Rendering a membrane protein soluble in water: a common packing motif in bacterial protein toxins

TIBS 18 - OCTOBER 1993

PROTEIN TOXINS, at first sight, appear to have little in common. They are produced by a variety of living organisms and are directed towards an equally diverse range of targets. Their toxic activities are expressed in different ways, from simple disruption of cell membranes through to inhibition of the cell's protein synthesis apparatus. Nevertheless, protein toxins share a common requirement - the need to insert into, and in many cases, translocate across, the cell membranes of the host. This raises the fundamental question of how water-soluble proteins insert into biological membranes. The three-dimensional structure of the pore-forming domain of the bacterial toxin colicin was solved by X-ray crystallography in 1989 (Ref. 1) revealing for the first time the structure of an 'inside-out' protein. Based on this structure and a variety of other experimental data, a mechanism of membrane insertion was proposed t-s. It was speculated that some of the basic ideas used to develop the model might be relevant to other toxins z. Surprisingly, the similarity has turned out to be much closer than was first thought, with the finding that insecticidal ~endotoxin 6 and diphtheria toxin 7 have a colicin-like domain.

Rendering a membrane protein solub!e in water: a common packing moti+fin bacter ial protein toxins

The recently determined structures of three different protein toxins by X-ray crystallography has unexpectedly revealed a common membraneinsertion domain. This domain consists of an (x-helical bundle of between seven and ten helices, some of which are hydrophobic. The three toxins, colicin, insecticidal &endotoxin and diphtheria toxin are directed towards different hosts, have different killing mechanisms and bear no sequence homology. The observation of a common membrane-insertion domain has implications for the design of therapeutic agents in combating disease.

swelling and lysis of the gut epithelium leading to death of the insect through starvation and septicaemia9. Diphtheria toxin is secreted from Corynebacterium diphtheriae as a single chain that cleaves before or soon after binding to the target cell to generate two fragments, A (the amino-terminal 21 kDa fragment) and B (the remaining 37 kDa fragment). The B fragment binds to a cell-surface receptor and after internalBackground The three toxins are produced by dif- ization somehow promotes the transfer ferent bacteria and are directed of fragment A to the cytoplasm where it towards different organisms. Colicins catalyses the ADP ribosylation of elonare plasrnid-encoded toxins (molecu- gation factor 2 and hence stops protein lar weight 60kDa), secreted from synthesis I°. A very similar multistep mechanism Otrobacter freundii and Escherichia coil strains, which kill closely related entero- appears to operate for the insertion/ bacteria. Most colicins (A, El, la, Ib, N internalization of each toxin. In the inand B) destroy target cells by forming itial step, the toxin recognizes the target pores in their membranes, and in doing cell's membrane by binding to a recepso destroy the cellular membrane tor. Like bacteriophages, colicins have potentiaP. &Endotoxins (molecular parasitized outer-membrane proteins of weight 70-135 kDa) belong to a family their susceptible bacterial targets, of toxins produced by Bacillus thurin- using them as receptors. &Endotoxins giensis during sporulation. They are bind to specific receptors on brushreleased as microcrystalline protoxins, border membranes9 whilst diphtheria which are solubilized in the midgut of a toxin binds to a heparin-binding epidervariety of feeding insects. Upon acti- mal growth factor-like growth factor vation by gut proteases, the mature precursor n. Nter binding to the receptoxin binds to brush border mem- tor, the toxin is inserted into, or transbranes to create pores, which cause located across, the membrane after which it expresses its toxic activity. In common with many other protein M. W. Parker is at the St. Vincent's Institute toxins, the receptor-binding, membrane of Medical Research,41 Victoria Parade. insertion/translocation and toxic activiFitzroy, Victoria 3065, Australia; and F. ties of all three toxins are associated Pattus is at the EuropeanMolecular BiologY with distinct protein domains as shown Laboratory, Meyerhofstrasse1, D-69012 in Fig. 1. Heidelberg, Germany. © 1993.ElsevierSciencePublishers.LUK)~%8-0004/93/$06.00

Colicins The three-dimensional fold of the pore-forming domain of colicin A (Refs 1,4) is shown diagrammatically in Fig. 2a. It consists of ten c( helices with the two outer layers sandwiching a middle layer of three helices (helices 8, 9 and 10), the first two of which are completely buried and consist of hydrophobic residues only. Three of the helices (1, 8 and 9) are sufficiently long to span a membrane. A mechanism for insertion of colicin into membranes was proposed on the basis of its structure as well as spectroscopic and biochemical data ~-S. The first step is the initial interaction with negatively charged headgroups on the membrane surface via the longrange effects ot an electrostatic field generated by a ring of eight positively charged sidechains that surround the hydrophobic hairpin (helices 8 and 9), which properly orientates the hairpin towards the membrane. Colicins are known to preferentially insert into negatively charged lipid bUayers at low pH. The low pH at the membrane suflace triggers the transistion of colicin A from its native state to a 'molten globule' state ~. The molten globule state is a partially unfolded but compact state characterized by a marked increase in sidechain flexibility whilst retaining a near-native secondary structure t2. The increased flexibility alforded by the molten globule state reduces the energy barrier for unmasking the hydrophobic hairpin.

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340

173

592

colicin A

57

290

500

~5.endotoxin 193

378 -

644 [

535

than 30 ,~ long and thus would be capable of spanning the hydrophobic core of a membrane bilayer. Little is known about the details of &endotoxin activity. The long hydrophobic and amphipathic helices of the pore-forming domain are likely components of a pore based

on current conceptions of membrane channel architecture. It is likely that the central helix plays an 252 364 404 613 important role in insertion exotoxin A as its residues are highly conserved throughout the 5-endotoxin family and mutations of nonpolar residues to polar residues in the helix inhibit pore I insertion/ ~ enzymatic ~ receptor-binding formation 14. Mutagenesis translocation results demonstrate the importance of a positive 1 outer membrane ['--7 unknown prosequence charge near the amino tertranslocation minus of helix 3 for membrane insertion 14. There is some evidence that insect Rgure 1 gut proteases cleave a Functional organization of the polypeptide chains of various toxins. The different stages of toxic acloop between helices 3 tivity are associated with distinct domains as indicated in the key. The disulphide bond in exotoxin A and 4 (Ref. 15). If so, the is indicatedby a black line. cleavage could be the trigger for exposing the The presence of the hairpin led to helical layers into the membrane and hydrophobic core of the domain to the

diphtheriatoxin

our earlier insertion models for colicin A in which interaction of the hydrophobic loop of the helical hairpin with the lipid bilayer was followed by the spontaneous insertion of the entire hairpin. Because the passage ol charged residues through the membrane is unfavourable on energetic grounds ~, the insertion of the hairpin necessitated the opening up of the colicin structure leaving the two outer layers (helices 1-2 and helices 4-7) embedded in the surface of the bilayer, in a process rather like the opening of an umbrella. However, recent fluorescent e~lergy transfer measurements s, partial proteolysis and engineering of disulphide bonds (D. Duch6, pers. commun.) suggest an alternative model in which the hydrophobic hairpin does not insert into the membrane, but separates from the amino-terminal helical layer so that the helices are splayed out on the surface of the membrane. Application of a trans-negative potential (i.e. making the opposite side of the membrane negative relative to the side on which the protein inserts) would cause the concerted movement of

392

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result in the formation of the pore. The low pH environment of the membrane surface would optimize the effect of the trans-negative potential by ensuring the helical layers have a net positive charge. Oligomerization would favour the insertion of charged residues into the membrane by screening the charges from the hydrophobic core of the bilayer. The conductance and permeability of colicin pores imply the need for toxin oligomerization although this is controversial4,8. Perpendicular insertion of the hydrophobic hairpin would occur during channel formation and might be retained after channel closing.

membrane.

DIpMherla toxin The translocation domain of diphtheria toxin consists of nine helices arranged in three layers ~ as shown in Fig. 2c. The middle layer consists of the carboxy-terminal helices, 8 and 9, both of which are hydrophobic and long enough to span a membrane. One of the

outer layers, consisting of helices 5-7, also has a hydrophobic pair of helices (6 and 7). The other flanking layer consists of helices I-4. It is noteworthy that two loops connecting the helices on one face of the helical barrel (loops between helices 5 and 6 and between Insecticidal ~.endotoxins helices 8 and 9) are very acidic, conThe structure of the pore-forming taining a total of six aspartate and glutadomain of insecticidal ~>-endotoxinG is mate residues. Out of 22 positively shown diagrammatically in Fig. 2b. The charged residues in the translocation domain is a seven-helix bundle with a domain, 21 reside on the opposite face central helix (helix 5) completely sur- to the acidic loops, thereby creating a rounded by the six outer helices. The large molecular dipole. central helix is not entirely hydroAfter binding to its receptor, diphphobic as there are a few polar theria toxin enters its target cell via residues, all of which are involved in endocytic vesicles where the low pH hydrogen bonds and salt bridges. Five triggers a conformational change. This is of the helices (helices 3-7). are more characterized by the appearance of pre-

TIBS 1 8 - OCTOBER1993

viously buried proteolysis (a) c sites, exposure of aromatic (b) residues and the ability of 4 _ the B fragment to bind to detergents and spontaneously insert into artificial membranes to form ionselective channels ~°. These characteristics are reminis3 cent of the molten globule state described for colicin. Reduction ~ " h ' , ~ ; , a i . disulphide bridge that becomes exposed in the low pH conformation leads to translocation of the A frag~ N ment into the cytoplasm. How the A fragment translocates is controversial but 5 most models suggest that the B fragment shields hy(e) (d) drophilic surfaces of the A 6 C fragment from the bilayer by forming a membrane pore /~ ~ 4 3 or cleft. Proteolysis experiments suggest the B fragment remains associated with the plasma membrane after translocation of the A 1 fragmentl°. Just as for colicin, negatively charged lipids and membrane potential may play a role in the insertion mechanism ~. Eisenberg and co-workers 7 have proposed a 2 model for membrane insertion based on the structure of diphtheria toxin and bioFlgure 2 chemical results. Helices Ribbon representations of the protein insertion/translocation domains of various toxins as determined 1-4 are very hydrophilic by X-ray cffstallography. (a) The pore-forming domain of colicin A (Refs 1, 4). (b) The pore-forming domain of insecticidal ~3-endotoxin6. (©) The translocation domain of diphtheria toxin ~, (d} The and thus are unlikely to translocation domain of exotoxin A (Ref. 17). The figures were produced by the computer program undergo membrane inserMOLSCRIPT24. Amino and carboxyl termini are marked and ~-helices are numbered. tion. They propose that the hydrophobic helical hairpins composed of helices 5 and 6 and aeruginosa as a single-chain protoxin toxin warrants inclusion in this discushelices 8 and 9 initiate membrane inser- (molecular weight 67 kDa) and kills cells sion because it does share some function. The low pH of the endosome by the same ribosylation activity as tional similarities with the other toxins. would neutralize the acidic loops on diphtheria toxin 16. It is also composed The mechanism by which exotoxin A one face of the helical barrel, rendering of distinct structural domains as- translocates across membranes is them more hydrophobic and lowering sociated with its various stages of known in some detail ~i. Like colicin, the energy costs of inserting the activity (Fig. 1). The translocation do- exotoxin A preferentially binds negahydrophobic helices into the mem- main of exotoxin A is composed of six ct tively charged lipids and forms pores in brane. If the pH on the trans face of the helices; two helices (2 and 5) are approxi- artilical bilayers. There is evidence membrane is neutral, as expected for mately 30 ]~ in length and are there- from mutagenesis studies that implithe cytosol, the acidic sidechains would fore long enough to span a membrane ~7 cates positively charged residues in the be deprotonated and the inserted heli- (Fig. 2d). A disulphide bridge links insertion mechanism TM. After binding, cal hairpins would anchor the B frag- helices 1 and 2. At first sight, the domain the toxin enters the cell by endocytosis ment to the membrane. shows far less resemblance to the and is exposed to low pH in the endocolicin A domain compared with the cytic vesicles. An acid-induced coniorExotoxin A other toxins. There is no three-layer mational state has been stu~lied in ~fitro. A fourth toxin shows some structural structure, there are no buried helices It is characterized by enhanced expoand functional similarity to colicins. and there are only short stretches of sure of aromatic residues, increased Exotoxin A is secreted by Pseudomonas hydrophobic sequence. However, the susceptibilty to proteolysis, strong

393

TIBS 18 - OCTOBER1993

binding to detergent and spontaneous insertion into artificial membranes tg. The transistion has all the hallmarks of the molten globule. Although the transition occurs at about pH 3.7, the inclusion of physiological proportions of acidic iipids in artificial membranes increased the pH threshold to about pH 5.0 (Ref. 19). An acid-activated protease cleaves the toxin in the loop between helices 1 and 2, to generate an aminoterminal 28 kDa fragment and a carboxy-ter.minal 37kDa fragment. The disulphide bond linking the two frag,,~ats is broken by reduction and the carboxy-terminal fragment is translocated into the cytosol to exert its ribosylation activity. The mechanism of translocation is complicated by the observation that an intact carboxyl terminus appears essential. The carboxy-terminal sequence shows strong homology to a retention signal found in proteins within the endoplasmic reticulure, suggesting the involvement of other proteins in translocation2°.

A commonmechanismof membrane Insertion The insertion/translocation folds of colicin, &endotoxin and diphtheria toxin bear a striking resemblance. The basic fold consists of a bundle of between seven and ten (x helices organized in a three-layer structure. Each layer is formed by two or more antiparallel helices, some of which are completely buried. In ;dl cases, there are at least two helices that are sufficiently long to span a membrane, It appears that exotoxin A can also be included in the family on the basis that its translocation domain consists of a helical bundle that .(.unctions in many respects like tb.?2~of the other toxins. The common fold represents a soluble form of packaging for the hydrophobic and amphipathic helices that are used to insert the toxin or translocate it across the membrane. The resemblance of the fold is not maintained at the primary sequence level suggesting it is a product of convergent evolution, The conversion from a water-soluble to a membrane form necessitates a large change in confo='mation. It s e e m s likely on the available evidence that colicin, diphtheria toxin and exotoxin convert into the membrane-bound conformation via a molten globule intermediate which would lower the energy barrier of the conversion3. The transistion to the molten globule state can be induced by means other than

394

low pH, such as high temperature, high pH and other destabilizing agents. Thus this transition may occur in vivo under less drastic conditions than those needed in vitro, so the insect toxin may also pass through a molten globule state in the high pH environment of the insect gut. Charged residues have been implicated in the insertion mechanism of each toxin, suggesting that electrostatics may play an important role. The flexibility of the molten globule state would increase the accessibility of the hydrophobic core of the insertion domain leading to its exposure within the membrane. The layered structure of the insertion domain would permit the spreading of helical layers onto the membrane surface. The insertion would require only local conformational changes in loop regions without disturbing the helices themselves. The fact that this occurs for colicin is well supported by experimenP-5. A striking feature exhibited by all four toxins is their ability to form pores in artificial membranes although its relevance in vivo is not yet firmly established for either diphtheria toxin or exotoxin2. The pore formation could be incidental to toxic activity and could simply be a consequence of their turning 'inside-out' on insertion into membranes. Many of the concepts and ideas developed by studies on toxins may have more general applicability. The large-scale conformational changes, involving rearrangement of helical segments whilst preserving the secondary structural elements, have also been proposed for the binding of apolipoproteins to membranes 2~ and the membrane-mediated viral assembly of the filamentous bacteriophage Pfl (Re[. 22).

chimeric toxins have been designed for use in the treatment of cancer, chronic infectious diseases, and autoimmune diseases such as AIDS. A good example is DAB3sg-IL-2, a diphtheria-toxin-related fusion protein in which the native receptor-binding domain of the t6xin was replaced with the cytokine interleukin-2 (Ref. 23). Although this work started before the crystal structure of the toxin was known, it has revealed the fusion junction that results in a chimera with complete receptor-binding activity. The crystal structure has since provided a framework on which to initiate structure/function studies of various chimeric constructs. The interested reader is directed to a recent review by Pastan and co-workers16. The discovery that some toxins use a common domain for entry into cells has further implications. The work on chimeric toxins could be extended by utilizing the colicin-like domain for transporting all types of foreign antigens into cells. Examples include drugs and cell markers. A deeper understanding of the functional differences between colicin-like domains could lead to the judicious selection of particular domain types for different applications. For example, an agent could be designed to form pores that open in r e s p o n s e to certain chemicals. The agent could be directed to selected target cells by linking it to an appropriate receptor-binding domain.

Acknowledgements We thank the following people for providing us with atomic coordinates:

D. Eisenberg for the diphtheria toxin model, J. Li for the insecticidal &endotoxin model and D. McKay for the exotoxin A i~o~d~;'We would particularly like to thank Demetrius Tsernoglou, in Implications for the design of chimeric whose lab the crystallographic work on toxins a5 therapeutic agents colicin h was carried out, for his advice The crystal structures of the four tox- and encouragement. M. W. P. is a ins demonstrate that the receptor-bind- Wellcome Australian Senior Research ing, insertion/translocation and toxic Fellow and acknowledges support from activities are associated with discrete, the Australian Research Council and compact domains. This observation has the National Health and Medical had important implications for the Research Council. rational design of novel therapeutic agents, Identification of the function References and boundaries of toxin domains has I Parker, M. W., Pattus, F., Tucker, A. D. and Tsemoglou, D. (1989) Nature 337, 93-96 led to the construction of chimeric toxins by recombinant DNA technology. In 2 Parker, M. W., Tucker, A. D., Tsernoglou, D. and Pattus, F. (1990) Trends Biochem. Sei. 15, these toxins, the receptor-binding 126-129 domain has been replaced by targetting 3 Van der Goot, F. G., Gonz~lez-Mafias, J. M., Lakey, J. H. and Pattus, F. (1991) Nature 354, molecules such as antibodies or growth 408-410 factors that preferentially bind to the 4 Parker, M. W. eta/. (1992) J. Mot Biol. 224, surface of chosen target cells. Already, 639-657

TIBS 1 8 - OCTOBER1993 5 Lakey, J. H. et al. (1993) J. Mol. Biol. 230, 1055-1067 6 Li, J., Carrol, J. and Ellar, D. J. (1991) Nature 353, 815-821 7 Choe, S. et aL (1992) Nature 357, 216-222 8 Lazdunski, C. et al. (1988) Biochire. Biaphys. Acta 947, 445-464 9 H6fte, H. and Whiteley, H. R. (1989) Micrebiol. Rev. 53, 242-255 10 London, E. (1992) Biochire. Biophys. Acta 1113, 25-51 11 Naglich, J. G., Metherall, J, E., Russell, D. W. and Eidels, L. (1992) Cell 69, 1051-1061 12 Ptitsyn, O. B, (1992) in Protein Folding

(Creighton, T. E., ed.), pp. 243-300, W. H. Freeman 13 Engelman, D. and Steitz, T. A. (1981) Cell 23, 411-422 14 Wu, D. and Aaronson, A. I. (1992) J. Biol. Chem. 267, 2311-2317 15 Carroll, J., Li, J. and Ellar, D. J. (1989) Biochem. J. 261, 99-105 16 Pastan, I., Chaudhary, V. and FitzGerald, D. J. (1992) Annu, Rev. Biochem. 61, 331-354 17 Allured, V. S., Collier, R. J., Carroll, S. F. and McKay, D. B. (1986) Proc, Natl Pced. Sci. USA 83, 1320-1324 18 Jinno, Y. et aL (1989) J, Biol, Chem. 264, 15953-15959

INTRACELLUIAR ORGANELLES of higher eukaryotic cells are defined by their characteristic morphology and the presence of resident membrane and lumenal marker proteins. Using these criteria, several organelles exist along the secretory and endocytic pathways (Fig. I). The secretory pathway can be considered to start at the endoplasmic reticulum (ER) into whose membrane or lumen nascent proteins are co-translationally translocated. Proteins then pass sequentially through an ordered series of membrane.bound intracellular compartments the ER to Golgi inter-

19 Jiang, J. X. and London, E. (1990) J. BioL Chem. 265, 8636-8641 20 Chaudhary, V. K., Jinno, Y., FitzGerald, D. and Pastan, I. (1990) Proc. Nat/Acad. Sci. USA 87, 308-313 21 Breiter, D. R, et al. (1991) Biochemistry 30, 603-608 22 Namudripad, R., Stark, W., Opella, S. J. and Makowski, L. (1991) Science 252, 1305-1308 23 van der Speck, J. C., Mindell, J. A., Finkelstein, A. and Murphy, J. R. (1993) J. Biol. Chem. 268, 12077-12082 24 Kraulis, P. J. (1991) J. Appl. Crystallngr. 24, 946-950

Eukaryotic membrane traffic: retrieval and retention mechanisms to achieve organelle residence .

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Th9 localization of integral membrane proteins to specific organelles is mediate compartment (ERG1C)t, the cis necessary to maintain the functional integrity of eukaryotic cells. Recent Golgi network (CGN), the cisternae studies indicate firstly, that retrieval and retention mechanisms, requiring of the Golgi stack and the trans Golgi specific primary sequence motifs, are used to ensure that proteins reside network (TGN)2 - before arrival at the in specific membranes of the secretory and endocytic pathways and plasma membrane. secondly, that these membranes exist as comloonents of two juxtaposed Endocytosis, the process by which systems separated by the cisternae of the Golg[ stack. macromolecules, including membrane proteins, are internalized and delivered to intracellular compartments, starts at the plasma membrane. Again there is that proteins never move elsewhere, as membrane protein of the TGN de. transfer of proteins between successive those of us who leave our residences to scribed, cloned and sequenced so far Lu N ~ ~,,,~,. 3). At . . . . . state, gu out to work and piay will know. of this protein is in the TGN, but it re. membrane to early endosomes to late cycles between this organelle and the endosomes to lysosomes, but other Juxtaposedmembranesystems Recent experiments have provided plasma membrane~ . When ceils are pathways also exist). All transfer between organe]les on both the secretory new insights into the relationship incubated with the fungal metabolite and endocytic pathways is mediated by between the Golgi stack, the TGN and brefeldin A (BFA), the contents of the membran¢~bound vesicles. Given the the plasma membrane, and have Golgi stack redistribute into the ER"q, large amount of soluble and membrane prompted a series of questions regard- whereas the TGN remains intact protein traffic through these pathways, ing the mechanisms by which organelle and functional, but collapses upon it is evident that proteins residing with- residence is achieved. The Golgi stack the microtubule organizing centre in the organelles of these pathways and the plasma membrane are morpho- (MTOC)l°.u. If the TGN were part of the require mechanisms to achieve resi- logically well defined in mammalian Golgi stack it would redistribute into dence. However, it is important to note cells, and the TGN is discernible as a the ER in response to BFA; it does not. that organelle residence does not imply tubuloreticular membrane network on The recycling of TGN38 between the the trans side of the Golgi stack (i.e. the TGN and the plasma membrane is exit side of the Golgi stack)2. It is a key essentially unaffected by treatment of J. P. Luzio is at the Department of Clinical sorting station, where proteins destined cells with BFA45. Biochemistry, University of Cambridge, The current view of the mechanism for regulated secretion and for lysoAddenbrookes Hospital, Cambridge, UK of action of BFA is welt described elsesomal delivery are separated from those CB2 2QR; and G. Banting is at the where°.12; its effects on membrane tralon the constitutive secretory pathway. Department of Biochemistry, University The only endogenous type I integral ficking pathways may be interpreted by of Bristol, Bristol, UK BS8 lTD. -

© 1993, Elsevier Science Publishers, (ILK) 0968--0004/93/$06.00

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