chemistry to install topological links in small molecules. By following these ...... mechanism as unifying theme in the folding of biomolecules. Theor. Chem.
Threading a peptide through a peptide: Protein loops, rotaxanes, and knots JOHN W. BLANKENSHIP1
AND
PHILIP E. DAWSON
Departments of Chemistry and Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA (R ECEIVED November 20, 2006; F INAL R EVISION February 27, 2007; ACCEPTED April 3, 2007)
Abstract Proteins adopt complex folds in nature that typically avoid conformations that are knotted or ‘‘threaded’’ through closed loops. Is this the result of fundamental barriers to folding, or have proteins simply evolved to avoid threaded conformations? Organic synthesis has been used in supramolecular chemistry to install topological links in small molecules. By following these principles, we now show that it is possible to assemble a topologically linked protein complex by threading a linear protein through a cyclic protein to form a [2]pseudo-rotaxane. Subsequent ring closure using native chemical ligation cyclizes the linear protein, forming a [2]heterocatenane. Although the kinetics of protein threading are slower than the folding kinetics of the native protein, threading appears to be a highly efficient process. Keywords: protein structure/folding; conformational changes; circular dichroism; fluorescence; forces and stability; thermodynamics, hydrodynamics; kinetics; synthesis of peptides and proteins Supplemental material: see www.proteinscience.org Proteins are typically composed of linear polypeptides that fold to a defined tertiary structure. These structures can be stabilized with cross-links such as disulfide bonds (Matsumura et al. 1989) or metal cofactors, and natural and engineered proteins with a circularized backbone have been investigated (Goldenberg and Creighton 1983; 1 Present address: Department of Protein Engineering, Genentech, South San Francisco, CA 94080, USA. Reprint requests to: Philip E. Dawson, Departments of Chemistry and Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., CB256-D, La Jolla, CA 92037, USA; e-mail: dawson@ scripps.edu; fax: (858) 784-7319. Abbreviations: p53tet, tetramerization domain of the p53 tumor suppressor; p53catdim, monomeric catenane designed from the p53tet M340E/L344K sequence; p53dimlin/ext, linear peptide incorporating the p53tet M340E/L344K sequence with extended termini; p53hetA, p53catdim thioester peptide with Tyr ! Nph 327 mutation; p53hetB, p53catdim thioester peptide with Asn ! Dab-Abz 331 mutation; p53hetC, p53catdim peptide with Asn ! Dab-Abz 331 mutation; m, the slope of the unfolding transition in relation to denaturant; DGuH2O, the free energy of unfolding extrapolated linearly to zero denaturant; D50%, the midpoint of denaturation; Nph, p-nitrophenylalanine; Dab, diaminoisobutyric acid; Abz, 2-aminobenzoic acid; GuHCl, guanidine hydrochloride. Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062673207.
Camarero and Muir 1999; Camarero et al. 2001). We have explored a stabilization strategy that is rarely utilized by natural proteins, topological linking or mechanical interlocking (Yan and Dawson 2001; Blankenship and Dawson 2003). These interlocking proteins, or catenanes, are based on an oligomerization motif from the transcription factor p53 that adopts a ‘‘bisecting U’’ folded conformation. Introduction of the topological link into these proteins rendered them highly stable to thermal and chemical denaturation as well as resistant to proteolysis (Blankenship and Dawson 2003). It remains unclear, however, why this strategy for stabilizing proteins is rarely exploited in the natural world (one notable exception is the bacteriophage HK97 capsid) (Wikoff et al. 2000) and whether or not proteins can easily ‘‘thread’’ through closed loops in other proteins, as a first step toward establishing a topological linkage. Although non-covalently ‘‘closed’’ loops between 20 and 50 residues in size are a common element in protein structure (Connolly et al. 1980; Berezovsky et al. 2000), and formation of longrange, stable tertiary contacts is an important step in the folding of many proteins, most do not have their main chain ‘‘threaded’’ through these closed loops. Since it is
Protein Science (2007), 16:1249–1256. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2007 The Protein Society
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known that ‘‘closed’’ loops are present in the folding process, it is reasonable to ask why more protein structures are not threaded or have significant populations of threaded intermediates in the folding process. One approach to resolving this question has been to assume that threaded intermediates are not sampled during the folding process (Connolly et al. 1980; Mansfield 1994). In this model, any threaded structures in proteins can arise from the loop folding ‘‘around’’ the thread, rather than the thread going through the loop. However, experimental studies on several proteins such as BPTI (Creighton 1978), human NGF (De Young et al. 1996), and the methyltransferase YibK (Mallam and Jackson 2005, 2006) suggest that loop threading (or unthreading) is a necessary part of the folding/unfolding process for some proteins. In addition, the structures of a growing number of additional proteins such as S-adenosylmethionine synthetase (Mansfield 1997), RNA 29-O-ribose methyltransferase (Nureki et al. 2002), plant acetohydroxy acid isomeroreductase (Taylor 2000), and the human ubiquitin hydrolases UCH-L1 (Das et al. 2006) and UCH-L3 (Misaghi et al. 2005) have all been shown to be linearly ‘‘knotted,’’ or are threaded through a ‘‘closed’’ loop in the protein structure (Lua and Grosberg 2006; Virnau et al. 2006). A knotted conformation can introduce an unusual degree of ‘‘topological frustration’’ (Thirumalai et al. 1997) into the folding process of these proteins; while the protein chain can theoretically sample a large number of conformations in the folding process, only a very limited subset can lead to a native-like conformation (Clementi et al. 2000). Is threading a rate limiting step for the folding process, or is threading even kinetically accessible on the same timescale as folding of individual protein domains? It is difficult to answer this question for proteins that have been identified as ‘‘threaded,’’ as they are typically large, multidomain proteins, and it is difficult to deconvolute the threading event from other kinetic processes and intermediates in the folding process (Mallam and Jackson 2006). One approach to answer these questions experimentally in an unambiguous fashion would be to develop a model system with a covalently closed loop that would require threading through the loop to fold. Although we have previously introduced topological links into proteins, no threading has occurred. In our previous study of a catenane (Blankenship and Dawson 2003) based on the dimeric M340E/L344K analog (Davison et al. 2001) of the p53tet domain, the chemoselective ligation (Dawson et al. 1994) that forms the topological link occurs after the linear peptides associate and fold. To adapt our existing system into one that requires threading to fold, we considered utilizing temporal control of the folding of the individual components. Cyclization of an individual p53 monomer before folding results 1250
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in a cyclic peptide that is unable to fold (Blankenship and Dawson 2003), as the p53tet domain requires dimerization in order to fold (Davison et al. 2001). However, if a linear p53 monomer could thread through a cyclic p53 monomer, it should be possible for the protein to fold, forming a [2]pseudo-rotaxane. However, this threading process would need to be efficient, as it would compete with dimerization of the linear dimer (Fig. 1B). In addition, an independent probe of the folding of the threaded state would be required to unambiguously distinguish between formation of the linear dimer and the threaded pseudo-rotaxane. The folded [2]pseudo-rotaxane could also provide a template to allow a second, irreversible chemoselective ligation reaction—closing the second ring and forming a [2]catenane (Fig. 1A). If the cyclic and linear components differ in linear sequence, this could also be used to effect the stepwise formation of a protein [2]heterocatenane, which would provide an additional proof of the threading process. Results Based on our earlier catenane design (p53catdim), which did not require threading (Blankenship and Dawson 2003), we have constructed both a protein pseudorotaxane and a heterocatenane that require protein threading to fold. To provide a unique and independent probe of the threaded state, we have introduced a collisionally quenched fluorescent pair through chemical synthesis, consisting of a fluorophore on one monomer and a nonfluorescent quencher on the other monomer, giving a sensitive readout of the threaded or folded state of the protein pseudo-rotaxane or protein catenane (Figs. 1A,B, 2). This design is essential to distinguish the folded linear p53 dimer from the threaded p53 proteins. The fluorescence quenching pair was placed in a contact area between the two monomers, with the quencher on the first cyclic component, and the fluorophore on the second (linear) component. In the pseudo-rotaxane model system, the protein should be fluorescent in the unthreaded state, and upon threading will become quenched (Fig. 1B). Similarly, the heterocatenane model system (incorporating the quencher on the first cyclic component, and the fluorophore on the second cyclic component) should also exhibit fluorescence quenching in the folded state, and fluorescence in the unfolded state. Residues Tyr 327 and Asn 331 were chosen as the modification sites from inspection of the p53 tet structure (Davison et al. 2001), since the side chains are proximal in the folded structure and are part of the antiparallel b sheet, which is dependent on interstrand contacts and hydrogen bonding to fold. Tyr 327 was changed to p-nitrophenylalanine on the first p53catdim sequence (hereafter referred to as p53hetA), and Asn 331 changed to diaminobutyric acid (Dab) on
Threading a peptide through a peptide
Figure 1. (A) Heterocatenane synthesis. Threading of the linear p53 thioester peptide through the cyclic p53 peptide results in folding of a p53 heterodimer, forming a pseudorotaxane (center). This folding brings the termini of the linear peptide in close proximity. Native chemical ligation cyclizes the second ring, and creates a heterocatenane (bottom). (B) Protein threading vs. protein folding. Folding of the linear monomer, bearing a fluorophore, has two possible paths. First, self-association and dimerization can occur (left), resulting in the formation of a linear dimer, which will still be fluorescent. Second, threading through the cyclic peptide, bearing a quencher, could occur (right), resulting in the formation of a pseudorotaxane, which will result in quenched fluorescence.
the second analog sequence (p53hetB) to serve as an attachment point for the 2-aminobenzoyl (Abz) fluorophore. Both peptides were synthesized on a thioester resin (Hackeng et al. 1999) to yield peptide thioesters. The second Abz-containing sequence was also synthesized as a nonthioester peptide (p53hetC) on a MBHA resin to yield a peptide amide. After cleavage and purification, dilute (;200 mM) solutions of p53hetA were quantitatively cyclized in 6 h using native chemical ligation in 6 M guanidine hydrochloride (GuHCl), 100 mM NaPi (pH 7), with 1%/1% (v/v) thiophenol and benzyl mercaptan as additives (Supplemental Fig. S1; Dawson et al. 1994; Blankenship and Dawson 2003). No polymerization or catenation was observed, and conversion was quantitative as monitored by HPLC. Threading to form the intermediate pseudo-rotaxane and subsequent ring closure to form the heterocatenane proceeded rapidly under folding conditions. Addition of the linear p53hetB peptide thioester to the cyclic p53hetA peptide resulted in rapid ligation (complete in under 1 h), with the p53het[A,B] heterocatenane as the primary product (Fig. 1A). The homocatenane, p53hom[B,B], could be observed as a side-product, arising from the folding and ligation of two linear p53hetB thioester peptides. This product was eliminated by adding a slight excess (25-fold at the www.proteinscience.org
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Figure 2. (A) Formation of additional structure upon rotaxane formation. CD spectra of the cyclic p53hetA peptide (10 mM), the linear p53hetC peptide (5 mM), and the combination of the cyclic p53hetA and the linear p53hetC peptides in solution (10 and 5 mM, respectively). All spectra were taken in 25 mM NaPi, 150 mM NaCl, 1 mM DTT (pH 7.0) at 4°C. (B) CD spectra of the p53het[A,B] heterocatenane (5 mM). (C) Efficient fluorescence quenching within the p53het[A,B] heterocatenane. The fluorescence of the Abz residue (420 nm) was highly sensitive to the folded state of the heterocatenane. Filled circles indicate 5 mM p53het[A,B] catenane, 100 mM phosphate buffer (pH 7), open circles indicate 5 mM p53het[A,B] catenane, 6M GuHCl, 100 mM phosphate buffer (pH 7). (D) The degree of fluorescence quenching reflects the folded state of the protein. The unfolding transition during guanidine denaturation is identical whether monitored by fluorescence at 420 nm (open circles) or by circular dichroism at 222 nm (filled diamonds).
emission maximum of 415 nm and is essentially unquenched. Using this assay to monitor equilibrium GuHCl denaturation at 25°C revealed that the unfolding transition is superimposable with the same transition monitored by CD, suggesting that the quenching is characteristic of the folded state (Fig. 2D). The thermodynamic parameters from the fluorescence experiments are within experimental error of those derived from CD of
the heterocatenane (Table 1). Folding of the linear p53hetC dimer alone showed no self-quenching, and emission was identical to the unfolded state. Since fluorescence quenching was efficient in the folded state of the heterocatenane, and absent in the linear folded homodimer, we reasoned that fluorescence could also be used to follow the kinetics of pseudorotaxane formation, assuming the pseudorotaxane exhibited similar
Table 1. Equilibrium denaturation data Sequence Catenanes p53catdim a p53het[A,B] p53het[A,B] Linear dimers p53dim (L344A) p53dim (L348A) p53dimlin/ext a p53hetC
b b
Method
Temperature (°C)
Concentration (mM)
M (kcal mol1M1)
D50% (M GuHCl)
DGuH2O (kcal mol1)
CD CD FL
25°C 25°C 25°C
5, 20 5 5
1.5 6 0.17a 1.2 6 0.16 1.2 6 0.09
5.0 6 0.11a 5.1 6 0.12 5.0 6 0.06
7.3 6 0.99a 5.9 6 0.72 5.8 6 0.50
CD CD CD CD
25°C 25°C 4°C 4°C
40 40 50 5
a
3.0 2.7 3.3 3.4
6 6 6 6
0.1 b 0.07b 0.40a 0.27
From Blankenship and Dawson (2003). From Mateu and Fersht (1998). Corrected number from Blankenship and Dawson (2003). d DGuH2O values reported for linear dimers are extrapolated to a reference concentration of 1 M peptide. b c
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1.11 0.82 1.0 0.8
6 6 6 6
0.01 b 0.01b 0.01a 0.2
9.3 8.2 8.7 9.4
6 6 6 6
0.2b,d 0.1b,d 0.2c,d 0.2d
Threading a peptide through a peptide
quenching in the folded state. In order to allow threading and pseudorotaxane formation to compete with linear dimerization and refolding (Fig. 1B), conditions were needed in which the linear homodimer would be unfolded or only partially folded. Fortunately, both the parent linear p53 dimer and the linear p53hetC peptide are both relatively unstable at 20°C, being partially folded (20% of the population) at low micrometer concentrations and being completely unfolded in 1 M GuHCl (data not shown). Analytical ultracentrifugation was also used to verify the oligomerization state of the linear p53hetC dimer, the cyclic p53hetA monomer, and the p53[A,C]pseudorotaxane (Supplemental Table S2). The linear p53hetC sedimented in a monomer/dimer equilibrium, with the majority of monomers at 56 mM and the majority of dimers at 150 mM. As expected, the cyclic p53hetA sedimented as a monomer at 84 mM. The p53[A,C]pseudorotaxane sedimented as a monomer at a concentration of 56 mM; no equilibrium between threaded and unthreaded states was observed. These studies suggest that only threaded proteins are stable at
