nascent chain by these species likely involved regions synthesized early, and might make use of structural elements that were present even when the protein was incompletely folded. One example would be c~helices. Furthermore, we anticipated that recognition sites for chaperone binding would present a surface that was no longer accessible once the protein had folded, e.g. a hydrophobic surface. Using these premises in the case of GroEL, we examined the protein rhodanese, for which a crystal structure is available, and selected an N-terminal c~helix that lies on the surface of the native protein as a potential region for GroEL binding. We synthesized a thirteen-residue peptide corresponding to this region, AC-STKWLAESVRAGKNH2, and applied a novel nmr method to analyze the peptide/GroEL interaction. The nmr approach used, measurement of transferred nuclear Overhauser effects (tr-nOes, Clore & Gronenborn 1982), relies on the large differential in size between the peptide and the chaperonin. Nuclear Overhauser effects (nOes), which arise when two protons are within 4 A, are most efficiently developed on the slower tumbling large molecule. Thus, when the peptide is bound to GroEL, its interproton nOes are rapidly evolved, and when the peptide is released from GroEL, the nOes are only slowly dissipated. In a situation of rapid exchange between bound and free peptide, the free population retains a 'memory' of the nOes characteristic of its bound state. For peptides the size of our rhodanese fragment, the nOes of the free peptide are very small. Consequently, we see few interactions for the peptide alone and addition of GroEL is expected to lead to substantial nOes if the peptide binds. Furthermore, the pattern of trnOes reveals close interproton approaches in the bound peptide. We can in favorable cases deduce the bound conformation of the peptide. As shown in Fig. 2, the rhodanese peptide indeed binds GroEL (Landry & Gierasch 1991). Additionally, the tr-nOe pattern is strongly indicative

of an c~-helical conformation for the bound peptide. We modified the peptide to reduce its intrinsic propensity to adopt an ct helical conformation. Its affinity for GroEL was reduced, but it still bound as an ~thelix. We are now testing a variety of sequence variants to learn the limits of recognition by GroEL. Binding of a peptide from the vesicular stomatitis virus (vsv) G-protein, KLI6VLSSLFRP~:,to DnaK was revealed using the same method of tr-nOes (Landry & Gierasch 1992). Interestingly, the pattern of tr-nOes is quite different from that seen in the rhodanese peptide and appears to fit best to an extended conformation for the DnaK-bound peptide. The same peptide from the vsv G-protein also will bind GroEL, but, as in the case of the rhodanese peptide, it adopts an cz helix upon binding to this chaperone. This results shows the conformational plasticity of short peptide sequences and introduces the concept that different classes of chaperone may bind polypeptide sequences based on a conformational bias. We are currently testing these ideas.

IV. Implications of biophysical studies for recognition of nascent chains The 'birth' of a protein, i.e. its emergence from the ribosome, initiates a complex series of events culminating with the attainment of the native threedimensional structure in the correct cellular compartment. Recent work has identified a class of proteins that facilitates these processes, the socalled molecular chaperones. This name is now intended to include the species that help localize the nascent chain as well as those that act primarily to facilitate its folding and prevent its aggregation. A common property of these chaperones is the ability to recognize a variety of proteins that lack regions of primary sequence identity and that are incompletely folded. Our efforts have been directed at understanding the biophysical aspects of these recognition steps. How can different sequences bind to the same site? What conformational features are recognized? How is the incompletely folded state distinguished from the native

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Fig. 2. Two-dimensional nOe spectra of the rhodanese peptide without (a) and with (b) GroEL. Note the appearance of numerous tr-nOe peaks in the presence of the chaperone, including several in the NH to NH region (between 8.5 and 7.5 ppm) indicating the helical conformation of the peptide when bound to the chaperone. Reproduced with permission from Landry & Gierasch 1991. 9 1991 American Chemical Society

protein? The results we have obtained to date lead to several possible answers to these questions. First, secondary structural similarity appears to be a central characteristic both of sequences that present targeting information and of sequences that are bound by chaperones for folding. Signal sequences not only have a high propensity to fold as ct helices in interfacial environments, but also have clear patterns of residue type within the helix. We cannot yet identify this sort of pattern in the sequences that bind GroEL or DnaK, but we are altering the peptide sequences studied so as to explore this question. Second, the recognition sequences for chaperones must be accessible in the context of the nascent chain. We suggest, largely by extrapolation from targeting sequences, that an N-terminal location is probable both for topological reasons, since the termini of proteins are generally more accessible than the interior regions, and for temporal reasons, since the N-terminal region would be available for interaction early in the synthesis of the protein.

Third, we find that two chaperones bind the same peptide, but in different conformations. This result suggests that substrate selection by chaperones may rely on conformational propensity (as might be concluded from the first point discussed above). Also, this result raises the possibility that the chaperone acts as a template which favors particular secondary structures in order to help the chain achieve its native structure. This active assistance of folding by chaperones has not been strongly argued in the literature, but appears to require continued consideration. Fourth, we can glean some hints from our results to date about the characteristics of the incompletely folded chain that are exploited for recognition by chaperones: Some secondary structure has been shown to form on a very rapid time scale in the folding of proteins in vitro (see for example: Matouschek et al. 1990; Bycroft et al. 1990). Helices and extended or [3-strands would thus be expected to be present quite early on in the life of a nascent chain. The folding of the chain to optimize packing and sequestration of hydrophobic surface area is,

98

by contrast, a slow step in vitro (Creighton 1990). Hence, the ability to discriminate an incompletely folded nascent chain from one that has attained its native fold could be realized by binding specificity for these early-forming secondary structures together with patches of hydrophobic surface. Our results are consistent with this hypothesis. Clearly, there is a need for more study of the recognition of peptide sequences by chaperones and of the mechanism whereby their binding may facilitate folding. Binding to a chaperone will help prevent aggregation of nascent chains, as has been postulated by several researchers (for example, Buchner et a|. 1991), but more active roles in facilitating folding may also be important.

References Akita M, Sasaki S, Matsuyama S & Mizushima S (1990) J. Biol. Chem, 265:8164-8160 Briggs MS, Gierasch LM, Zlotnick A, Lear J & DeGrado WF (1985) In vivo function and membrane binding properties are correlated for E. coli LamB signal peptides. Science 228: 1096-1097 Bruch MD, Gierasch LM (1990) Comparison of helix stability in wildtype and mutant LamB signal sequences. J. Biol. Chem. 265:3851-3858 Bruch MD, McKnight CJ & Gierasch LM (1989) Helix formation and stability in a signal sequence. Biochemistry 28: 85548561 Buchner J, Schmidt M, Fuchs M, Jaenicke R, Rudolph R, Schmid FX & Kiefhaber T (1990) GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30: 1586-1591 Bycroft M, Matouschek A, Kellis JT, Jr Serrano L & Fersht AR (1990) Detection and characterization of a folding intermediate in barnase by NMR. Nature 346:488--490 Clore GM & Gronenborn AM (1982) Theory and applications of the transferred nuclear Overhauser effect to the study of the conformations of small ligands bound to proteins. J. Magn. Res. 48:402-417 Creighton TE (1990) Protein folding. Biochem. J. 270:1-L6 Eilers M & Schatz G (1986) Binding of a specific ligand inhibits import of a purified precursor into mitochondria. Nature 322: 228-232 Ellis RJ & van der Vies SM (1991) Molecular chaperones. Ann. Rev. Biochem. 60:321-347 Emr SD & Silhavy TJ (1983) Importance of secondary structure in the signal sequence for protein secretion. Proc. Natl. Acad. Sci. USA 80:4599-4603 Fikes JD, Bassford PJ, Jr (1989) Novel secA alleles improve

export of maltose-binding protein synthesized with a defective signal pcptide. J. Bacteriol. 171:402-409 Fischer G & Schmid FX (1990) The mechanism of protein folding. Implications of in vitro refolding models for de novo protein folding and translocation in the cell. Biochemistry 29: 2205-2212 Gierasch LM (1989) Signal sequences. Biochemistry 28: 923930 Goldstein J, Lehnhardt S & Inouye M (1990) Enhancement of protein translocation across the membrane by specific mutations in the hydrophobic region of the signal peptide. J. Bacteriol. 172:1225-1231 Goldstein J, Lehnhardt S & Inouye M (1991) Effect of asparagine in the hydrophobic region of the signal sequence. J. Biol. Chem. 266:14413-14417 Goloubinoff P, Christeller JT, Gatenby AA & Lorimer GH (1989) Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP. Nature 342:884--889 Hoyt DW, (1991) Ph.D. dissertation, Univ. of Texas Southwestern Med. Centre Hoyt DW & Gierasch LM (1991) A peptide corresponding to an export-defective mutant OmpA signal sequence with asparagine in the hydrophobic core is unable to insert into model membranes. J. Biol. Chem. 266:14406-14412 (1992) Hydrophobic content and lipid interactions of wildtype and mutant OmpA signal peptides correlate with their in vivo function. Biochemistry (in press) Jones JD, McKnight CJ & Gierasch LM (1990) Biophysical studies of signal peptides: imlications for signal sequence functions and the involvement of lipid in protein export. J. Bioener. Biomemb. 22:213-232 Kyte J & Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132 Laminet A A , Ziegelhoffer T, Georgopoulos C & Plueckthun A (1990) The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the 13-1actamase precursor. EMBO J. 9:2315-2319 Landry SJ & Gierasch LM (1991) Recognition of nascent polypeptides for targeting and folding. TIBS 16:159-163 - - (1992) The chaperonin GroEL binds a polypeptide in an co-helical conformation. Biochemistry 30:7359-7362 - - (1992) Recognition of peptides by the E. coli molecular chaperones, GroEL and DnaK. In: Smith JA (Ed) Peptides: Chemistry, Structure, and Biology. ESCOM Science Publishers, Leiden, The Netherlands Lehnhardt S, Pollitt S & Inouye M (1987) The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide. J. Biol. Chem. 262:1716-1719 Liu G, Topping TB & Randall LL (1989) Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein. Proc. Natl. Acad. Sci. USA 86:9213-9217 Martin J, Langer T, Boteva R, Schramel A, Horwich AL & -

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99 Hartl F-U (1991) Chaperonin-mediated protein folding at the surface of groEL through a "'molten globule"-like intermediate. Nature 352:3(~-42 Matouschek A, Kellis JT Jr, Serrano L, Bycroft M, Fersht AR (19911) Transient folding intermediates characterized by protein engineering. Nature 346:440--445 McKnight C J, Briggs MS & Gierasch LM (19891 Functional and nonfunctional Lamb signal sequences can be distinguished by their biophysical properties. J. Biol. Chem. 264:17293-17297 McKnight C J, Rafalski M & Gierasch LM (19911 Fluorescence analysis of tryptophan-containing variants of the Lamb signal sequence upon insertion into a lipid bilayer. Biochemistry 31): 6241-6246 McKnight CJ, Stradley SJ, Jones JD & Gierasch LM (1991) Conformational and membrane-binding properties of a signal sequence are largely unaltered by its adjacent mature region, Proc. Natl. Acad. Sci. USA 88:5799-58113 Mendoza JA, Rogers E, Lorimer GH & Horowitz PM, (1991)

Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J. Biol. Chem. 266:13044-13049 Neupert W, Hartl F-U. Craig EA, Pfanner N (1990) How do polypeptides cross the mitochrondrial membrane? Cell 63: 447-45(1 Simons SM & Blobel G (1991) A protein-conducting channel in the endoplasmic reticulum. Cell 65: 371-38(I Skowyra D, Georgopoulos C & Zylicz M (19911) The E. coil dnaK gene product, the hsp70 homolog, can reactivate heatinactivated RNA polymerase in an ATP hydrolysis-dependent manner. Cell 62:939-944 Stader J. Benson SA & Silhavy TJ (1987) Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export. J. Biol. Chem. 261:15075-15(/80