Crystal structure of TTHA1429, a novel metallo-β-lactamase superfamily protein from Thermus thermophilus HB8

INTRODUCTION

TTHA1429 is a metallo-β-lactamase superfamily protein from an extremely thermophilic bacterium, Thermus thermophilus HB8. The metallo-β-lactamases, first identified as class B β-lactamases, possess an αββα-fold and a di-metal binding site.1, 2 Other enzymes with this fold include glyoxalase II, rubredoxin oxygen:oxidoreductase, phosphorylcholine esterase, and tRNA maturase. A BLAST search revealed that homologues of TTHA1429 are present in a wide range of bacteria and archaea. Therefore, although its function remains unknown, TTHA1429 may be an essential gene for prokaryotes. To analyze the structural properties of TTHA1429, we have determined the 2.1-Å crystal structure of TTHA1429. TTHA1429 exhibits a unique putative substrate binding pocket with a glyoxalase II-type metal coordination.3

MATERIALS AND METHODS

Cloning, expression, and purification

The gene of TTHA1429 from T. thermophilus HB8 (gi: 55981398) was amplified by PCR using T. thermophilus HB8 genomic DNA as template. The PCR primers were 5′-GGAATTCCATATGAAGGCCCTCCTGC-3′ (including an Nde I site) and 5′-GAAGATCTTATTAGCGCCGGAAGTACC-3′ (including a Bgl II site). Thirty cycles of PCR were performed using KOD-plus (Toyobo, Japan) with the melting phase at 94°C for 30 s, annealing phase at 53°C for 30 s, and polymerization phase at 68°C for 60 s. The PCR product and the vector plasmid pET-11a(+) (Novagen) were digested by Nde I and Bgl II, and ligated with Ligation High (Toyobo). The DNA sequence of the TTHA1429-encoding region of the resulting plasmid was verified. The protein without any tag was overexpressed in E. coli BL21(DE3) (Novagen). Harvested cells were resuspended in 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl, and then disrupted by sonication. The lysate was centrifuged at 40,000g at 4°C for 30 min, and the resulting supernatant was subjected to a heat treatment at 70°C for 10 min. The resulting supernatant was subjected to ammonium sulfate fractionation with a final concentration of 1.5M (NH₄)₂SO₄. The resulting supernatant contained TTHA1429, which was purified by four steps of column chromatography: (1) Resource ISO (GE Healthcare Bioscience) with a linear gradient elution of 1.5–0M (NH₄)₂SO₄ in 50 mM sodium phosphate buffer (pH 7.0); (2) Resource Q (GE Healthcare Bioscience) with a linear gradient elution of 0–500 mM NaCl in 20 mM Tris-HCl buffer (pH 8.0); (3) hydroxyapatite CHT10 (Bio-Rad) with a linear gradient elution of 10–100 mM sodium phosphate (pH 8.0); (4) HiLoad 16/60 Superdex 75 pg (GE Healthcare Bioscience) with an isocratic elution of 20 mM Tris-HCl (pH 8.0) and 0.15M NaCl. The purified TTHA1429 was desalted with a HiPrep 26/10 Desalting column (GE Healthcare Bioscience) equilibrated with 20 mM Tris-HCl (pH 8.0). The purified protein showed a single band with a molecular weight of 35 K in SDS-PAGE, which was consistent with the predicted molecular weight of the protein (35.4 K).

Crystallization and data collection

The sitting drop vapor diffusion method was used for crystallization. Two microliters of purified TTHA1429 solution (13.7 mg/mL) and 2 μL of reservoir solution were mixed to prepare a crystallization drop, and the drop was equilibrated against 100 μL of reservoir solution. Crystals of TTHA1429 were grown at 293 K with the reservoir solution of 100 mM Bicine-NaOH buffer (pH 8.0), 25% (w/v) PEG 4000 (Hampton Research), 5% (v/v) 2-propanol, and 5 mM zinc acetate. The crystals grew to a final dimension of 0.07 × 0.07 × 0.4 mm³ within 4 days. The crystals were picked up with mounting loops and frozen directly in liquid nitrogen without adding any cryoprotectants. X-ray diffraction data of TTHA1429 crystals were obtained at beamline BL26B2 at SPring-8 (Harima, Japan). The diffraction data were obtained to 2.1-Å resolution at three wavelengths determined from an X-ray fluorescence spectrum—the zinc peak (1.2822 Å), edge (1.2829 Å), and remote (1.0000 Å)—for the zinc multiple-wavelength anomalous dispersion (MAD) method. The crystals belonged to the space group P2₁2₁2₁ with unit cell dimensions of a = 55.3 Å, b = 62.9 Å, and c = 87.5 Å.

Structure solution and refinement

The diffraction data were processed with the program package HKL20004 and the CCP4 suite.5 Zinc sites were determined with the program SOLVE6 using the MAD datasets. The resulting phases were improved with the programs RESOLVE6 and ARP/wARP.7 Refinement of the initial model against the remote data set was performed using CNS,8 Coot,9 and Refmac5.10 The stereochemistry of the structure was checked by the program PROCHECK.11

Oligomeric state analysis

The purified TTHA1429 was loaded onto a Superdex 75 HR 10/30 (GE Healthcare Bioscience) column pre-equilibrated with 50 mM Tris-HCl buffer (pH 8.0) and 150 mM NaCl, and eluted with the same buffer at a flow rate of 0.5 mL/min at room temperature. The molecular weight of TTHA1429 was estimated by comparing its elution volume with those of standard proteins: transferrin (MW 81 K), ovalbumin (43 K), myoglobin (17.6 K), ribonuclease A (13.7 K), and aprotinin (6.5 K).

RESULTS AND DISCUSSION

The crystal structure of TTHA1429 in the zinc-bound form was determined at 2.1-Å resolution by the zinc MAD method. The refined structure includes a monomer of TTHA1429 (residues 3–124, 132–277, and 285–317 out of 317 residues of the protein), 281 water molecules, and two Zn²⁺ ions in the asymmetric unit. No electron density was observed for the remaining residues. The Matthews coefficient (V_m)12 was 2.14 ų/Da, and the estimated solvent content was 42.5%. In the Ramachandran plot, 92.4, 6.4, 0.4, and 0.8% of the residues were located in the most favored regions, additionally allowed regions, generously allowed regions, and disallowed regions, respectively. Two residues, Lys20 and Asp37, were in the disallowed regions of the Ramachandran plot. Asp37 corresponds to the buried Asp residues of metallo-β-lactamase superfamily proteins which lie in the disallowed regions of the Ramachandran plot. Data collection and refinement statistics are summarized in Table I. The gel filtration chromatogram of TTHA1429 confirmed that it is monomeric in solution. The TTHA1429 monomer contains 14 β-strands, six α-helices, and two 3₁₀-helices [Fig. 1(A)]. TTHA1429 has a ββ sandwich with helices on each external face, as in other metallo-β-lactamase superfamily proteins. The di-metal binding site is located on one edge of the ββ sandwich, with two zinc ions (Zn1 and Zn2) located 3.3-Å apart from each other [Fig. 1(B)]. The residues involved in the metal coordination are identical to those in glyoxalase IIs (Table II). His70, His72, His171, and a water molecule participate in the tetrahedral coordination for Zn1, while Asp74, His75, Asp190, His233, and a water molecule participate in the distorted trigonal bipyramidal coordination for Zn2. Unlike glyoxalase IIs, Asp190 does not bridge two zinc ions [Fig. 1(B)].

Figure 1. A: Overall structure of TTHA1429. The N- and C-termini are labeled as N and C, respectively. β-strands (β1–14), α-helices (α1–6), and 3₁₀-helices (η1–2) are shown in yellow, red, and blue, respectively. Two zinc ions in the putative active site are presented with gray spheres. B: The putative active site of TTHA1429. Zn1 and Zn2 are located 3.3-Å apart from each other. Orange lines show the tetrahedral coordination to Zn1 and the distorted trigonal bipyramidal coordination to Zn2. Wat1 stands for the coordinated water molecule. Note that Asp191 is not bridging two zinc ions (gray dotted line). Distances between two atoms in angstroms are labeled on the dotted line. C: Superposed diagram of TTHA1429 (shown in pink), 1A7T (green), and 1SML (blue). The unique region of TTHA1429 is highlighted in magenta. D: Cartoon diagrams of TTHA1429 (left), 1A7T (center), and 1SML (right). The unique region of TTHA1429 is highlighted in magenta. E: Surface diagram of TTHA1429. The unique regions of TTHA1429 are highlighted in magenta. This figure was drawn with PyMOL.13

Table I. Summary of Data Collection and Refinement Statistics

	Zn edge	Zn peak	Zn remote
Values in parentheses are for the highest resolution shell. a Rmerge = Σhkl [(Σi |Ii – 〈I〉|)/Σi |Ii|]. b Rfactor = (Σhkl ‖Fo| − |Fc‖)/Σhkl |Fo|. Rfree was calculated with 5% of the data excluded from refinement.
Data collection
Wavelength (Å)	1.2829	1.2822	1.0000
Resolution range (Å)		50.00–2.10 (2.18–2.10)
Number of observed reflections	127,275	127,042	128,963
Number of unique reflections	18,471	18,434	18,442
Data completeness (%)	99.2 (97.1)	99.2 (97.2)	99.7 (99.7)
Rmergea	0.048 (0.124)	0.050 (0.104)	0.036 (0.077)
〈I〉/〈σ(I)〉	11.4 (5.7)	10.7 (6.4)	14.1 (11.0)
Space group		P212121
Unit cell parameters		a = 55.3 Å, b = 62.9 Å, c = 87.5 Å
Refinement
Resolution range used for refinement (Å)		20.0–2.1
Rfactor (%)b		19.8
Rfree (%)b		25.7
Number of reflections used for refinement		17,356
Protein residues modeled		301 of 317
Number of protein atoms modeled		2,399
Number of water molecules modeled		281
Mean overall B value (Å^2)		17.5
RMSD bond angle (°)		1.034
RMSD bond length (Å)		0.006
Ramachandran plot
Residues in most favored regions (%)		92.4
Residues in additionally allowed regions (%)		6.4
Residues in generously allowed regions (%)		0.4
Residues in disallowed regions (%)		0.8

Table II. Metal Binding Sites of Metallo β-Lactamase Superfamily Proteins

Classification	Source	Protein name	Residues involved in coordination
Undefined	T. thermophilus	TTHA1429	Site 1: H70, H72, H171
			Site 2: D74, H75, D190, H233
Glyoxalase II	Human	GLX2-2	Site 1: H54, H56, H110
			Site 2: D58, H59, D134, H173
	Arabidopsis thaliana	GLX2-2	Site 1: H54, H56, H110
			Site 2: D58, H59, D133, H172
		GLX2-5	Site 1: H54, H56, H112
			Site 2: D58, H59, D131, H169
	Trypanosoma brucei	GLX2	Site 1: H71, H73, H131
			Site 2: D75, H76, D156, H211
	Salmonella typhimurium	GLX2	Site 1: H53, H55, H110
			Site 2: D57, H58, D127, H165
MBL subclass B1	Bacillus cereus	BcII	Site 1: H116, H118, H196
			Site 2: D120, C221, H263
MBL subclass B2	Aeromonas hydrophila	CphA	Site 1: N116, H118, H196
			Site 2: D120, C221, H263
MBL subclass B3	Stenotrophomonas maltophilia	Fez-1	Site 1: H116, H118, H196
			Site 2: D120, H121, H263
Rubredoxine oxygen:oxidoreductase	Desulfovibrio gigas	Roo	Site 1: H79, E81, H146, D165
			Site 2: D83, D165, H226
Summerization of DALI results
1A7T	Bacteroides fragilis	CfiA (Metallo-β-lactamase)	Site 1: H82, H84, H145
			Site 2: D86, C164, H206
1SML	Stenotrophomonas maltophilia	L1 (Metallo-β-lactamase)	Site 1: H84, H86, H160
			Site 2: D88, H89, H225

A structural similarity search was performed with the atomic coordinates of TTHA1429 using the DALI server,14 and the top five hits were Desulfovibrio gigas rubredoxin oxygen:oxidoreductase (PDB code: 1E5D),15Bacterioides fragilis metallo-β-lactamase (PDB code: 1A7T),16Xanthomonas maltophilia metallo-β-lactamase (PDB code: 1SML),17Thermotoga maritima flavoprotein (PDB code: 1VME), and Pseudomonas aeruginosa metallo-β-lactamase (PDB code; 2FHX).18 The superimpositions of TTHA1429 with 1A7T and 1SML, di-zinc containing structures among these structurally similar enzymes, were performed by DaliLite.19 Features of 1A7T and 1SML are summarized in Table II. The DALI output reveals that the αββα-folds of the metallo-β-lactamase superfamily proteins are similar, but α5, α6, α9, α10, β12, and β13 do not fit with the corresponding elements of other proteins [Fig. 1(C,D)]. The region unique to TTHA1429 forms a putative substrate binding pocket with the metal ions at the bottom [Fig. 1(E)]. Residues 125–131 and 278–284, whose electron densities are not observed, are located at the entrance of the pocket.

The present results reveal that TTHA1429 exhibits a unique putative substrate binding pocket with a glyoxalase II-type metal coordination mode. The atomic coordinates and structural factors have been deposited in the Protein Data Bank with the PDB code 2ZO4.

Acknowledgements

This work was performed under the Structural-Biological Whole Cell Project led by Dr. Seiki Kuramitsu at the RIKEN SPring-8 Center. The synchrotron-radiation experiments were performed at BL26B2 at SPring-8 (Harima, Japan).