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Phage Tailspike Protein: A fishy tale of protein folding
PHAGE TAILSPIKE PROTEIN PHAGE TAIISPIKE PROTEIN
D.P. GOLDENBERG AND T.E. CREIGHTON D.R GOLDENBERG AND T.E. CREIGHTON
A fishy tale of protein folding The crystal structure of bacteriophage P22 tailspike protein reveals a striking fold with a distinctive, fish-like appearance, and helps explain many of the properties of this unusual molecule and its folding pathway. The biological and physical properties of proteins are determined by their three-dimensional structures, which, in turn, are specified by their amino-acid sequences. Unfortunately, however, we still understand relatively little about the relationships between protein structure and function; even after a protein structure has been determined, it is often not obvious why that particular structure has been selected by evolution to carry out a particular function. A notable exception, where a protein structure explains many of its biological properties, is the tailspike protein of bacteriophage P22, the structure of which was recently solved by Steinbacher et al. [1]. The folding of this protein and its assembly into the mature phage have been studied extensively, and many biochemical and genetic results are now explained by the structure. The structures of bacteriophages are remarkably intricate (Fig. 1), and their assembly is no mean feat. How can such a rigid and stable structure self-assemble? How can a protein molecule be made suitably rigid to serve as the various architectural elements in a phage? Genetic approaches were used to great effect in the 1960s to map out several phage assembly pathways. Not surprisingly, in retrospect, accessory scaffold proteins and chaperones were found to be involved, and the currently popular GroE chaperonin system of the host bacterium Escherichia coli was first identified because of its role in phage assembly.
the strands and connecting turns vary along the length of the helix, leading to a tapering of the overall structure towards the carboxy-terminal end. Only a cursory examination of the lattice of hydrogen bonds of the 3 helix is necessary to see that it is ideally suited to forming a rigid, elongated structure. Packing of the three 3 helices along the length of the trimer (Fig. 3a) presumably further enhances the rigidity of the structure. The carboxy-terminal 'caudal fin' domain has a topology that is even more remarkable than that of the 13 helix. Like the main body, the caudal fin is composed primarily of 3 strands, but here the polypeptide chains are extensively twisted around one another to create a single structural unit (Fig. 3b). Reflecting the overall three-fold symmetry of the molecule, this unit contains three copies of two 3 sheets, one composed of seven strands, the other three. In the large 3 sheet, five of the strands
The tailspike protein of phage P22 is a trimer of identical 666-residue polypeptide chains, and is about 200A long and 60A wide; six spikes are attached to each phage capsid (Fig. 1). The crystal structure was determined [1] of a form of the tailspike protein lacking 108 residues from the amino terminus, which appear to be involved primarily in attachment to the capsid [2,3]. The overall shape of each subunit bears an uncanny resemblance to a fish, with domains corresponding to a main body, dorsal fin and caudal fin (Fig. 2). The overall path of each of the chains is parallel to the long three-fold symmetry axis of the trimer. The main body of each subunit is made up of a remarkable large '3 helix', a recently discovered structural motif in which parallel 13strands are coiled into a right-handed helix. The 13helix of the tailspike is composed of 13 complete helical turns, with an axis parallel to that of the trimer. All the turns of the helix have a similar triangular conformations with three 3 strands, but the lengths of 1026
Fig. 1. The bacteriophage P22 virion. The tailspike proteins are shown as elongated protrusions at the base of the icosahedral capsid, and are distinct from the longer fiber emerging from the base. (Based on a drawing kindly provided by Sherwood Casjens.)
© Current Biology 1994, Vol 4 No 11
DISPATCH
Fig. 2. Ribbon drawing of the tailspike monomer (a) and trimer (b) [1]. Drawn using the program MOLSCRIPT by Per Kraulis from coordinates kindly provided by Stefan Steinbacher.
come from one of the polypeptides, but the other two come from each of the other two symmetry-related chains. Over the length of the caudal fin domain, each polypeptide chain makes an almost complete turn around the three-fold axis and portions of the other two chains. This interdigitation of the caudal fin domains undoubtedly contributes to the extreme stability of the tailspike trimer, which is resistant to proteolytic digestion and to denaturation by the detergent sodium dodecyl sulphate (SDS) at room temperature; thermal denaturation requires temperatures in excess of 80 C. We have suggested [4] that a cardboard box with four interleaved flaps is a good model to describe the energetic cooperativity of folded proteins, but Nature appears to have used a physical, three-flap form first! This architecture helps account for the folding and assembly pathway of the tailspike trimer, which has been studied both in vivo [5] and in vitro [6] and does not appear to require chaperones. Intermediates in the process include partly folded monomers and an unstable 'protrimer', in which the chains have associated but are not completely folded. Because folding was found to be completed only after the subunits associate, it was proposed some time ago [7] that portions of the polypeptide chains might wrap around one another in the native
structure, exactly as now found in the caudal fin domain. In contrast, the -helix domain of each subunit is relatively independent of the other chains (Fig. 3a), and here the subunits interact in a more typical way, by complementarity of side chains on the surface of each monomer. Consequently, the helices could be imagined to fold prior to subunit association, followed by trimerization of the partly folded monomers and, finally, interdigitation of the carboxy-terminal sequences. The tailspike protein was chosen by Jonathan King and co-workers for a genetic analysis of protein folding because. of the ability to isolate mutants that were temperature-sensitive for folding - designated tsf mutants - rather than temperature-sensitive for stability of the native structure [7,8]. The tsf mutations prevent formation of the native trimer at the restrictive temperature, but once assembled at a lower permissive temperature, the mutant tailspike trimers have stabilities similar to that of the native wild-type protein. In contrast to the high stability of the mature trimer, the process of forming this structure is very sensitive to perturbation. Only about 80% of the wild-type polypeptide chains synthesized at 30°C reach the native conformation; at 390 C, this fraction is reduced to 20% [5]. The
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Current Biology 1994, Vol 4 No 1 1
Fig. 3. Ribbon drawings of the three-fold symmetry axis.
-helix domain (a) and the caudal fin domain (b) of the tailspike trimer viewed along the molecular
inactive wild-type or mutant molecules are sensitive to proteolytic digestion and to SDS, and form large aggregates in vivo [7]. It thus appears that there is a kinetic partitioning between the productive folding and subunit assembly pathway and a competing pathway leading to aggregates of inactive polypeptides. Elevated temperature and the tsf amino-acid replacements act synergistically to favor the non-productive pathway, most likely by altering the stability of an intermediate preceding the protrimer [6,7].
destabilized by the tsf mutations [6]. Whereas the tsf mutations and their suppressors are restricted to the main body and dorsal fin domains, several substitutions that prevent formation of native trimers at both low and high temperature have been identified in the carboxy-terminal caudal fin domain, as well as in the 13 helix [10]. This pattern may indicate that the folding of the carboxyterminal domain, which is presumably completed after subunit association, is even more sensitive to amino-acid replacements than the other domains.
All of the tsf mutations are now found to be located in the dorsal fin and 3-helix domains. It seems likely that the mutations act by destabilizing one or more monomeric folding intermediates in which the 13helix and dorsal fin have formed. As assembly requires association of three subunits, any decrease in the yield of assembly-competent monomers is expected to have a large effect on the yield of native trimers. Once the subunits have associated and formed their interlocking interactions, however, the tsf mutations have only small effects on the stability of the native trimer [6].
The region of the tailspike that is now least well understood is the amino-terminal 108-residue segment that was deleted from the protein used in the crystal structure determination. There are, however, tantalizing suggestions that the structure of this region will be as interesting as that of the other.domains. The amino-terminal domain is involved in attachment of the tailspike trimer to the phage capsid, and its removal destabilizes the native trimer [2,3,10]. The amino-terminal seven residues from the three chains of the crystallized fragment form a three-helix bundle that has the appearance of a neck at the top of the 13 helix. We anxiously look forward to seeing the head that it supports!
The effects of the tsf mutations can be suppressed by amino-acid replacements at other sites in the polypeptide [9]. These suppressor mutations, which are able to suppress the effects of many different tsf mutations, are also found to alter residues located in the 3-helix and dorsal fin domains, consistent with evidence that they act by stabilizing the same intermediate structures that are
References 1. Steinbacher S, Seckler R, Miller S, Steipe B, Huber R, Reinemer P: Crystal structure of P22 tailspike protein: interdigitated subunits in a thermostable trimer. Science 1994, 265:383-386. 2. Chen BL, King J: Thermal unfolding pathway for the thermostable P22 tailspike endorhamnosidase. Biochemistry 1991, 30:6260-6269.
DISPATCH 3. Danner M, Fuchs A, Miller S, Seckler R: Folding and assembly of phage P22 tailspike endorhamnosidase lacking the N-terminal, head-binding domain. EurJ Biochem 1993, 215:653-661. 4. Goldenberg DP, Creighton TE: The energetics of protein structure and folding. Biopolymers 1985, 24:167-182. 5. Goldenberg DP, King J: Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase
folding pathway of the phage P22 tail spike endorhamnosidase. Proc Natl Acad Sci USA 1984, 81:6584-6588.
9. Mitraki A, Fane B, Haase-Pettingell C, Sturtevant J, King J: Global suppression of protein folding defects and inclusion body formation. Science 1991, 253:54-58.
of bacteriophage P22. Proc Natl Acad Sci USA 1982,
10. Schwarz JJ, Berget PB: Characterization of bacteriophage P22 tailspike mutant proteins with altered endorhamnosidase and capsid assembly activities. J Biol Chem 1989, 264:20112-20119.
79:3403-3407. 6. Danner M, Seckler R: Mechanism of phage P22 tailspike protein folding mutations. Protein Sci 1993, 2:1869-1881. 7. Goldenberg DP, Smith DH, King J: Genetic analysis of the folding pathway for the tail spike protein of phage P22. Proc Natl Acad Sci USA 1983, 80:7060-7064. 8. Yu M-H, King J: Single amino acid substitutions influencing the
David P. Goldenberg, Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA. Thomas E. Creighton, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany.
THE JANUARY 1995 ISSUE (VOL. 5, NO. 1) OF CURRENT OPINION IN STRUCTURAL BIOLOGY will include the following reviews, edited by Chris Dobson, on Folding and Binding: Theories of protein folding by M. Karplus and A. Sali Structures of folding intermediates by 0. Ptitsyn Intermediates and transition states of folding by A. Fersht Protein folds and folding by J. Thornton Disulphide bonds in vitro and in vivo by R. Freedman Chaperones and folding pathways by E Hartl Protein-protein interactions by I. Campbell Protein-peptide interactions by I. Wilson
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D.P. GOLDENBERG AND T.E. CREIGHTON D.R GOLDENBERG AND T.E. CREIGHTON
A fishy tale of protein folding The crystal structure of bacteriophage P22 tailspike protein reveals a striking fold with a distinctive, fish-like appearance, and helps explain many of the properties of this unusual molecule and its folding pathway. The biological and physical properties of proteins are determined by their three-dimensional structures, which, in turn, are specified by their amino-acid sequences. Unfortunately, however, we still understand relatively little about the relationships between protein structure and function; even after a protein structure has been determined, it is often not obvious why that particular structure has been selected by evolution to carry out a particular function. A notable exception, where a protein structure explains many of its biological properties, is the tailspike protein of bacteriophage P22, the structure of which was recently solved by Steinbacher et al. [1]. The folding of this protein and its assembly into the mature phage have been studied extensively, and many biochemical and genetic results are now explained by the structure. The structures of bacteriophages are remarkably intricate (Fig. 1), and their assembly is no mean feat. How can such a rigid and stable structure self-assemble? How can a protein molecule be made suitably rigid to serve as the various architectural elements in a phage? Genetic approaches were used to great effect in the 1960s to map out several phage assembly pathways. Not surprisingly, in retrospect, accessory scaffold proteins and chaperones were found to be involved, and the currently popular GroE chaperonin system of the host bacterium Escherichia coli was first identified because of its role in phage assembly.
the strands and connecting turns vary along the length of the helix, leading to a tapering of the overall structure towards the carboxy-terminal end. Only a cursory examination of the lattice of hydrogen bonds of the 3 helix is necessary to see that it is ideally suited to forming a rigid, elongated structure. Packing of the three 3 helices along the length of the trimer (Fig. 3a) presumably further enhances the rigidity of the structure. The carboxy-terminal 'caudal fin' domain has a topology that is even more remarkable than that of the 13 helix. Like the main body, the caudal fin is composed primarily of 3 strands, but here the polypeptide chains are extensively twisted around one another to create a single structural unit (Fig. 3b). Reflecting the overall three-fold symmetry of the molecule, this unit contains three copies of two 3 sheets, one composed of seven strands, the other three. In the large 3 sheet, five of the strands
The tailspike protein of phage P22 is a trimer of identical 666-residue polypeptide chains, and is about 200A long and 60A wide; six spikes are attached to each phage capsid (Fig. 1). The crystal structure was determined [1] of a form of the tailspike protein lacking 108 residues from the amino terminus, which appear to be involved primarily in attachment to the capsid [2,3]. The overall shape of each subunit bears an uncanny resemblance to a fish, with domains corresponding to a main body, dorsal fin and caudal fin (Fig. 2). The overall path of each of the chains is parallel to the long three-fold symmetry axis of the trimer. The main body of each subunit is made up of a remarkable large '3 helix', a recently discovered structural motif in which parallel 13strands are coiled into a right-handed helix. The 13helix of the tailspike is composed of 13 complete helical turns, with an axis parallel to that of the trimer. All the turns of the helix have a similar triangular conformations with three 3 strands, but the lengths of 1026
Fig. 1. The bacteriophage P22 virion. The tailspike proteins are shown as elongated protrusions at the base of the icosahedral capsid, and are distinct from the longer fiber emerging from the base. (Based on a drawing kindly provided by Sherwood Casjens.)
© Current Biology 1994, Vol 4 No 11
DISPATCH
Fig. 2. Ribbon drawing of the tailspike monomer (a) and trimer (b) [1]. Drawn using the program MOLSCRIPT by Per Kraulis from coordinates kindly provided by Stefan Steinbacher.
come from one of the polypeptides, but the other two come from each of the other two symmetry-related chains. Over the length of the caudal fin domain, each polypeptide chain makes an almost complete turn around the three-fold axis and portions of the other two chains. This interdigitation of the caudal fin domains undoubtedly contributes to the extreme stability of the tailspike trimer, which is resistant to proteolytic digestion and to denaturation by the detergent sodium dodecyl sulphate (SDS) at room temperature; thermal denaturation requires temperatures in excess of 80 C. We have suggested [4] that a cardboard box with four interleaved flaps is a good model to describe the energetic cooperativity of folded proteins, but Nature appears to have used a physical, three-flap form first! This architecture helps account for the folding and assembly pathway of the tailspike trimer, which has been studied both in vivo [5] and in vitro [6] and does not appear to require chaperones. Intermediates in the process include partly folded monomers and an unstable 'protrimer', in which the chains have associated but are not completely folded. Because folding was found to be completed only after the subunits associate, it was proposed some time ago [7] that portions of the polypeptide chains might wrap around one another in the native
structure, exactly as now found in the caudal fin domain. In contrast, the -helix domain of each subunit is relatively independent of the other chains (Fig. 3a), and here the subunits interact in a more typical way, by complementarity of side chains on the surface of each monomer. Consequently, the helices could be imagined to fold prior to subunit association, followed by trimerization of the partly folded monomers and, finally, interdigitation of the carboxy-terminal sequences. The tailspike protein was chosen by Jonathan King and co-workers for a genetic analysis of protein folding because. of the ability to isolate mutants that were temperature-sensitive for folding - designated tsf mutants - rather than temperature-sensitive for stability of the native structure [7,8]. The tsf mutations prevent formation of the native trimer at the restrictive temperature, but once assembled at a lower permissive temperature, the mutant tailspike trimers have stabilities similar to that of the native wild-type protein. In contrast to the high stability of the mature trimer, the process of forming this structure is very sensitive to perturbation. Only about 80% of the wild-type polypeptide chains synthesized at 30°C reach the native conformation; at 390 C, this fraction is reduced to 20% [5]. The
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Current Biology 1994, Vol 4 No 1 1
Fig. 3. Ribbon drawings of the three-fold symmetry axis.
-helix domain (a) and the caudal fin domain (b) of the tailspike trimer viewed along the molecular
inactive wild-type or mutant molecules are sensitive to proteolytic digestion and to SDS, and form large aggregates in vivo [7]. It thus appears that there is a kinetic partitioning between the productive folding and subunit assembly pathway and a competing pathway leading to aggregates of inactive polypeptides. Elevated temperature and the tsf amino-acid replacements act synergistically to favor the non-productive pathway, most likely by altering the stability of an intermediate preceding the protrimer [6,7].
destabilized by the tsf mutations [6]. Whereas the tsf mutations and their suppressors are restricted to the main body and dorsal fin domains, several substitutions that prevent formation of native trimers at both low and high temperature have been identified in the carboxy-terminal caudal fin domain, as well as in the 13 helix [10]. This pattern may indicate that the folding of the carboxyterminal domain, which is presumably completed after subunit association, is even more sensitive to amino-acid replacements than the other domains.
All of the tsf mutations are now found to be located in the dorsal fin and 3-helix domains. It seems likely that the mutations act by destabilizing one or more monomeric folding intermediates in which the 13helix and dorsal fin have formed. As assembly requires association of three subunits, any decrease in the yield of assembly-competent monomers is expected to have a large effect on the yield of native trimers. Once the subunits have associated and formed their interlocking interactions, however, the tsf mutations have only small effects on the stability of the native trimer [6].
The region of the tailspike that is now least well understood is the amino-terminal 108-residue segment that was deleted from the protein used in the crystal structure determination. There are, however, tantalizing suggestions that the structure of this region will be as interesting as that of the other.domains. The amino-terminal domain is involved in attachment of the tailspike trimer to the phage capsid, and its removal destabilizes the native trimer [2,3,10]. The amino-terminal seven residues from the three chains of the crystallized fragment form a three-helix bundle that has the appearance of a neck at the top of the 13 helix. We anxiously look forward to seeing the head that it supports!
The effects of the tsf mutations can be suppressed by amino-acid replacements at other sites in the polypeptide [9]. These suppressor mutations, which are able to suppress the effects of many different tsf mutations, are also found to alter residues located in the 3-helix and dorsal fin domains, consistent with evidence that they act by stabilizing the same intermediate structures that are
References 1. Steinbacher S, Seckler R, Miller S, Steipe B, Huber R, Reinemer P: Crystal structure of P22 tailspike protein: interdigitated subunits in a thermostable trimer. Science 1994, 265:383-386. 2. Chen BL, King J: Thermal unfolding pathway for the thermostable P22 tailspike endorhamnosidase. Biochemistry 1991, 30:6260-6269.
DISPATCH 3. Danner M, Fuchs A, Miller S, Seckler R: Folding and assembly of phage P22 tailspike endorhamnosidase lacking the N-terminal, head-binding domain. EurJ Biochem 1993, 215:653-661. 4. Goldenberg DP, Creighton TE: The energetics of protein structure and folding. Biopolymers 1985, 24:167-182. 5. Goldenberg DP, King J: Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase
folding pathway of the phage P22 tail spike endorhamnosidase. Proc Natl Acad Sci USA 1984, 81:6584-6588.
9. Mitraki A, Fane B, Haase-Pettingell C, Sturtevant J, King J: Global suppression of protein folding defects and inclusion body formation. Science 1991, 253:54-58.
of bacteriophage P22. Proc Natl Acad Sci USA 1982,
10. Schwarz JJ, Berget PB: Characterization of bacteriophage P22 tailspike mutant proteins with altered endorhamnosidase and capsid assembly activities. J Biol Chem 1989, 264:20112-20119.
79:3403-3407. 6. Danner M, Seckler R: Mechanism of phage P22 tailspike protein folding mutations. Protein Sci 1993, 2:1869-1881. 7. Goldenberg DP, Smith DH, King J: Genetic analysis of the folding pathway for the tail spike protein of phage P22. Proc Natl Acad Sci USA 1983, 80:7060-7064. 8. Yu M-H, King J: Single amino acid substitutions influencing the
David P. Goldenberg, Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA. Thomas E. Creighton, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany.
THE JANUARY 1995 ISSUE (VOL. 5, NO. 1) OF CURRENT OPINION IN STRUCTURAL BIOLOGY will include the following reviews, edited by Chris Dobson, on Folding and Binding: Theories of protein folding by M. Karplus and A. Sali Structures of folding intermediates by 0. Ptitsyn Intermediates and transition states of folding by A. Fersht Protein folds and folding by J. Thornton Disulphide bonds in vitro and in vivo by R. Freedman Chaperones and folding pathways by E Hartl Protein-protein interactions by I. Campbell Protein-peptide interactions by I. Wilson
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