Use of a non-rigid region in T4 lysozyme to design an adaptable metal-binding site

Jonathan W. Wray1, Walter A. Baase, Gerard J. Ostheimer, Xue-jun Zhang2 and Brian W. Matthews3

Institute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, 1229 University of Oregon, Eugene,OR 97403-1229, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is not easy to find candidate sites within a given protein where the geometry of the polypeptide chain matches that of metal-binding sites in known protein structures. By choosing a location in T4 lysozyme that is inherently flexible, it was possible to engineer a two-histidine site that binds different divalent cations. Crystallographic analysis shows that the geometry of binding of zinc is distorted tetrahedral while that of cobalt and nickel is octahedral. Insofar as spectroscopic data can be measured, they indicate that similar modes of coordination are retained in solution. The two substitutions, Thr21 -> His and Thr142 -> His, lie, respectively, on the surface of the N- and C-terminal domains on opposite sides of the active site cleft. The design takes advantage of hinge-bending motion which allows the binding site to adapt to the most favorable ligand geometry for the metal. Introduction of the two histidines increases the melting temperature of the protein by 2.0°C at pH 7.4. Metal binding further increases the melting temperature, but only by a small amount (up to 1.5°C). A third substitution, Gln141 -> His, which could act as a third ligand in principle, does not do so, demonstrating the difficulty in mimicking naturally occurring metal-binding sites.

Keywords: divalent metals/flexibility/hinge-bending/protein stability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Metals bind to many proteins and confer properties related to catalysis, redox chemistry, ligand binding and stability. Therefore, metal–protein interactions represent an integral aspect of protein chemistry, and the understanding of these interactions is essential to further our ability to redesign or engineer proteins with enhanced or novel functions.

Excluding polyhistidine `His-tags', the most prevalent metal-binding site to be introduced into proteins has been designed to accommodate the tetrahedral coordination geometry of Zn(II). Most successful designs have relied on the introduction of one or more cysteines (e.g. Corey and Schultz, 1989; Regan and Clarke, 1990; Ippolito and Christianson, 1993; Lu and Valentine, 1997; Pinto et al., 1997; Wisz et al., 1998). Craik and co-workers introduced a histidine proximal to the active site histidine of rat trypsin to obtain an enzyme whose activity is inhibited in the presence of zinc and copper (Higaki et al., 1990Go). Three-histidine sites have been introduced into a variety of proteins, and protein motifs, such as zinc fingers, immunoglobins, scorpion toxin and four-helix bundles (Pessi et al., 1993Go; Müller and Skerra, 1994Go; Regan, 1995Go; Lu and Valentine, 1997Go). A particularly successful example is the three-histidine site introduced into retinol-binding protein, which had an association constant for Zn(II) of 2.8 x 107 M–1 and increased stability towards chemical denaturation in the presence of the metal ion (Müller and Skerra, 1994Go).

In general terms, a high-affinity metal-binding site should consist of a set of rigidly held ligands whose geometrical arrangement is poised to match perfectly the preferred coordination of the metal ion. In practice, as is illustrated below, it is difficult if not impossible to find candidate sites at which ligands with such ideal geometry can be introduced. As a compromise, we have explored a site which is flexible, and which can adjust to provide the requisite binding geometry. Specifically, we introduced histidines at sites 21 and 142 in T4 lysozyme. These two residues can change their relative positions by `hinge-bending' movement of the molecule as a whole. It has previously been shown that cysteines introduced at these two sites form a disulfide bridge that stabilizes the protein (Matsumura and Matthews, 1989Go). The two-histidine mutant, designated H2, binds different divalent cations in either tetrahedral or octahedral geometry and, in so doing, slightly stabilizes the protein.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutagenesis and protein purification

Mutations were introduced into the T4 lysozyme gene borne by M13 phage by the directed mutagenesis method of Kunkel et al. (1987). Mutant lysozyme genes were subcloned and expressed in plasmid vector pHN1403. Escherichia coli strain RR1 was transformed with the plasmids and ampicillin-resistant transformants were isolated. Clones bearing the mutant lysozymes were identified, grown in liquid culture, and protein produced as described (Muchmore et al., 1989Go; Poteete et al., 1991Go; Baldwin et al., 1993Go). The mutant protein H2 was constructed from the gene for pseudo-wild-type T4 lysozyme, C54T/C97A (`WT*'), in which the two cysteine residues have been replaced with Thr and Ala respectively. This cysteine-free lysozyme has structure and functional characteristics similar to the wild-type, but displays better reversibility in thermal denaturation experiments (Matsumura and Matthews, 1989Go). The full designation of the mutant protein H2 is T21H/C54T/C97A/T142H.

Protein stability

Thermal unfolding experiments were done for WT* and H2 protein in 20 mM glycine–HCl, 1 mM EDTA, pH 3.0, and in 0.10M KCl, 0.016M 3,3-dimethyl glutarate, pH 7.4, at a scanning rate of +2°C min and with protein concentrations of 0.7–0.9 µM. Stability changes were estimated using van't Hoff analysis of the change in circular dichroism (CD) at 223 nm upon heating as described previously (Eriksson et al., 1993Go). Transition curves at both values of pH appeared to be two-state. At pH 3 restoration of the CD signal upon cooling was in excess of 95%, but at pH 7.4 it was between 40 and 60%.

To maximize the reversibility at pH 7.4, Suprasil optical cells (QS-111 from Hellma) were cleaned with RBS-35 (Pierce Chemical) and water and at least two subsequent 10 min incubations at 62°C with 20 mM potassium phosphate, pH 3. Incubations were done inside the spectrophotometer to clean all parts of the instruments normally in contact with protein solution and to confirm directly removal of protein film.

Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) (Wiseman et al., 1989Go) was done for NiCl2, CoCl2 and ZnCl2 binding to H2 protein in 0.10 M NaCl, 20 mM HEPES, pH 7.8. An OMEGA titration calorimeter (Microcal) was operated using a 43.2 µl/in injection syringe at 29°C and 299 rpm with a feedback setting of `4' and a data collection interval of 3 min per injection. Twenty-four injections of 6 µl each were typically done with the protein in the sample cell at about 0.1 mM and the ligand in the syringe adjusted such that protein : ligand stoichiometry was 1 : 1 at about 40% of the way through the titration. Baseline mixing heats were determined by injection of buffer into protein samples immediately prior to injection of ligand. Reaction heat profiles were obtained from raw heats by subtraction of the baseline heats. Reaction heat profiles were fit to the single binding site model using the ITC worksheet of ORIGIN, version 4.10 (Microcal Software). Solutions were outgassed at one-third atmosphere with controlled stirring (using an outgassing station from Microcal) for at least 10 min. This appeared to help prevent the precipitation of what might have been a carbonate, which was problematic for Zn(II) titrations. Control titrations using WT* (C54T/C97A) were done using otherwise identical conditions.

T4 lysozyme concentration was determined optically in 0.55 M NaCl, 0.10 M NaPO4, 0.02% sodium azide, pH 6.5, using a molar absorptivity at 280 nm of 23 900 M–1 cm–1 (Elwell and Schellman, 1975Go). Deionized water was used for all solutions. Solutions were clarified as needed using 0.2 µm Acrodisc B syringe filters (Gelman Sciences) or centrifugation.

UV–visible spectroscopy

Absorbance spectra were recorded in a 0.1 or 1 cm pathlength cell using a Varian Cary 3E UV–visible spectrophotometer. T4 lysozyme samples were dialyzed into 0.10 M NaCl, 20 mM HEPES, pH 7.8, in Spectra/Por 6000–8000 dialysis membrane. Spectra of protein and protein–metal complexes were recorded at 20°C with the protein present at 0.15–0.2 mM and with divalent metal ion concentrations varying from 0.0 to 0.5 mM.

The molar absorptivities of the metal ions in this buffer and also that of the H2 protein itself in this region of the spectrum were directly determined. The molar absorptivities of the protein–metal complexes were determined from absorbance data and from the mole fractions of unbound metal ions, unbound H2 protein and the metal ion–H2 complexes calculated from the binding constants and heats of binding determined under essentially identical solvent conditions by means of ITC. The limits of uncertainty are relatively high for this reason and because of the low absolute magnitude of the raw absorbance.

Crystallography

Protein crystals in space group P3221 were obtained by vapor diffusion using the hanging-drop method. The well buffer contained 200–300 mM NaCl, 20 mM HEPES, pH 7.0, 12–16% PEG 8000, 10% 2-propanol and 2 mM metal ion as the chloride or acetate salt. Hanging drops were made by mixing 5 µl of well buffer with an equal volume of a 1 mM protein solution in 20 mM HEPES, pH 7.0. Crystals were obtained at 4°C in the presence of Zn(II), Ni(II), Co(II) and Cu(II), although in the last case they were not of sufficient quality to permit X-ray data collection. Apo-H2 crystals could not be obtained under these conditions, and instead were grown by the same method using a well buffer of 1.8–2.0 M Na/K phosphate, pH 6.5–7.0, 5 mM oxidized BME and a protein solution of 1 mM protein in 200 mM NaCl, 100 mM phosphate, pH 5.5 (Eriksson et al., 1993Go). X-ray diffraction data were collected using a Xuong–Hamlin multiwire detector (Hamlin, 1985Go). Structures were refined with the program TNT (Tronrud et al., 1987Go) and crystallographic models were built with the program O (Jones, 1985Go). Except for initial rigid-body positional adjustment, the metal–ligand bond lengths and angles were not restrained during refinement. Coordinates have been deposited in the Protein Data Bank for immediate release (I.D. codes 257L–260L, 1EPY).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Structures of the metal-binding sites

The high-resolution structure (Table IGo) shows that H2 accommodates zinc in a distorted tetrahedral geometry using the nitrogens N{varepsilon}2 of His21 and His142, a single carboxylate oxygen (O{varepsilon}2) of Glu22 and a water molecule (Figure 1AGo and B; Table IIGo). The His21(N{varepsilon}2)–Zn(II)–His142(N{varepsilon}2) angle is 103°. In contrast, H2 binds Co(II) and Ni(II), both with their preferred octahedral geometry, but utilizes only the two histidines as direct ligands. In each case the angle subtended by the histidines at the metal is close to 90° (Table IIGo). The two histidines, which ligand the metal in equatorial positions through their N{varepsilon}2 nitrogens, undergo only small shifts compared with their positions in the zinc structure. The remaining ligands are water molecules (Figures 2A and BGo and 3A and BGo).


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Table I. X-ray data collection and refinement statistics
 



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Fig. 1 (A) Electron density maps showing the binding of zinc(II) to the mutant H2. The map drawn with thin lines has coefficiencts 2Fo-Fc where Fo is the observed structure amplitude and Fc that calculated from the refined complex with the metal detected. The resolution is 1.8 Å and the map is contoured at 1{sigma}, where {sigma} is the root-mean-square density throughout the unit cell. The thick line shows the electron density in a map with coefficients Fo-Fc, contoured at 10{sigma}, resolution 1.8 Å. Figures were rendered with MOLSCRIPT and BOBSCRIPT (Kraulis, 1991Go; Esnouf, 1997Go). (B) Schematic drawing illustrating the coordination of zinc bound to mutant H2. The longer dashed lines show the salt-bridge interaction between Glu22 and Arg137 which is present in WT* lysoxyme and is retained in the metal-bound mutants.

 

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Table II. Stereochemistry of the ligand-binding sites
 



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Fig. 2 (A) Electron density maps showing the binding of cobalt(II) to mutant H2 at 19 Å resolution. All details are as in Figure 1AGo. (B) Schematic drawing showing the octahedral coordination of cobalt bound to mutant H2.

 



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Fig. 3. (A) Electron density maps showing the binding of nicke(II) to mutant H2 at 1.8 Å resolution. All details are as in Figure 1AGo. (B) Schematic illustration showing octahedral coordination of nickel bound to to mutant H2.

 
Structural changes associated with metal binding

The metal site bridges the active site cleft, and metal-binding induces a positive hinge-bending angle which corresponds to a widening of the active site cleft by ~1 Å (Table IIGo). In addition to the global hinge-bending motion, there are also localized changes on binding the different metals. Figure 4BGo shows the large rotamer shift which takes place when His142 is liganded to Zn(II). In Apo-H2 the {chi}1 and {chi}2 angles of His142 (308 and 328°) correspond to the most populated rotamer in well determined protein structures (Blaber et al., 1994Go). In the Zn(II) complex this residue undergoes a conformational change to {chi}1 = 334° and {chi}2 = 84°, which does not correspond to any of the commonly observed histidine side-chain rotamers. Hence the mutant protein does adjust so as to bind zinc with close to ideal tetrahedral geometry but at the expense of an unfavorable conformational of one of the liganding histidines. This no doubt contributes to the relatively weak binding (Table IIIGo) (cf. Müller and Skerra, 1994). The planes of the imidazole rings undergo rotations when binding the different metals, most notably His21, the ring plane of which rotates 34° in the nickel structure compared with its position when liganding zinc. The remaining two equatorial ligands are water. Although the oxygen O{varepsilon}1 from Glu22 could, in princple, make a direct ligand contact, as seen in the zinc structure, a water is interposed. This solvent molecule (Wat501) is hydrogen bonded to the glutamate oxygen in both the nickel and cobalt complexes with distances of 2.3 and 2.4 Å and B values of 17.8 and 15.0 Å2, respectively.




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Fig. 4 (A) Stereo pair comparing the structure of mutant H2 in complex with zinc (drawn solid) with the structure of apo-protein (drawn open). The change of ~1 Å in the backbone separation of residues 21 and 142 is made possible by an overall change in the hinge-bending angle between the two domains. The non-connectivity between the individual amino acids and the backbone is a consequence of the smoothing algorithm used to draw the latter. (B) Superposition of selected residues from the zinc-bound (black) and zinc-free (open bonds) structure of mutant H2. The superposition is based on minimization of the root-mean-square difference between the core residues of the N- and C-terminal domains, i.e. 15–60 plus 80–160. Although the backbone atoms in the vicinity of residues 21 and 142 move ~1.0 Å further apart on binding zinc, the Arg137–Glu22 salt bridge (shown as long, dashed lines) is conserved.

 

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Table III. Binding of metal ions to mutant H2 at 29°C
 
An intermolecular nickel-binding site

In the crystal structure of wild-type T4 lysozyme, two molecules are related by a crystallographic 2-fold axis forming an extensive back-to-back interface (Weaver and Matthews, 1987Go; Zhang et al., 1995Go). The ability of lysozyme to form these dimers has been shown to be an important step in nucleation of crystallization (Heinz and Matthews, 1994Go). During refinement of the H2–Ni crystal structure, a large positive density peak (10{sigma}) was seen in the FoFc maps. This corresponds to a second nickel ion which is liganded, with octahedral geometry, to the nitrogen of Met1, Wat601, Wat602, Wat603, and a carboxylate oxygen of Glu64 of a symmetry-related lysozyme molecule (Figure 5AGo and 5BGo). No obvious ligand is seen at the remaining octahedral site (Figure 5AGo), although we cannot exclude poorly ordered solvent in this vicinity. This site is formed by the interface between symmetry-related molecules well away from the engineered metal-binding site, which suggests that it should also be present in crystals of WT*. We tested this hypothesis by determining the structure of WT* crystallized under the same conditions as H2–Ni. The difference density map (not shown) had a large positive peak (10{sigma}) at the expected site. None of the other divalent metals were, however, observed to bind to this adventitious site, either in WT* or in H2. Because this Ni(II) binding site has only two proteinaceous ligands, each of which comes from a different molecule in the crystal lattice, it is unlikely to bind metals when the protein is in solution. This is also indicated by the observations that Ni(II) binds to the intermolecular site in crystals of WT* lysozyme but has no effect on the melting temperature of the WT* protein in solution.




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Fig. 5 (A) Electron density showing the binding of nickel at a site of crystal contact. All details are as in Figure 1AGo. The results shown are for the mutant H2 crystallized in the presence of 2 mM NiCl2. Virtually identical results were obtained for WT* lysozyme crystallized under the same conditions (not shown). (B) The coordination of nickel when bound between Met1 and Glu64 at a site of intermolecular contact in crystals of T4 lysozyme. Atoms 601, 602 and 603 are presumed water molecules.

 
Effect of ions on stability

At pH 3.0, the mutant H2 is slightly (0.6 kcal/mol) less stable than WT* whereas at pH 7.4 it is 0.7 kcal/mol more stable (Figure 6Go). In the absence of divalent ions its melting temperature (Tm) at pH 7.4 is 61.5°C. In the presence of 0.06 mM Co(II), Zn(II) and Ni(II), the Tm of H2 increased by 0.3, 0.7 and 1.5°C respectively. The increases in Tm are clear cut for Ni(II) (Figure 6Go) and Zn(II) but less so for Co(II). Repeated trials, however, support this ranking. The Tm for WT* is 59.5°C and, in the presence of the same concentration of metal ion, did not change by more than the standard deviation of the measurement, i.e. ±0.2°C. This lends credence to the idea that the new histidines at sites 21 and 142 in the H2 mutant are indeed the reason for the increases in Tm seen in the presence of these divalent ions. The van't Hoff enthalpies of unfolding of H2 with and without the metals were, within experimental error, essentially the same as WT*.



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Fig. 6. Comparison of the thermal unfolding, at pH 7.4, of WT* lysozyme, the two-histidine mutant, H2, and the mutant in the presence of 60 µM Ni(II). Unfolding is monitored by the change in circular dichroism at 223 nm.

 
Isothermal titration calorimetry

Isothermal titration at 29°C at pH 7.4 showed the Kb for Ni(II) to be 100 000 M–1, that for Zn(II) to be 70 000 M–1 and that for Co(II) to be 20 000 M–1 (Table IIIGo). The result for Zn(II) must be considered tentative because of the low heat of binding for Zn(II) and the necessity to use a relatively low protein concentration to avoid what appeared to be carbonate formation in the buffer. The thermal upshift measurements, done at 300-fold lower protein concentration and at higher temperature, appear to avoid the precipitation problem. Under these conditions the Tm increases are modest in absolute terms, but dramatic considering the small amounts of divalent ion which are present. Hence, while the calorimetric data are least reliable for Zn(II) binding, when taken together with the thermal upshift results, the data show divalent ion binding in solution to the folded form of H2 with order of preference Ni(II) > Zn(II) > Co(II).

Spectroscopic analysis of coordination in solution

Figure 7AGo shows the molar absorptivity of the Ni(II)-H2 complex and also that of NiCl2 alone (inset) as a function of wavelength. The spectrum for Ni(II) alone is essentially identical with that for Ni(H2O)62+ (Cotton and Wilkinson, 1972Go), supporting the idea that the Ni(II) is principally coordinated by water in the protein-free solution. Upon binding to H2, the molar absorptivity increases as Ni(II) is added and the peak at 396 nm appears to shift slightly to 410 nm. The broad transition at 720 nm in the metal alone appears to shift to 650 nm in the complex but is somewhat obsfucated by noise and by lack of data above 860 nm. Irrespective of shifts in the peak positions, the low values of both spectra are consistent with octahedral coordination of the nickel ion both with and without the presence of H2 protein.




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Fig. 7. (A) Molar absorptivity of the nickel–H2 complex and of NiCl2 (inset) in 0.10 M NaCl, 20 mM HEPES, pH 7.8, at 20°C. The uncertainty in the magnitude for the complex is roughly ±10% since the mole fraction of complex was calculated from the Kb determined by ITC. (B) Molar absorptivity of the cobalt–H2 complex and of CoCl2 (inset) in 0.10 M NaCl, 20 mM HEPES, pH 7.8, at 20°C. The uncertainty in the magnitude for the complex is roughly ±20% since the mole fraction of complex was calculated from the Kb determined by ITC.

 
Figure 7BGo shows the molar absorptivity of the Co(II)–H2 complex and also that of CoCl2 alone (inset) as a function of wavelength. The molar absorptivity of Co(II) is similar to that shown for Co(H2O)62+ but of only 50% of the published magnitude (Cotton and Wilkinson, 1972Go). Upon incremental addition of Co(II) to H2, the absorbance increases incrementally near 340 nm but the spectrum remains relatively featureless above 350 nm. Using a 0.1 cm cell, we estimate the molar absorptivity of the Co(II)–H2 complex to be not more than 2000 M–1 cm–1 greater than that of H2 itself in the 265–295 nm region (not shown). Nevertheless, the low molar absorptivities for both spectra in the visible region are still consistent with octahedral coordination of the cobalt ion both with and without the presence of H2 protein.

Ligand field stabilization effects for Zn2+ are not expected owing to filled d orbitals (Cotton and Wilkinson, 1972Go). Indeed, neither ZnCl2 itself nor Zn(II) complexed with H2 protein appeared to have detectable absorbance peaks in the visible region (not shown).

Enzymatic activity

As estimated by halo size (Streisinger et al., 1966Go), the catalytic activity of H2 is roughly one-tenth to one-twentieth that of WT*. No further change in lysozyme activity with H2 was observed in the presence of up to 100 mM metal ions in the plate media.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Use of adaptability in designing binding sites

In order to achieve a high-affinity engineered metal-binding site, it is desirable (and possibly essential) that the participating residues on the protein be pre-organized into a rigid framework with liganding geometry matched to the preferred coordination of the metal. In practice this is not easy to achieve. Our inititial approach to this problem was to search systematically for any possible site on T4 lysozyme that could be changed to resemble any metal-binding site of known proteins. For example, the zinc ion in carbonic anhydrase is bound by His94, His96 and His119 (Eriksson et al., 1988Go). We therefore asked if there was any combination of three residues in T4 lysozyme that could be replaced with histidines to mimic this site geometrically. Specifically, we attempted to superimpose the C{alpha} and Cß atoms of His94, His96 and His119 from the structure of carbonic anhydrase on the C{alpha} and Cß atoms of all sets of three residues from the structure of T4 lysozyme (Weaver and Matthews, 1987Go). Assuming no steric interference from neighboring parts of the structure, if the C{alpha} and Cß atoms of the set of three residues matched, then three histidines introduced at these sites in T4 lysozyme would be able to adopt the same conformation as the three histidines of carbonic anhydrase.

As can be seen from the representative results in Table IVGo, however, there is no putative site in T4 lysozyme that closely matches a known site in the proteins listed or, for that matter, in other proteins not shown. Indeed, even for the `best' candidate sites the structural agreement is poor, approaching 1 Å. Since the C{alpha} and Cß atoms do not correspond well, it implies that the C{alpha}–Cß bonds are pointing in different directions, and that distal parts of the side chains are likely to have even greater discrepancies. Hellinga et al. (1991) and Gregory et al. (1993) have also discussed the geometrical difficulties in designing metal-binding sites in proteins.


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Table IV. A search for combinations of residues in T4 lysozyme that match the geometry of known metal-binding sites
 
A possible way around this dilemma is to utilize sites at which the protein is flexible and can adjust to bind the ligand in its preferred geometry. In the present case, hinge-bending motion (Zhang and Matthews, 1995Go) allows the N- and C-terminal domains to rotate, varying the distance between sites 21 and 142 (Figure 4AGo). The same strategy was very effective in designing stabilizing disulfide bridges (Matsumura et al., 1989Go; Jacobson et al., 1992Go). Similarly, the flexibility and adaptability of the hypervariable loops that comprise antibody combining sites may have contributed to the successful use of such modules in engineering metal binding sites (Pessi et al., 1993Go; Crowder et al., 1995Go; Roberts and Getzoff, 1995Go).

This general philosophy is also illustrated by the construction of a variant of the H2 protein, in which Gln141 was replaced by a histidine in order to create a potential three-histidine site. This protein (H3; Table IGo) also binds zinc, cobalt and nickel, but the X-ray structure (Table IGo) reveals that, for the engineered binding site, only the same two histidines that are seen with H2 are used for metal binding (not shown). Although the third histidine is, in principle, available to contribute as a ligand, it does not do so. This is because the imidazole of His141 is not optimally positioned, and the local structure is too rigid to allow it to move into the correct position. This simple example further illustrates that the mimicking of naturally occurring metal-containing proteins is not easily accomplished.

Yet another example is provided by the intermolecular metal-binding site that occurs at a crystal contact. This site was observed to bind nickel but not the other metals tested. It shows that, in order to bind a given metal ion, a designed site must either closely match the preferred coordination of the ion, or must be capable of relaxing to achieve the required geometry.

Another challenge in engineering metal-binding sites is to avoid substitutions that substantially destabilize the protein. This is more likely to occur when core residues are replaced. In the present case the use of sites on the surface of the protein not only avoided destabilization but slightly increased stability, at least at pH 7.4 (Figure 6Go). In the mutant protein the side chains of His21, His142 and Arg145 are all close together. Therefore, it is not surprising that at pH 3.0, where all will be positively charged, the protein is slightly destabilized relative to WT*. It is not immediately obvious, however, why the mutant protein is slightly more stable than the wild-type at pH 7.4.

The order of the binding strength [Ni(II) > Zn(II) > Co(II)] follows the stability of complexes of a variety of different ligands with dipositive metal ions (Irving and Williams, 1953Go). This suggests that the strength of binding is not greatly influenced by any pre-organized structure of the binding ligands, and is consistent with the design philosophy which allows the ligands to move easily to adopt optimal binding geometry. At the same time, there may be a cost in using part of the binding energy to organize the binding site. This presumably explains the relatively low binding affinity achieved in the present experiments and highlights the fundamental dilemma in designing a high affinity site. As noted above, it is extremely difficult to find sites on a protein that will provide the requisite geometry for tight binding. On the other hand, to the degree that one introduces flexibility to allow the site to relax to the required binding geomery, affinity will be lost via the energy of organizing (or reorganizing) the site.

Use of metals to regulate catalytic activity

Higaki et al. (1990) used an engineered histidine proximal to the active site to inhibit the activity of trypsin. In the present case, the introduction of the two histidines reduced activity, presumably because they partially occupy the active site cleft (Figure 4AGo). Addition of metals, however, did not further reduce activity, even though the metal-binding site bridges the active site cleft. This may at first seem puzzling, but it may be noted that Glu22 and Arg137 form a salt bridge across the active site cleft of wild-type lysozyme (Weaver and Matthews, 1987Go). As estimated by the decrease in pKa of Glu22, the strength of interaction appears to be ~1 kcal/mol in 0.1 M NaCl (Anderson et al., 1993Go). Clearly, the presence of this salt bridge does not inactivate the native enzyme. When metals are bound to the mutant H2, the {alpha}-carbon atoms of Glu22 and Arg137 move apart (Figure 4BGo). Because of compensating adjustments in the side chains, however, the salt bridge remains intact. At the same time, Glu22 becomes a metal ligand. As with the original salt bridge, the binding of a metal ion (which is relatively weak) does not appear to interfere with catalytic activity. This suggests that if a metal-binding site is to be used to control activity, it needs to be a high-affinity site.


    Notes
 
1 Present address: Advanced Medicine Inc., 901 Gateway Boulevard, South San Francisco, CA 94080, USA Back

2 Present address: Crystallography Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA Back

3 To whom correspondence should be addressed Back


    Acknowledgments
 
We are most grateful to Hong Xiao for help with protein purification and crystallization, Larry Weaver for refining the H3-Co structure and Joel Lindstrom for stability measurements. Helpful discussions with Dale Tronrud are gratefully acknowledged, as are comments from one of the referees on metal-binding affinity. This work was supported in part by NIH Fellowship F32GM17434 to J.W.W. and NIH grant GM21967 to B.W.M.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received May 1, 1999; revised January 1, 2000; accepted February 20, 2000.