Metal ions‐binding T4 lysozyme as an intramolecular protein purification tag compatible with X‐ray crystallography

Abstract

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein‐coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions‐binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized‐metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X‐ray crystallography.

Short abstract

PDB Code(s): 5I14

Introduction

The lysozyme of enterobacteria phage T4 (also known as endolysin, muramidase, hereafter referred to as T4 lysozyme) is a protein capable of degradation of host peptidoglycans by hydrolysis of the linkages between N‐acetylmuramic acid and N‐acetylglucosamine residues. It participates with a second lysis factor, a small membrane protein designated as holin, in the sequential events which lead to the host cell lysis releasing the mature viral particles.1

T4 lysozyme is a well folded soluble protein that crystallizes under many different conditions.2 In biochemistry and structural biology, it has served as a model molecule for studying the factors that determine the structure and stability of proteins.2 Recently, it has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein‐coupled receptors,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 sterol binding proteins,13, 14 or type II phosphatidylinositol 4‐kinases.15, 16, 17 In this strategy, usually the flexible intracellular loops of the target proteins are replaced by T4 lysozyme, nevertheless, fusing T4 lysozyme to the amino terminus of the target protein could also facilitate crystallogenesis.18 A remarkable feature of T4 lysozyme is its tolerance of changes in the amino acid sequence.2 The enzymatic activity is preserved upon single substitutions of more than half of the amino acid residues of T4 lysozyme.19

In this study, we present a structural and biochemical characterization of its novel, metal ions‐binding mutant (mbT4L). Specifically, we present a crystal structure of mbT4L in complex with Ni²⁺ ions at resolution of 1.8 Å. In addition, we show that mbT4L can be utilized as an intramolecular purification tag of recombinant proteins in the immobilized‐metal affinity chromatography (IMAC). Compared to the commonly used N‐terminal hexahistidine tag, mbT4L is not affected by degradation of the intrinsically disordered N‐termini of the target proteins resulting in their higher yields, binds to the immobilized metal ions with stronger affinity resulting in higher purity of purified proteins, and improves the solubility of the target proteins. In addition, upon crystallization of the mbT4L‐fusion protein the anomalous signal of the bound metal ions may be utilized for phasing along with the known structure of the mutant lysozyme. Taken together, our data provide a structural rationale for a novel intramolecular protein purification tag compatible with both the IMAC and X‐ray crystallography techniques.

Results

Design and crystal structure of the metal ions‐binding T4 lysozyme

Wild type T4 lysozyme (wtT4L) is composed of an N‐terminal alpha helix α1, N‐terminal lobe formed by three beta strands β1‐β3 and one alpha helix α2, and a C‐terminal lobe formed by nine alpha helices α3‐α11 [Fig. 1(A,B)]. The substrate binding groove is located between the lobes.22 The flexibility between the lobes is required for the enzymatic activity of T4 lysozyme,23 nevertheless, in some cases it may contribute to poor crystal quality of T4L‐fusion proteins.20

Figure 1.

Crystal structure of the metal ions‐binding T4 lysozyme. (A) Multiple alignment of the wild‐type phage T4 lysozyme (wtT4L), T4 lysozyme with a truncated N‐terminal lobe as described by Thorsen et al.20 (trT4L), and metal ions‐binding T4 lysozyme presented in this study (mbT4L). Blue areas represent the glycine/serine linker (‐GGSGG‐) replacing the N‐terminal lobe in trT4L and mbT4L; the red areas represent amino acid residues mutated to histidines in mbT4L, while the yellow area represents the methionine residue mutated to alanine in mbT4L. The numbers indicate amino acids positions. The secondary structures are indicated above the sequences. (B) The overall fold of mbT4L originates from the fold of wtT4L. The protein backbone of mbT4L in cartoon representation is depicted in rainbow colors from blue (N terminus) to red (C terminus), and is superimposed with wtT4L (PDB entry 3FA0)²¹ depicted in grey. The metal ions‐binding site of mbT4L is shown in stick representation. (C) The mbT4L crystals contained two molecules of mbT4L binding four nickel ions per asymmetric unit. The protein backbones in cartoon representation as well as the semi‐transparent surfaces of the first and second mbT4L molecule in the asymmetric unit are colored in yellow and red, respectively. The Ni²⁺‐binding sites are shown in stick representation. The nickel ions are represented as green spheres. (D) Detailed view of the Ni²⁺‐binding sites of the mbT4L molecules in complex with nickel ions depicted as in C. The anomalous scattering was analyzed using a crystallographic dataset at the wavelength of the absorption peak of the nickel atoms (1.4859 Å), and the anomalous map contoured at 3 sigma is shown.

To design a protein with desired properties, we choose truncated T4 lysozyme (trT4L) as described by Thorsen et al.20 as our starting molecule [Fig. 1(A)]. In this “minimal version” of T4 lysozyme, the flexible N‐terminal lobe (residues R12 to K60) is replaced with a short glycine/serine linker (‐GGSGG‐), which leads to the enzymatic inactivity of the trT4 lysozyme along with a limited flexibility and improved crystallization properties of the mutant protein.

To generate a metal ions‐binding mutant of T4 lysozyme (mbT4L), several point mutations to trT4 lysozyme were introduced. The commonly used metal ions‐binding protein purification tags usually contain 6‐10 histidines which are sterically capable of forming coordinate covalent bonds with metal ions such as Co²⁺, Cu²⁺, Ni²⁺, or Zn²⁺.24 Therefore, we decided to introduce at least six to‐histidine point mutations to trT4 lysozyme. The candidate amino acid residues for mutagenesis had to meet several criteria as follows: they should be solvent accessible (preferentially surface‐exposed polar residues), they should be located distally to the N‐ and C‐ termini of the molecule (preferentially within the α7 and α8 helices of trT4 lysozyme) to facilitate a subsequent utilization of the mutant lysozyme in the fusion protein strategy, and they should be sterically capable of forming coordinate covalent bonds with the metal ions. Homology modeling of T4L mutants based on a previously known T4L structure (PDB entry 3FA021) suggested that the suitable candidates for mutagenesis could be the residues N73, R76, Q80, R82, E85, and N89 [Fig. 1(A)]. In addition to these to‐histidine mutations we introduced an M77A mutation to prevent the methionine sidechain from sterical interference with the coordinate covalent bonds formed by metal ions and introduced histidines. The resulting construct was expressed in E. coli at high levels and could be purified to homogeneity using a simple immobilized‐metal (Ni²⁺) affinity chromatography.

To evaluate the effect of the T4 lysozyme truncation combined with the mutagenesis mentioned above, we determined the structure of the mbT4L protein in complex with the nickel ions using protein crystallography. We obtained crystals that diffracted to 1.8 Å and belonged to the orthorhombic P2₁2₁2₁ space group with two molecules per asymmetric unit. The structure was subsequently solved by molecular replacement using the truncated T4 lysozyme (PDB entry 4U15)20 as a search model and refined to R‐work = 21.28% and R‐free = 24.55% (Table 1). We were able to trace the entire polypeptide chain from M1 to L121 [Fig. 1(B)]. Superimposition of the mbT4L and wtT4L proteins revealed that the overall fold of mbT4L originates from the fold of wtT4L with minimal dissimilarities besides truncation of the N‐terminal lobe [Fig. 1(B)].

Table 1.

Data Collection and Refinement Statistics

Dataset	mbT4L/native	mbT4L/anomalous
PDB acc. code	5I14
Data collection and processing
Space group	P 21 21 21	P 21 21 21
Cell dimensions—a, b, c (Å)	60.1, 61.2, 69.8	60.4, 60.9, 68.9
Cell dimensions—α, β, γ (°)	90, 90, 90	90, 90, 90
Diffraction source	BESSY ID 14‐1	BESSY ID 14‐1
Wavelength (Å)	0.9796	1.4859
Resolution range (Å)	46.02–1.75	42.88–2.38
	(1.81–1.75)	(2.47–2.38)
No. of unique reflections	26668 (2498)	10526 (942)
Mean I/σ(I)	22.46 (1.33)	11.24 (2.03)
Completeness (%)	99.45 (95.31)	99.07 (90.84)
Multiplicity	7.1 (6.7)	12.1 (12.0)
Structure solution and refinement
R‐work (%)	21.28 (34.06)	24.31 (31.79)
R‐free (%)	24.55 (32.50)	25.88 (28.87)
No. of non‐H atoms	2069	1909
R.m.s. deviations—bonds (Å)	0.006	0.003
R.m.s. deviations—angles (°)	0.94	0.91
Average B factors (Å2) ‐ protein	36.4	49.4
‐ ligands	34.8	69.0
‐ solvent	43.6	31.5
Ramachandran favored/outliers (%)	99/0	100/0

Statistics for the highest‐resolution shell are shown in parentheses.

The asymmetric unit of the mbT4L/Ni²⁺ crystals contained two molecules of mbT4 lysozyme and four nickel ions [Fig. 1(C)]. Two Ni²⁺ ions formed coordinate covalent bonds with histidines H73, H85, and H89 from the same mbT4L molecule, while the other two Ni²⁺ ions formed coordinate covalent bonds symmetrically with histidines H76 and H80 from one mbT4L molecule and H82 from the other mbT4L molecule from the asymmetric unit, inducing dimerization of mbT4L under conditions within the crystal [Fig. 1(D)].

Next, we analyzed the anomalous scattering from the nickel atoms. We collected a crystallographic dataset at the wavelength of the absorption peak of the nickel atoms (1.4859 Å, Table 1). The anomalous scatterers were clearly located at the positions of the nickel atoms [Fig. 1(D)], however, we were not able to solve the entire mbT4L structure using just the anomalous scattering of the nickel atoms and the single‐wavelength anomalous dispersion (SAD) technique. Nevertheless, we concluded that co‐crystallization of a target mbT4L‐fused protein with nickel ions may facilitate crystallogenesis through formation of the Ni²⁺ ions‐dependent crystal contacts of mbT4L. In addition, the molecular replacement/single‐wavelength anomalous dispersion (MR‐SAD) technique using the mbT4L structure as a search model and the anomalous scattering from the nickel atoms can be applied to solve the crystal structure of this mbT4L‐fused protein.

Metal ions‐binding T4 lysozyme as an intramolecular protein purification tag

Immobilized‐metal affinity chromatography (IMAC) is the most widely used affinity chromatography technique in protein fractionation.24 It is based on the affinity of metal ions such as Co²⁺, Cu²⁺, Ni²⁺, and Zn²⁺ to histidine and cysteine residues and formation of the specific coordinate covalent bonds. The most important application of IMAC is based on a combination of a tetradentate ligand (such as nitrilotriacetic acid, NTA) ensuring strong immobilization of metal ions such as Ni²⁺ or Co²⁺ that leave two coordination sites free for interaction with biopolymers, and oligohistidine extended (His‐tagged) recombinant proteins.

In this section, we investigated utilization of the metal ions‐binding T4 lysozyme as an intramolecular protein purification tag in the IMAC technology, and compared its advantages and drawbacks with the commonly used N‐terminal hexahistidine (His₆) tag. As a model molecule, we choose a chimeric protein composed of the kinase domain of the phosphatidyl inositol 4‐kinase IIalpha (PI4K2A) and truncated T4 lysozyme. PI4K2A is associated with various cellular membranes through its specific cysteine‐rich motif (‐LCCPCCF‐) post‐translantionally modified with palmitoyl moieties.25 For the purpose of biochemical studies and particularly protein crystallography, this palmitoylated segment of PI4K2A provides a perfect target for replacement with T4 lysozyme. Recently, we successfully employed such T4 lysozyme fusion strategy to facilitate crystallization of both human type II PI4‐kinases PI4K2A¹⁵ and its relative PI4K2B.16

Here, we generated a construct composed of the N‐terminal His₆ tag followed by a GB1 folding tag and the kinase domain of PI4K2A with the cysteine‐rich loop replaced by the truncated T4 lysozyme (His‐PI4K2A‐trT4L). Next, we generated another construct lacking the N‐terminal His₆ tag and containing the metal‐ions binding T4 lysozyme instead of the truncated T4 lysozyme mutant (PI4K2A‐mbT4L) [Fig. 2(A)]. A model of the PI4K2A‐mbT4L chimeric protein based on our previously published crystal structure of PI4K2A‐wtT4L¹⁵ and the mbT4L crystal structure presented in this study indicates that the metal ions‐binding site of mbT4L within the PI4K2A‐mbT4L construct can be freely accessed by immobilized metal ions such as NiNTA‐immobilized nickel ions as expected [Fig. 2(B)].

Figure 2.

Metal ions‐binding T4 lysozyme as an intramolecular protein purification tag. (A) Schematic representation of the wild‐type PI4K2A lipid kinase and the chimeric constructs used for further experiments. The N‐lobe and C‐lobe of PI4K2A are colored in gold and aquamarine, respectively; the palmitoylated motif of PI4K2A (replaced by T4L in the chimeric constructs) is depicted in orange. The N‐terminally His₆‐tagged GB1 folding tag (His₆GB1) and the metal ions‐binding T4 lysozyme (mbT4L) are colored in red, while the GB1 protein lacking the N‐terminal His₆‐tag and the truncated T4 lysozyme lacking the to‐histidine mutations (trT4L) are depicted in grey. (B) Model of the PI4K2A‐mbT4L chimeric protein based on our previously published crystal structure of the PI4K2A‐wtT4L chimeric protein (PDB entry 4PLA)15 and the mbT4L crystal structure presented in this study. The protein backbone is shown in cartoon representation. The PI4K2A N‐lobe, C‐lobe, and mbT4 lysozyme are colored in gold, aquamarine, and red, respectively. The metal ions‐binding site of mbT4L is shown in stick representation. (C) Bar graph presents the mean values of the yields of the chimeric proteins from A expressed and purified from E. coli BL21 (see Materials and methods for more detail); error bars are standard errors of the mean based on three independent experiments. (D) Analysis of the affinity of the chimeric proteins from A to immobilized Ni²⁺ ions. Upper panel, elution profiles of the chimeric proteins bound to a HisTrap HP column and eluted with a gradient of imidazole ranging from 0 to 400 mM, monitored by the absorbance at 280 nM. Lower panel, SDS‐PAGE analysis of the collected fractions followed by Coomassie Blue staining.

Both His‐PI4K2A‐trT4L and PI4K2A‐mbT4L constructs expressed well in E. coli with high yields within the range of approximately 10 mg of protein per 1 L of bacterial culture. However, the yields of the PI4K2A‐mbT4L chimeric protein were significantly higher than the yields of the His‐PI4K2A‐trT4L construct [Fig. 2(C)]. Next, we examined the affinity of both His‐PI4K2A‐trT4L and PI4K2A‐mbT4L chimeric proteins to immobilized nickel ions. Both proteins were sequentially bound to a HisTrap HP (Ni²⁺ Sepharose High Performance) column and eluted with a gradient of imidazole (pH 8.0) ranging from 0 to 400 mM. Elution of these proteins was monitored by the absorbance at 280 nM along with the SDS‐PAGE analysis of the collected fractions followed by Coomassie Blue staining [Fig. 2(D)]. The PI4K2A‐mbT4L chimeric protein was eluted at significantly higher concentration of imidazole than the His‐PI4K2A‐trT4L construct, indicating the higher affinity of nickel ions to PI4K2A‐mbT4L compared to the affinity to His‐PI4K2A‐trT4L.

Discussion

Phage T4 lysozyme is a well folded and highly soluble protein which has been used in the fusion protein strategy to facilitate crystallization of various proteins. One of its noteworthy attributes is its tolerance of changes in the amino acid sequence. In this study, we present a structural and biochemical characterization of its novel, metal ions‐binding mutant, and investigated its utilization as an intramolecular protein purification tag in the immobilized‐metal affinity chromatography.

The metal ions‐binding property of this mutant was achieved by introduction of six histidines into the α7 and α8 helices of the “minimal version” of T4 lysozyme described by Thorsen et al.20 Structural characterization of the resulting mutant using protein crystallography confirmed that this metal‐ions binding mutant of T4 lysozyme (mbT4L) still folds well with minimal dissimilarities compared to the wild‐type T4 lysozyme (wtT4L), besides truncation of the N‐terminal lobe. All six introduced histidines are located at the surface of mbT4L within a small region ‐ the Cα atoms of all these residues are located within a region with a radius of approximately 7 Å. Side chains of these residues are solvent accessible and thus capable of binding of metal ions such as Ni²⁺. Within the crystal structure of mbT4L co‐crystallized with Ni²⁺ ions, one nickel ion forms coordinate covalent bonds with three histidines of mbT4L. However, in case of mbT4L bound to NTA‐immobilized Ni²⁺ ions we expect a stoichiometry of one nickel ion per two histidines of mbT4L, given the tetradenticity of the nitrilotriacetic acid leaving two Ni²⁺ coordination sites free for interaction with the mbT4L histidines.

Oligohistidine tags are common tools for purification of recombinant proteins expressed in E. coli or other prokaryotic expression systems by the IMAC technique. In this study, we compared utilization of the commonly used N‐terminal hexahistidine tag with application of an intramolecular fusion of the target protein with the mbT4 lysozyme. Intramolecular mbT4L fusion may provide several advantages compared to the N‐terminal His₆‐tag or compared to the intramolecular wtT4L fusion as follows:

• MbT4L is not affected by degradation of the intrinsically disordered N terminus of the target mbT4L‐fused protein by N‐terminal peptidases. This implies that knowledge of exact domain boundaries for the expression of the target protein in a prokaryotic expression system is not essential. Negligible degradation of the purification tag also contributes to higher yields of the purified protein.

• MbT4L‐fusion improves the solubility of the target protein.

• Target mbT4‐fused protein binds to the immobilized metal ions with stronger affinity than the His₆‐tagged protein, resulting in its higher purity upon purification by the IMAC technique.

• Co‐crystallization of a target mbT4L‐fused protein with metal ions may facilitate crystallogenesis through formation of specific metal ions‐dependent crystal contacts of mbT4L.

• MR‐SAD technique using the mbT4L structure as a search model and the anomalous scattering from the metal atoms can be applied to solve the crystal structure of a target mbT4L‐fused protein.

In case that a high concentration of imidazole (or its basic pH) is disadvantageous for the target protein, the imidazole concentration during elution can be modulated by pH of the elution buffer, salt concentration, addition of EDTA or other chelating agents, etc., similarly as in case of elution of the N‐terminally histidine‐tagged proteins.24, 26

The obvious practical drawback of utilization of the intramolecular mbT4L fusion, similarly to the intramolecular wtT4L fusion, is the right positioning of mbT4L within the target protein, especially in case when the structure of the target protein (or its homologue) is not known and is difficult to predict. In addition, even in case that the lysozyme position appropriate for protein expression and purification is achieved (such as within the palmitoylated loop of type II PI4‐kinases15, 16 or within the third intracellular loop of multiple G protein‐coupled receptors20), generation of tens of mutants with slightly shifted lysozyme positions is usually required to achieve crystallization.

In summary, we designed a novel, metal ions‐binding mutant of phage T4 lysozyme and presented its high resolution crystal structure in complex with Ni²⁺ ions. Our study provides a structural rationale and methodological insights for its utilization as an intramolecular protein purification tag for purification of recombinant proteins by the IMAC technique.

Materials and Methods

Plasmids

Wild‐type phage T4 lysozyme was cloned into NdeI and KpnI restriction sites of the pRSFD vector (Novagen) using routine restriction cloning. Truncated and metal ions‐binding T4L mutants were generated using the Phusion Site‐Directed Mutagenesis Kit (Thermo Scientific). Human PI4K2A residues 76–467 with a palmitoylation motif replaced by wtT4L was cloned into pRSFD vector with an N‐terminal His₆‐tag followed by a GB1 folding tag as described previously15 and further mutated using the Phusion Site‐Directed Mutagenesis Kit. All DNA constructs were verified by sequencing.

Protein expression and purification

Metal ions‐binding T4 lysozyme and PI4K2A‐T4L chimeric proteins were expressed in E. coli BL21 Star cells using the ZY5052 autoinduction medium. The bacterial cells were grown at 37°C to OD600 of 0.7 and then cultivated at 18°C for another 20 h. Then, they were harvested by centrifugation and lysed in the lysis buffer (50 mM Tris, 300 mM NaCl, 3 mM β‐mercaptoethanol, 30 mM imidazole, 10% glycerol, pH 8.0). The lysate was incubated with the Ni‐NTA resin (Macherey‐Nagel) and then extensively washed with the lysis buffer. The protein was eluted with the lysis buffer supplemented with 300 mM imidazole (pH 8.0). The proteins were further purified using the size exclusion chromatography at Superdex 75 or Superdex 200 columns (GE Healthcare) in the SEC buffer (10 mM Tris, 200 mM NaCl, 3 mM β‐ME, pH 8.0). The molecular weight and purity of all proteins was verified by SDS‐PAGE and Matrix‐Assisted Laser Desorption/Ionisation (MALDI). Purified proteins were concentrated, aliquoted, flash frozen in the liquid nitrogen, and stored at −80°C until needed.

Crystallization and data collection

Crystals were grown at 20°C in a sitting drop consisting of a 1:1 mixture of the protein supplemented with 2mM NiCl₂ and a well solution (20% PEG 6000, 100 mM MES pH 5). The crystals were cryoprotected in the well solution supplemented with 20% glycerol, flash frozen in the liquid nitrogen, and analyzed. Measurements were carried out at the synchrotron BESSY II (MX14.1 beamline) at Helmholtz‐Zentrum Berlin.27 The crystallographic datasets were collected from single frozen crystals.

Structure determination and refinement

The crystallographic data were integrated and scaled using XDS.28 The structure of the metal ions‐binding T4 lysozyme was solved by molecular replacement using the truncated T4 lysozyme (PDB entry 4U15)²⁰ as a search model. The initial MR model was obtained with Phaser 29 from the Phenix package.30 The model was further improved using automatic model refinement with Phenix.refine 31 and manual model building with Coot.32 Structural figures were generated with PyMol.33

Data Deposition

Coordinates and structure factors of the metal ions‐binding T4 lysozyme presented in this study have been submitted to the Protein Data Bank (www.pdb.org) and assigned the identifier 5I14.

Conflict of Interest

The authors declare no conflict of interest.