Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing

The crystal structure of bacteriophage T4 Spackle as determined by sulfur SAD phasing reveals a monomeric protein with a helical bundle fold and a disc-like overall shape. Spackle has an intramolecular disulfide bond and a bipolar surface-charge distribution, which are consistent with its proposed role as a lysozyme inhibitor that functions in the Escherichia coli periplasm.

Abstract

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

1. Introduction  

The product of bacteriophage T4 early gene 61.3, known as Spackle, confers the infected host bacteria with immunity against secondary phage infections (Cornett, 1974 ▸; Lu & Henning, 1994 ▸; Obringer, 1988 ▸). The available genetic data suggest that Spackle achieves superinfection exclusion by localizing to the periplasm and inhibiting the activity of tail lysozyme, which is a key component of the baseplate of the phage tail, thereby preventing penetration by the tail tube of incoming phages (Emrich, 1968 ▸; Emrich & Streisinger, 1968 ▸; Kao & McClain, 1980a ▸,b ▸). Although the phenomenon of genetic exclusion in bacteriophages has been known for more than half a century, Spackle has not been studied biochemically or structurally. In this study, we have characterized the basic biophysical properties of purified T4 Spackle and determined its crystal structure in order to help to better understand this unique viral protein.

2. Materials and methods  

2.1. Protein expression and purification  

T4 gene 61.3 encoding the Spackle protein was amplified from bacteriophage T4 (ATCC) by polymerase chain reaction using the forward primer 5′-CGGAATTCCATATGaaaaaattcatctttgctacaatttttgctttag-3′ and reverse primer 5′-CACCGATTCTCGAGttcacctaccacttcagcgatgatatttttgttattaaag-3′ (upper case, overhang; lower case, coding region). The amplified gene was inserted into pET-24a expression vector between the NdeI and XhoI sites, so that the expressed full-length protein has a 6×His tag (LEHHHHHH) at the C-terminus. The protein was overexpressed in Escherichia coli strain BL21(DE3) by induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside and was purified from the cleared total lysate using nickel-affinity chromatography and Superdex 75 size-exclusion chromatography (SEC). The eluted protein in 20 mM Tris–HCl pH 7.4, 0.5 M NaCl was concentrated by ultrafiltration, flash-frozen in liquid nitrogen and stored at −80°C.

2.2. Crystallization and structure determination  

Purified T4 Spackle concentrated to 26 mg ml⁻¹, as determined based on UV absorption and the theoretical extinction coefficient calculated from the amino-acid sequence, was subjected to crystallization screening. Initial broad crystallization screening was performed in sitting-drop vapor-diffusion mode on a Crystal Phoenix (Art Robbins) with 0.1 µl protein solution and 0.1 µl reservoir solution. Crystals were obtained in condition F4 of the SaltRx screen (Hampton Research), which consists of 2.5 M ammonium sulfate, 0.1 M sodium acetate buffer pH 4.6. The crystals grew as clusters of thin rods. Optimizing the crystallization condition, in particular the inclusion of 2.5–5% glycerol, allowed the growth of single crystals with improved morphology. Larger crystals suitable for data collection were obtained in hanging drops by mixing 1.0 µl protein solution and 1.0 µl reservoir solution. The optimized well condition consisted of 2.5 M ammonium sulfate, 0.05 M sodium acetate buffer pH 4.5, 4%(v/v) glycerol. Crystals were cryoprotected by transferring them to reservoir solution supplemented with 1.0 M Tacsimate pH 5.0, mounted on nylon loops (Hampton Research, Aliso Viejo, California, USA) or MicroMeshes (MiTeGen, Ithaca, New York, USA) and flash-cooled in liquid nitrogen. X-ray diffraction data were collected on the Northeastern Collaborative Access Team (NE-CAT) beamlines of the Advanced Photon Source (APS). The data were processed using XDS (Kabsch, 2010 ▸). The initial crystals suffered from high mosaicity in one direction, and therefore sulfur SAD phasing was unsuccessful. After screening more crystals, one batch of crystals were found to have better diffraction spots. A SAD data set of 3600 frames (720°) was collected to 1.69 Å resolution on beamline 24-ID-C using an X-ray wavelength of 1.653 Å (7000 eV). The data set was examined and truncated to 3300 frames (660°; the first 300 frames were discarded). The sulfur substructure was solved by AutoSol (Terwilliger et al., 2009 ▸) at a resolution cutoff of 2.2 Å with 12 S atoms located, which gave a figure of merit of phasing of 0.36 before density modification. AutoSol produced a readily interpretable electron-density map (Fig. 1 ▸ d). Further density modification and automated model building by AutoBuild (Terwilliger et al., 2008 ▸) gave a complete model. The final model was refined against a higher resolution data set collected at a wavelength of 0.979 Å. A summary of X-ray data-collection, phasing and model-refinement statistics is shown in Table 1 ▸. The structure figures were generated using PyMOL (https://pymol.org/2/).

Figure 1.

Structure determination of Spackle by sulfur SAD phasing. (a) Coomassie Brilliant Blue-stained SDS–PAGE analysis of purified T4 Spackle, with size standards on the left. (b) Crystals obtained in the optimized condition. (c) Anomalous difference Fourier map contoured at 4.0σ (red mesh) overlaid on the C^α trace of the refined model of a Spackle monomer in magenta. The methionine and cysteine side chains are shown as blue sticks, with S atoms in yellow. (d) Experimental electron-density map from AutoSol contoured at 1.1σ shown as a blue mesh and overlaid with the refined model shown as orange sticks. The orientation is the same as in (c).

Table 1. Data-collection and refinement statistics.

Values in parentheses are for the highest resolution shell.

	Native Spackle (refinement)	Native Spackle (phasing)
Data collection
Wavelength (Å)	0.979	1.653
Resolution range (Å)	40.1–1.52 (1.57–1.52)	39.7–1.69 (1.75–1.69)
Space group	I222	I222
a, b, c (Å)	44.42, 79.82, 93.23	43.85, 79.67, 93.32
Total reflections	240190 (22989)	388377 (19478)
Unique reflections	25903 (2499)	17774 (1379)
Multiplicity	9.3 (9.1)	21.9 (14.1)
Completeness (%)	97.88 (98.38)	94.95 (75.26)
〈I/σ(I)〉	31.82 (1.89)	32.61 (9.76)
R merge (%)	4.28 (109)	6.23 (22.8)
R meas (%)	4.54 (115)	6.37 (23.7)
R p.i.m. (%)	1.47 (37.1)	1.32 (6.08)
CC1/2	0.999 (0.891)	0.999 (0.983)
Phasing
Sites located (S atoms)		12
FOM		0.37
Refinement
No. of reflections	25396 (2497)
No. of reflections for R free	1243 (112)
R work (%)	15.29 (31.65)
R free (%)	19.25 (36.30)
No. of non-H atoms
Total	1353
Macromolecules	1221
Ligands	4
Solvent	128
No. of protein residues	150
R.m.s.d., bond lengths (Å)	0.004
R.m.s.d., bond angles (°)	0.99
Ramachandran plot
Favored (%)	100.0
Allowed (%)	0.00
Outliers (%)	0.00
MolProbity score	0.84
Clashscore	1.25
Average B factor (Å2)
Overall	37.74
Macromolecules	36.77
Ligands	83.41
Solvent	45.60

2.3. Mass spectrometry  

Prior to matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, 50 pmol of the sample was desalted using a C4 ZipTip (Millipore Sigma, Billerica, Massachusetts, USA). The manufacturer’s standard protocol for protein desalting was followed. After elution from the ZipTip, 1 µl of the sample (10 pmol) was spotted onto a MALDI-TOF 384-spot stainless-steel target (Bruker Daltonics, Billerica, Massachusetts, USA) and mixed with 1 µl sinapinic acid matrix (15 mg ml⁻¹ sinapinic acid in 50% acetonitrile). The spot was allowed to dry at room temperature. The target was placed in a Bruker Autoflex Speed MALDI-TOF mass spectrometer (Bruker Daltonics) equipped with an Nd:YAG 355 nm pulsed laser. The data were collected in linear mode, positive polarity, with an accelerating potential of 19.5 kV. External calibration was performed using 0.5 µl sinapinic acid and 0.5 µl Protein Standard I (catalog No. 8206355, Bruker Daltonics). The Bruker data file was converted to an mzXML file using the MSConvertGUI tool in the ProteoWizard open-source software.

2.4. Size-exclusion chromatography with multi-angle light scattering (SEC-MALS)  

The light-scattering data were collected using Superdex 75 and Superdex Peptide 10/300 size-exclusion chromatography (SEC) columns in tandem (GE Healthcare, Piscataway, New Jersey, USA) connected to an Agilent 1200 high-performance liquid-chromatography (HPLC) system (Agilent Technologies, Wilmington, Delaware, USA) equipped with an autosampler. The elution from SEC was monitored by a photodiode array (PDA) UV–Vis detector (Agilent Technologies), a differential refractometer (OPTI-Lab rEx; Wyatt, Santa Barbara, California, USA) and a static and dynamic multi-angle laser light-scattering (LS) detector (HELEOS II with QELS capability; Wyatt). The SEC-UV/LS/RI system was equilibrated with buffer (20 mM Tris–HCl pH 7.4, 0.5 M NaCl) at a flow rate of 0.5 ml min⁻¹. Two software packages were used for data collection and analysis: the ChemStation software (Agilent Technologies) controlled the HPLC operation and data collection from the multi-wavelength UV–Vis detector, while the ASTRA software (Wyatt) collected the data from the refractive-index detector and the light-scattering detector and recorded the UV trace at 280 nm sent from the PDA detector. The weight average molecular masses, MW, were determined across the entire elution profile at intervals of 1 s from static LS measurements using ASTRA as described previously (Folta-Stogniew & Williams, 1999 ▸). During the data analysis a refractive-index increment (dn/dc) value of 0.188 ml g⁻¹ was used as it proved to be satisfactory during analyses of the protein standards BSA (66 kDa) and cytochrome c (12.3 kDa) under the same conditions (Supplementary Fig. S1).

3. Results  

3.1. Mature Spackle is free from the signal peptide  

T4 gene 61.3 encodes a protein of 97 amino acids, including an N-terminal 22-residue signal peptide that shows significant homology to the periplasmic pectate lyase B (PelB) leader peptide (Kai et al., 1999 ▸). We recombinantly expressed full-length T4 Spackle with an additional C-terminal affinity tag in E. coli and purified it from the total soluble lysate using nickel-affinity chromatography and Superdex 75 SEC. Purified T4 Spackle appeared as a single band with an apparent mass of ∼10 kDa on Coomassie Blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE; Fig. 1 ▸ a) and eluted as a single peak in SEC. An analysis by MALDI-TOF MS showed the mass of the purified T4 Spackle to be 9742 Da (Supplementary Fig. S2), closely matching the theoretical mass of 9742.9 Da including the additional C-terminal His-tag sequence (LEHHHHHH) after processing into the mature form by a signal peptidase-mediated cleavage between Ala22 and Gly23 and losing two H atoms to form a disulfide bond.

3.2. Structure determination by native SAD phasing  

Purified T4 Spackle was crystallized by the hanging-drop vapor-diffusion method in an optimized condition containing ammonium sulfate as the precipitant and sodium acetate buffer pH 4.5 (Fig. 1 ▸ b). The structure was determined by the native SAD phasing approach using sulfur anomalous signal. The mature 83-residue (including the His tag) Spackle is naturally rich in sulfur, containing four methionine and two cysteine residues in ∼9.74 kDa. A SAD data set was collected to 1.69 Å resolution at an X-ray wavelength of 1.653 Å (7500 eV). The sulfur substructure was solved and the protein phases were calculated in Phenix (Liebschner et al., 2019 ▸), generating a readily interpretable electron-density map (Figs. 1 ▸ c and 1 ▸ d). The final model was refined against a 1.52 Å resolution data set collected at an X-ray wavelength of 0.979 Å to an R _work and R _free of 15.3% and 19.3%, respectively. There are two Spackle monomers in the asymmetric unit of the crystal, and well defined electron density was observed for Gly23–Val95 and Gly23–Glu97 in chains A and B, respectively.

3.3. Structure of T4 Spackle  

The refined crystal structure shows that the T4 Spackle monomer consists of a bundle of five α-helices, having a relatively flat, disc- or slab-like overall shape with an average thickness of ∼15 Å (Figs. 2 ▸ a and 2 ▸ b). All five helices run parallel to the flat face of the protein. The short α4 and α5 helices are both capped at their N-terminus by a turn of 3₁₀-helix and show a smooth bending away from each other (diverging in the middle). Hydrophobic residues, including Tyr75, Val78, Leu79, Phe85, Ile90 and Val94, occupy the space between these helices. All helices contribute to the formation of the hydrophobic core, which is centered around Ile44 and Ile48 from the longest α-helix, α2 (Fig. 2 ▸ b). The protein fold is further stabilized by a disulfide bond formed between Cys29 from the α1 helix and Cys81 immediately following the α4 helix, consistent with the presumed function of Spackle in the periplasmic space of E. coli. The surface electrostatic potential of Spackle exhibits a strikingly bipolar charge distribution (Figs. 2 ▸ c–2 ▸ f). A cluster of positively charged residues (Lys26, Lys53, Arg54, Arg56, Lys60, Lys65, Lys68 and Lys88) form a highly basic strip on one side of the protein. The rest of the protein surface is highly acidic and is rich in negatively charged Asp and Glu residues.

Figure 2.

Crystal structure of T4 Spackle. (a) The overall structure of the Spackle monomer shown as a ribbon model colored in a gradient of blue to red from the N-terminus to the C-terminus. (b) A view after an ∼60° rotation about a horizontal axis from that in (a), highlighting the hydrophobic residues in the interior of the protein. (c) Ribbon model with the side chains of positively (Arg and Lys) or negatively (Glu and Asp) charged amino-acid residues shown as sticks. (d) Surface electrostatic potential (red, −4 kT e⁻¹, to blue, +4 kT e⁻¹) of the Spackle monomer oriented as in (c). (e, f) Views after a 180° rotation about a vertical axis from the views in (d) and (c), respectively.

The two Spackle molecules in the asymmetric unit have essentially identical structures, with a root-mean-square deviation of 0.3 Å for the main chain of 73 residues. A search using the DALI server (Holm & Laakso, 2016 ▸) for structurally similar proteins in the Protein Data Bank identified other helical bundle proteins, including a domain from the bacterial SecA ATPase (PDB entry 2ipc, chain A; Z-score = 5.1; Vassylyev et al., 2006 ▸) and the human death domain-associated protein DAXX (PDB entry 5y6o, chain D; Z-score = 4.1; Hoelper et al., 2017 ▸) (Supplementary Table S1 and Fig. S3). The functional significance of these structural similarities is unclear.

3.4. Spackle is a monomer in solution  

In the crystal lattice, two Spackle molecules in the asymmetric unit form a head-to-head dimer related by pseudo-twofold rotational symmetry (Supplementary Fig. S4). To test the relevance of this dimerization observed in crystallo, we determined the absolute mass of purified T4 Spackle in solution by SEC–multi-angle light scattering (SEC-MALS) analysis. The obtained average mass for the single, symmetrical SEC peak upon injection of purified Spackle at 6.7 mg ml⁻¹ was 1.000 × 10⁴ Da (±0.516%), corresponding to the monomeric form of mature Spackle (Fig. 3 ▸). Thus, the functional unit of Spackle in solution is likely to be a monomer.

Figure 3.

SEC-MALS analysis of purified Spackle. Solid line: UV absorbance at 280 nm in arbitrary units. Dots: weight-average molecular masses (MW) measured across the eluting peak.

4. Discussion  

The proposed biochemical activity of T4 Spackle is to inhibit the glycosyl hydrolase activity of the baseplate-associated T4 tail (gp5) lysozyme for its role in superinfection exclusion. Notably, the helical bundle structure of Spackle determined in this study shows no structural homology to any of the previously structurally characterized lysozyme inhibitors, which are β-strand-rich proteins (Abergel et al., 2007 ▸; Calle­waert et al., 2012 ▸; Leysen et al., 2013 ▸, 2015 ▸; Um et al., 2013 ▸; Yum et al., 2009 ▸). Thus, it will be interesting to examine whether Spackle indeed functions as a lysozyme inhibitor and, if it does, to determine its mode of inhibition through structural analysis. Based on the observed bipolar surface-charge distribution of Spackle and its proposed function in the periplasm of E. coli, it is tempting to speculate that the basic patch of Spackle interacts with the phospholipid head group of the plasma membrane, while its acidic surface interacts with lysozyme proteins, which are highly basic overall (Supplementary Fig. S5). In addition, the lysis-inhibition (LIN) defect caused by the original S12 frame-shift mutation in the T4 Spackle gene, which leads to the replacement of nine amino acids at the C-terminus with an unrelated sequence (Abedon, 1999 ▸, 2019 ▸; Emrich, 1968 ▸; Kai et al., 1999 ▸), may suggest that the last α-helix (α5) of Spackle is important for lysozyme interaction. Phage T4 has a cytoplasmic soluble (gpe) lysozyme that is responsible for degrading the cell wall of the host to release progeny particles. The two (gpe versus gp5) lysozymes share 43% sequence identity and have very similar tertiary structures (Kanamaru et al., 2002 ▸; Matthews & Remington, 1974 ▸). Thus, it will also be interesting to study whether Spackle inhibits gp5 lysozyme selectively or whether it inhibits both T4 lysozymes or perhaps even lysozymes from other organisms.

Supplementary Material

PDB reference: bacteriophage T4 Spackle, 6x6o

Funding Statement

This work was funded by National Institutes of Health, National Institute of General Medical Sciences grant R35-GM118047 to Hideki Aihara.