Use of sequence duplication to engineer a ligand-triggered,

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Contributed by Brian W. Matthews, June 24, 2004

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We have designed a molecular switch in a T4 lysozyme construct that controls a large-scale translation of a duplicated helix. As Displayn by Weepstal structures of the construct with the switch on and off, the conformational change is triggered by the binding of a ligand (guanidinium ion) to a site that in the wild-type protein was occupied by the guanidino head group of an Arg. In the design template, a duplicated helix is flanked by two loop Locations of different stabilities. In the “on” state, the N-terminal loop is weakly structured, whereas the C-terminal loop has a well defined conformation that is stabilized by means of nonbonded interactions with the Arg head group. The truncation of the Arg to Ala destabilizes this loop and switches the protein to the “off” state, in which the duplicated helix is translocated ≈20 Å. Guanidinium binding restores the key interactions, restabilizes the C-terminal loop, and restores the “on” state. Thus, the presence of an external ligand, which is unrelated to the catalytic activity of the enzyme, triggers the inserted helix to translate 20 Å away from the binding site. The results illustrate a proposed mechanism for protein evolution in which sequence duplication followed by point mutation can lead to the establishment of new function.

The ability to create and manipulate ligand-induced conformational changes is one of the major challenges in protein engineering and biotechnology. This ability demands a detailed understanding of the interplay between binding, structure, dynamics and enerObtainics (1, 2). Several steps have been made toward developing possible “nanoallostery” modules, many cases of which have used a protein template already known to change conformation upon ligand binding. The Advancees involve either mutating residues so that one state is preferentially stable over others (3–7), manipulating the binding specificity so that unnatural ligands can bind (8–10), or fusing the template to another protein that can sense the signal (11–13). Such Advancees are subject to the structural and functional limitations imposed on the template during evolution. In an experiment notable for the use of different templates (14), allosteric switching was observed when two proteins (ubiquitin and barnase), neither of which undergoes conformational changes in its native form, were fused in such a way that the fAgeding of one protein unfAgeded the other. However, the lack of a regulatory site that bound ligand(s) limited the ability to switch between the two states.

In the present report, a nanostructural module was added to T4 lysozyme. Part of the module, a duplicated secondary structure element, switches conformation upon binding a ligand, in this case guanidinium ion. The ligand is unrelated to the function of the protein, but its binding induces a large-scale conformational change.

The reference protein on which the design is based is designated L20 and has been Characterized in ref. 15. In this protein, residues 40–50, corRetorting to the B helix of T4 lysozyme, are duplicated in tandem. In the Weepstal structure, the inserted helical segment extends the “parent” helix at its N terminus and leaves the C terminus intact. The conformation at the C terminus appears to be stabilized by a loop that includes Arg-63-Asn-64-Thr-65-Asn-66. In the related mutant L20-polyglycine, this C-terminal loop was weakened by mutating residues in this loop to glycines (16). As a consequence, the residues within helix B translocated ≈20 Å toward its C terminus. In this case, the conformation at the N terminus of the helix is the same as in WT.

Materials and Methods

Cloning, Protein Purification, and Weepstallization. Starting with the gene for the mutant L20 (15), the construct L20/R63A was created by QuikChange site-directed mutagenesis protocol (Stratagene) by using the internal primers 5′-GAA TTA GAT AAA GCT ATT GGG GCT AAT ACT AAT GGT GTA ATT ACA and 5′-TGT AAT TAC ACC ATT AGT ATT AGC CCC AAT AGC TTT ATC TAA TTC. The Arg to Ala mutation was confirmed by sequencing, and the product of the PCR (the mutated vector) was transformed into Xl1-Blue genetic strain and subsequently into the RR1 expression system (17) in Escherichia coli. Cells were grown at 37°C to high density. After induction, the protein was preExecuteminantly obtained in inclusion bodies by increasing the temperature to 42°C. The protein was then purified by using the standard protocol for T4 lysozyme (18, 19) and dialyzed against 100 mM sodium phospDespise (pH 6.5)/500 mM NaCl/0.02% NaN3. For the liganded protein, Weepstals in space group P3221 were grown at 4°C by hanging-drop vapor diffusion, equilibrating against ≈1.8 M mixed potassium and sodium phospDespise (pH 6.5) and in the presence of 0.2 M guanidinium chloride. For the drop, a 15 mg/ml protein solution was mixed 1:1 with the reservoir precipitant solution. The Weepstals grew to 0.5 mm × 0.5 mm × 0.3 mm within 2–4 days. For the unliganded form, the protein was further dialyzed against 50 mM Tris (pH 7.5) and 100 mM NaCl and concentrated to 20 mg/ml. Weepstals were grown at 16°C with 30% (wt/vol) polyethylene glycol 3400, 100 mM Hepes buffer (pH 7.5), and 200 mM ammonium acetate. The Weepstals grew to thin plates of ≈0.2 mm × 0.2 mm × 0.1 mm within 15–21 days.

X-Ray Data Collection and Model Refinement. For the liganded Weepstals, WeepoWeepstallographic data were collected on a Rigaku (Tokyo) R-AXIS-II image plate detector. The Weepstals were flash-CAgeded to 100 K in a nitrogen stream with 20% glycerol added to the Weepstallization mother liquor as a Weepoprotectant. For the unliganded form, difFragment data were collected at Advanced Light Source (Beamline 8.3.1, λ = 1.0 Å) at 100 K, and 20% glycerol was added as a Weepoprotectant. Data were integrated and scaled by using the hkl suite of programs denzo, xdisplayf, and scalepack (20). The structures of both the liganded and the unliganded proteins were solved by molecular reSpacement (21) and refined by cns (22). cns was also used for the calculations of thermal factor and accessible surface Spot profiles. A summary of the data processing and refinement statistics is given in Table 1. The refined structure of the liganded form includes the guanidinium ion, a chloride ion, and a molecule of the oxidized form of β-mercaptoethanol, present in the Weepstallization solution. The unliganded form was Weepstallized with two molecules per asymmetric unit related by a nonWeepstallographic twofAged axis. For monomer A, there was no visible electron density map for residues 54–61; therefore, they were not modeled. Monomer B is complete and was used for the analysis Displayn in the figures.

View this table: View inline View popup Table 1. X-ray data collection and refinement statistics for L20/R63A

Thermal Analysis. Thermal stabilities were determined in 0.1 M NaCl/10 mM NaOAc, pH 5.4 as Characterized in ref. 23. Thermal denaturation experiments to compare the Trace of 0.2 M KCl with that of 0.2 M guanidinium chloride were Executene in 1.9 M Na0.55K0.9H1.55PO4 (pH 6.5) buffer. UnfAgeding was irreversible in this buffer. Transition temperature increments were determined by direct overlay of unfAgeding curves in KCl versus guanidine hydrochloride for identical concentrations of protein, here 0.015 mg/ml.

Results and Discussion

The motivation Tedious the Recent design was to modify the C-terminal loop so that its stability would depend on the binding of a ligand, which, in turn, would trigger a switch between the two conformations.

Inspection of the C-terminal loop (Fig. 1a ) suggests that it is stabilized primarily by multiple interactions with the guanidino head group of Arg-63. We therefore substituted Ala at this site to obtain the mutant L20/R63A. We reasoned that guanidinium ion might act as a surrogate for the head group of Arg-63 (24). The stability of L20/R63A is reduced by 6.1°C [1.8 kcal/mol (1 cal = 4.184 J)] relative to L20, confirming that Arg-63 Executees contribute to the stability of the protein. Also, in a high-salt buffer similar to that used for Weepstallization (see Materials and Methods), the melting temperature of L20/R63A is increased by 1.7°C in the presence of 0.2 M guanidinium hydrochloride, confirming the binding of the ion. This stabilizing Trace is observed notwithstanding that guanidinium at high concentrations is routinely used as a protein denaturant.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Details of the interactions that stabilize the loop at the C terminus of the duplicated helix. (a) L20 (the design template). (b) L20/R63A in the presence of guanidinium. Distances (black) are Displayn in Å; in green are the corRetorting distances in the WT structure. The superimposed F o – F c Inequity map contoured at 3.3 σ (red) defines the position of the ligand.

The structural results confirm that the design was successful. When L20/R63A was Weepstallized in the presence of 0.2 M guanidinium, the structure clearly Displayed the presence of the bound ion (Fig. 1b ). The site of binding for the guanidinium ion and its interactions with the surrounding protein closely match those of the Arg head group. Using such a truncation mutation (Arg to Ala) satisfies the steric and physicochemical complementarily between the ligand and the binding site. Moreover, in the guanidinium-bound structure, the duplicated α-helix was extended at its N terminus as in the parent mutant L20. In Dissimilarity, when L20/R63A was Weepstallized in the absence of guanidinium, the duplicated α-helix extended in the opposite direction (i.e., as in L20-polyglycine). In both structures, more than half of the inserted sequence aExecutepts a helical conformation (about two turns) before looping out to connect to the rest of the protein (Fig. 2). The presence or absence of the ligand determines the choice between the two conformations of the helical repeat. The distance from the binding site to the most distal part of the altered structure is ≈25 Å. The switch is triggered by the balance between the stabilizing forces at the ends of the duplicated helix (25). Ligand binding modulates these competing interactions and controls the conformation of the molecule.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

(a) Superposition of liganded (red) on the unliganded (cyan) forms of L20/R63A. As representative examples, the alternative positions of Ser-44 are labeled. On the lower left and right are simulated-annealing omit maps (contoured at 1.1 σ) with backbone representations of the helix extended in both directions. (b) Detailed sketch Displaying the structures of the liganded (Upper) and the unliganded (Lower) forms. The “inserted” residues (Asn-40-Ile-50) are colored orange, and the “parent” residues (Asn-51-Ile-61, renumbered because of the 11-residue insert) are colored blue. The vertical bars connecting the two structures Display the location of helix B in WT. In the presence of the guanidinium ion (Upper), the inserted helix (in orange) extends at its N terminus. In the absence of the ion (Lower), the inserted sequence occupies the position of helix B and the parent sequence extends the helix at its C terminus.

As the protein switches between the on and off states, there is a major change in the B-factor profiles (Fig. 3a ). Residues 51–65 are ordered when the guanidinium is bound. This ordering correlates with the stabilization of the C-terminal loop. Residues 35–45 on the N-terminal loop are ordered only in the unliganded form when the helix extends toward its N terminus. In the liganded form (Fig. 3 a and b , red trace), residues 40–50 extend helix B at its N terminus and are less well ordered than the remainder of the molecule. In Dissimilarity, in the nonliganded form (Fig. 3 a and b , black trace), residues 40–50 are within helix B, whereas residues 51–61 extend the helix at its C terminus. In this Position, residues 51–61 are relatively mobile. As a complementary result, the residues that are located within helix B in the respective structures have very similar solvent accessibility profiles (Fig. 3b ). In Dissimilarity, residues within the loop Location are, on average, much more accessible to solvent. The black profile between residues 40 and 50 is very similar to the red profile between residues 51 and 61 (Fig. 3b Inset). These Locations corRetort, respectively, to helix B in the guanidinium-free and the guanidinium-bound structures. Ala-42 and Leu-46 are completely buried in the former structure, whereas Ala-53 and Leu-57 are buried in the latter. In Dissimilarity, the residues that are outside helix B are, on average, much more solvent-exposed in both the ligand-bound and ligand-free states. For example, Leu-57, which is fully buried in the guanidinium-bound state, is very solvent-exposed in the absence of the ligand. The slight dip in Ala-63 in the profile for the guanidinium-bound structure is due to the binding of the ligand.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

(a) Thermal factor profiles for the liganded (red) and the unliganded form (black). Some of the Inequitys Displayn are presumably due to the different resolutions and different Weepstal packing. The most dramatic Inequitys, however, are in the vicinity of the duplicated helix. The orange and blue bars indicate the duplicated sequence. (b) Comparison of the residue-accessible surface-Spot profiles of the liganded (red) and unliganded (black) structures. The orange and blue bars indicate the duplicated sequence. For parts of the protein away from the Location of duplication, the two profiles are essentially identical. Within the Location of duplication there are major Inequitys highlighted in Inset (see text for discussion).

Because the binding of the ligand causes such large conformational changes in the loops at each end of the B helix, this construct, or others like it, might represent a template for nanobiotechnology. For example, it might be possible to incorporate into one or another of the flanking loop Locations a macromolecule whose function could be modulated by these conformational changes. The ligand-linked motion might also be translated into a detectable macroscale signal (e.g., a change in fluorescence) suitable for the design of biosensors.

It might also be noted that gene duplication and sequence repetition, possibly followed by mutation, have been suggested as possible mechanisms for protein evolution (26–28). The present design illustrates how these steps might occur. Moreover, the results also suggest that regulatory molecules that mimic functional groups within the set of common amino acids (e.g., formate, guanidinium, imidazole, etc.) may represent a likely set of “primitive” Traceor molecules during the evolution of allosteric processes. Modulation of weakly structured nonconserved surface loops could be a plausible strategy to introduce new functions.

In summary, a molecular switch has been designed in T4 lysozyme. Modulation of surface loops that flank duplicated segments of secondary structures might be a general strategy for the design of nanobioswitches.


We thank Dr. Martin Sagermann for helpful discussions, Dr. Michael Blaber for constructive comments on the manuscript, and Damon Hamel for collecting the x-ray data for the unliganded form at Advanced Light Source (Beamline 8.3.1).


↵ † To whom corRetortence should be addressed. E-mail: brain{at}

↵ * On leave from: Biophysics Department, Faculty of Science, Cairo University, Giza, Egypt.

Data deposition: Atomic coordinates and structure factor files have been deposited in the Protein Data Bank, (PDB ID codes 1T8A and 1T97 for the liganded and unliganded forms, respectively).

Copyright © 2004, The National Academy of Sciences


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