Elucidating the pH-Dependent Structural Transition of T7

Article pubs.acs.org/biochemistry

Elucidating the pH-Dependent Structural Transition of T7 Bacteriophage Endolysin Meenakshi Sharma,† Dinesh Kumar,§ and Krishna Mohan Poluri*,†,‡ †

Department of Biotechnology and ‡Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India § Centre of Biomedical Research, SGPGIMS, Lucknow 226014, Uttar Pradesh, India S Supporting Information *

ABSTRACT: Bacteriophages are the most abundant and diverse biological entities on earth. Bacteriophage endolysins are unique peptidoglycan hydrolases and have huge potential as effective enzybiotics in various infectious models. T7 bacteriophage endolysin (T7L), also known as N-acetylmuramoyl-L-alanine amidase or T7 lysozyme, is a 17 kDa protein that lyses a range of Gram-negative bacteria by hydrolyzing the amide bond between N-acetylmuramoyl residues and the Lalanine of the peptidoglycan layer. Although the activity profiles of several of the T7 family members have been known for many years, the molecular basis for their pH-dependent differential activity is not clear. In this study, we explored the pH-induced structural, stability, and activity characteristics of T7L by applying a variety of biophysical techniques and protein nuclear magnetic resonance (NMR) spectroscopy. Our studies established a reversible structural transition of T7L below pH 6 and the formation of a partially denatured conformation at pH 3. This low-pH conformation is thermally stable and exposed its hydrophobic pockets. Further, NMR relaxation measurements and structural analysis unraveled that T7L is highly dynamic in its native state and a network of His residues are responsible for the observed pH-dependent conformational dynamics and transitions. As bacteriophage chimeric and engineered endolysins are being developed as novel therapeutics against multiple drug resistance pathogens, we believe that our results are of great help in designing these entities as broadband antimicrobial and/or antibacterial agents.

B

glyacan hydrolase/enzymatic catalytic domain (ECD) at the Nterminus and a cell wall binding domain (CBD) located at the Cterminus.17,18 In contrast, a majority of endolysins infecting Gram-negative bacteria generally represent 15−20 kDa singledomain globular proteins consisting of a single catalytic domain.10 Endolysins such as T7L, T4L, λ phage lysozyme, K11, KP32, etc., are the major representatives of the singledomain family.19−22 The CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain is also often found in association with the EBD and CBD in several endolysins, thus contributing to multiple peptidoglycan hydrolase activities in a single polypeptide chain.23,24 Among several endolysins, amidases (also called N-acetylmuramoyl-L-alanine amidases) are known for their rapid lysis activity caused by destabilization of the peptidoglycan by separating the glycan polymer from the stem peptide.2 They serve as specific amidohydrolases that cleave a critical amide bond between the glycan moiety (MurNAc) and the peptide moiety (L-alanine) of the peptidoglycan.2 Bacteriophages such as T7, T3, K11,

acteriophages are obligate intracellular parasites that lack an independent metabolism and require a bacterial host to replicate themselves. They make use of some or all the host biosynthetic machinery to propagate and cause the complete lysis of susceptible host bacteria for their maturation. Just before the phage release, phage-encoded endolysin accumulates in the host cell alone or with holins (small hydrophobic membranespanning proteins) as dictated by the specificity and nature of the bacterial strain to gain access to the peptidoglycan layer.1,2 Endolysins degrade the cell membrane until the cell can no longer withstand the internal osmotic pressure, thus triggering the cell burst and release of mature phage particles.3,4 Endolysins are regarded as potential antibacterial agents in food science (biopreservatives),5,6 agriculture (against phytopathogenic bacteria),2,7 biotechnology and therapeutics (protein antibiotics or enzybiotics)8−13 removal of vaginal and oropharyngeal bacterial colonization,14 and biocontrol (antibiofilm therapy and disinfectants).15,16 Endolysins have been categorized into four classes: glycosidases (muramidase), endopeptidases, amidohydrolases (amidase), and lytic transglycosylases.2 Endolysins that infect Grampositive bacteria have two distinct polypeptide modules for the substrate recognition and enzymatic hydrolysis, i.e., a peptido© XXXX American Chemical Society

Received: March 16, 2016 Revised: July 28, 2016

A

DOI: 10.1021/acs.biochem.6b00240 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry Thermus scotoductus phage vB_Tsc2631, T. scotoductus MAT2119 bacteriophage Ph2119, Bacillus anthracis prophage, etc., contain endolysins that belong to the single-domain amidase family.19,25−31 In general, these proteins share the conserved α−β fold as described for T7L.19,26 Functional studies of these endolysins from different phages such as T7, K11, ph2119, etc., established their maximal lysis activity in the pH range of 6.5− 8.5.26,27,32−34 Further, these studies also demonstrated that their functionality is very stringent toward pH changes. All these enzymes are reported to be inactive in lysis assays below pH 6.0. However, their pH-dependent conformational changes have not been studied. In the study presented here, we have chosen T7 endolysin (T7L) to delineate the pH-dependent structural fluctuations of the amidase family of bacteriophage endolysins. T7L is a 151residue bifunctional zinc amidase protein with a pI of 8.8.19,35 It has three α-helices and five β-sheets (Figure 1). Earlier reports of

NP_041973.1), and the protein was purified as described previously.19 The T7L-His plasmid was produced by subcloning the T7L gene with a C-terminal His tag. For expression of T7L protein(s), the cells were then inoculated into 10 mL of LB medium containing 100 μg/mL ampicillin to grow an overnight seed culture. The large culture (1000 mL) was grown at 37 °C and 220 rpm to an OD600 of 0.4−0.5 using the saturated overnight 10 mL seed culture, induced with 0.5 mM isopropyl βD-1-thiogalactopyranoside (IPTG), and incubated at 37 °C and 220 rpm for an additional 4 h. Cells were harvested by centrifugation at 6000 rpm for 10 min, resuspended in 50 mL of lysis buffer [50 mM Tris and 500 mM NaCl (pH 8.0)] and treated with PMSF (100 mM) and BME (1 mM). Cells were sonicated and then centrifuged at 14000 rpm for 1 h at 4 °C. For T7L protein (His tag) purification, affinity chromatography was used; the supernatant was applied directly to the Ni-NTA column (bead volume of 5 mL), equilibrated with lysis buffer, and passed three or four times through the column to allow the binding of protein. The nonspecific protein impurities were removed by washing the column with an increasing gradient of imidazole in lysis buffer (10, 30, and 50 mM; 50 mL each). The adsorbed protein was eluted with 30−50 mL of 400 mM imidazole. Fractions (10 mL) were collected and analyzed by electrophoresis on a 12% SDS−PAGE gel. Protein fractions were dialyzed overnight in 20 mM Tris-acetate/20 mM glycinephosphate buffer (pH 7.0) containing 0.1 M NaCl and 5 mM EDTA. T7L proteins with and without the His tag were further purified by size exclusion chromatography (SEC) using a 120 mL HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) on an AKTA prime FPLC system (GE Healthcare) with a flow rate of 1 mL/min. The protein concentartion was meaured by UV absorbance at 280 nm using a molar extinction coefficient of 26470 M−1 cm−1. For NMR experiments, T7L was grown in M9 minimal medium containing 15NH4Cl and [13C]glucose and purified as described above. Both the untagged and His-tagged T7L proteins behaved similarly in all the biophysical experiments, suggesting no influence of His tag on the structural properties of T7L. We have specifically used untagged T7L exclusively for the experiments involving Zn [for Zn-dependent CD experiments (no EDTA) and cell lysis assays as explained below]. Circular Dichroism (CD) Spectroscopy. For CD and fluorescence experiments, 20 μM [20 mM glycine-phosphate buffer, 0.1 M NaCl, and 5 mM EDTA (pH 7 to 3)] HEL and T7L were used. The chemical denaturation was achieved by adding urea buffer [20 mM glycine-phosphate buffer, 0.1 M NaCl, 5 mM EDTA, and 10.5 M urea (pH 3)] to the protein samples. Urea concentrations were calculated using the refractive index method. Twenty microliters of T7L was added in 400 μL of 10.5 M saturated urea buffer to make 10 M urea protein samples. CD experiments with zinc-loaded T7L were performed in the presence of 100 μM ZnSO4 (without EDTA). CD measurements were taken on a Jasco J-1500 CD spectrophotometer at 25 °C form 190 to 250 nm using a 1 mm path length quartz cuvette. Thermal denaturation of T7L was monitored in the temperature range of 20−90 °C and then allowed to refold when the sample was cooled from 90 to 20 °C at regular step intervals of 10 °C with a gradient of 1 °C/min and an equilibration time of 5 min at every resting temperature. The secondary structural content of the T7L spectra was calculated using the online tool DICROWEB-K2D (http://dichroweb. cryst.bbk.ac.uk/html/home.shtml).36

Figure 1. Three-dimensional structural representation of T7L (PDB entry 1LBA). The structural elements (α-helices colored pink and βsheets colored cyan) are marked, and the cofactor zinc is shown as a sphere.

pH-dependent amidase activity analysis suggested that T7L has optimal lytic activity around pH 7−7.5. The residual activity decreases to 50% at pH 6.0 and drops drastically with a further decrease in pH.19,32 To unravel the structural contribution of the protein for such a huge-pH dependent variation in activity measurements, we have performed various biophysical experiments, including CD, fluorescence, size exclusion chromatography, and NMR techniques such as 15N relaxation and hydrogen exchange studies. Our studies suggested that T7L in the native state at pH 7 exhibits a fair amount of conformational dynamism, and at low pH (pH 70% of the resonance assignments. The backbone NH assignments for the assigned residues are labeled in Figure 9A, and the summary of the assignments is given in Figure 9B. The chemical shift details of the assigned residues are provided in Table S2. Upon close observation, we noticed that the residues missing in the sequence specific assignments are the stretches that are hanging on either side of the His residues. T7L comprises seven His residues with varied surface exposure (see Discussion). The conformational exchange of the protonated and deprotonated states of these His residues depending on their side chain pKa values can potentially contribute to an enhanced conformational exchange, which in turn resulted in the variable intensities and/or broadening of the HSQC peaks as well as the loss of sequence specific connections in the 3D spectrum. Such a dynamic phenomenon can be readily monitored using the 15N NMR relaxation experiments. Dynamics measurements using NMR relaxation experiments provide detailed sequence specific information along the

polypeptide chain at various time scales. In general, the longitudinal relaxation rate (R1), the transverse relaxation rate (R2), and the steady-state heteronuclear NOE measurements (het-NOE) provide motions that are sensitive to various time scales. The values for the longitudinal relaxation rate, R1, are sensitive to both low- to high-frequency motions (nanosecond to picosecond time scale motions). However, the longitudinal relaxation (R1) rate by itself does not discriminate effectively between faster and slower motions, whereas the heteronuclear [1H]−15N NOEs are typically most sensitive to higher-frequency motions (picosecond time scale motions) of the backbone. Their lower values indicate the increased local flexibility of the polypeptide.48,49 Similarly, the transverse relaxation (R2) rates are more sensitive to the contributions from slower millisecondto-microsecond exchange processes to the observed conformational dynamics. To assess the dynamic characteristics of T7L, we have performed the relaxation experiments. The relaxation rate parameters, R1, R2, and NOE, were reliably obtained for ∼90 residues, and a summary of the results is given in Figure 10A−C. From the figure, it is evident that T7L shows a significant chaindependent conformational dynamics in its R1 and R2 values, whereas the steady-state NOE shows a constant pattern over the polypeptide chain with an average of 0.79 ± 0.04 (Figure 10C), thus evidencing a characterstic folded structural conformation. The NOE pattern clearly revealed that the N-terminus is more flexible than its C-terminus. We further analyzed the R2 values in detail, which provides information about the residue specific conformational exchange. The average of the R2 values obtained is ∼22 ± 1.5 s−1. A group of residues such as S25, H37−G41, K71, G72, V83−D87, L107, K142, and T147 showed large R2 values (Figure 10B). As R2 values serve as useful monitors of local conformational H

DOI: 10.1021/acs.biochem.6b00240 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

initial HX spectrum of T7L is shown in Figure 11A, and the structural locations of the protected residues are marked in

Figure 11. (A) Hydrogen−Deuterium (H/D) exchange HSQC spectrum of T7L. The disappearance of NH peaks from the 15N−1H HSQC spectrum is monitored at 25 °C, by redissolving lyophilized T7L (pH 7) in D2O. (B) Residues present in the HSQC spectrum after H/D exchange mapped on the T7L structure.

Figure 11B. Of 151 amino acids, only ∼32 amino acids were protected and are major contributors to the structural elements (Figure 11B). Among the protected residues, helix α2 makes a major contribution with 14 residues followed by sheets β2 and β4 accounting for six residues, suggesting that these structural elements make major contributions to the stability of the native state. All the NMR-based structural, dynamics, and stability studies point to the fact that the native state of T7L is conformationally dynamic and the segments around the His residues make major contributions to such dynamic behavior. Several residues of helix α2 and sheets β2 and β4 of T7L provide core stability for the native structure.

Figure 10. Summary of the residue-wise backbone 15N relaxation parameters of T7L recorded on a 800 MHz NMR instrument: (A) longitudinal relaxation rates (R1), (B) transverse relaxation rates (R2), and (C) steady-state heteronuclear NOE (het-NOE). The corresponding secondary structure elements are also indicated. The red horizontal line represents the average value of the particular relaxation parameter.

■

transitions occurring on the millisecond-to-microsecond time scale and the conformational exchange present at particular residue sites results in conspicuously enhanced R2 values, the residues and/or segments mentioned above are regarded as the source of the T7L dynamic behavior. It is interesting to note that most of the residues flank either side of the unassigned stretches of histidine segments. Comparative analysis of the peak intensities of the HSQC spectra of pH 7 and 6 (Figure 3) along with the observed R2 pattern at pH 7 (Figure 10B) suggested that several of the broadened residues at pH 6 are identified as being in the same group or vicinity of residues that showed enhanced R2 values at pH 7. A further decrease in pH to ≤5 results in the complete protonation of several of these His residues, thus resulting in collapse of the native conformation (Figure 3). These observations altogether clearly emphasize the role of the His residues and/or His protonation as a source of the observed conformational dynamics. We further probed the conformational stability of T7L in its native-state ensemble using the hydrogen exchange experiment. NMR-based native-state hydrogen/deuterium exchange is a powerful technique for calculating residue-wise stabilities of proteins. In principle, the exchange rates of backbone amide protons depend on their accessibility to the solvent deuterons, which can be correlated to the stability of secondary structural elements and/or tertiary and/or quaternary interactions. The

DISCUSSION Mechanistic Insights into the pH-Dependent Structural Delicacy of T7 Endolysin. The functional characteristics of a protein can be directly correlated to its structural integrity. To assess the complete enzymatic activity profile of a protein, it is essential to understand the stability and nature of its conformational transitions. Environmental factors such as temperature, pressure, pH, etc., are known to alter the structure−function relationship of protein to a significant extent.50−52 Structure− function relationships are well-established for the peptidoglycan hydrolases such as T4 endolysin (T4L) and hen egg lysozyme (HEL).53−55 Despite their structural similarity, these enzymes exhibit striking differences in terms of their enzymatic characteristics. HEL shows broad spectrum activity and exhibits optimal activity at pH 9.2,53,56 whereas bacteriophage T4L exhibits a pHdependent enzymatic profile similar to that of T7 and K11 amidases, with its highest activity at pH 7.4.53,57 However, no such structure−function relationship studies were available for the amidase family of proteins. In this work, we present the comprehensive details regarding the pH-dependent structural delicacy of the bacteriophage T7 endolysin, which is considered as a model protein for peptidoglycan amidases.25,58 All our biophysical and NMR experiments delineated the fact that pH plays a major role in maintaining the structural integrity of the I

DOI: 10.1021/acs.biochem.6b00240 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

in T7L is far from the rest of the His and/or positively charged residues and did not show any noticeable side chain repulsive interactions in the surrounding environment (6 Å radius). From all these attributes, we exclude the H37, H75, and H124 residues as the source of the structural collapse. However, such fully and/ or partially exposed His residues are the key players that contributed to the observed T7L (pH 7) native-state conformational dynamics. We then analyzed the behavior of the four remaining His residues (H18, H48, H69, and H123). These His residues are highly conserved in members of the T7 family, and their accessible surface area calculations (not shown) for the His residues in T7L suggested that these four His residues are completely and/or partially buried in the hydrophobic core of the protein. Moreover, they are present in the proximity of each other in a limited conformational space (Figure 12C). We have observed several of the distances between the Nδ and Nε atoms of each of the His residues with their neighboring imidazole ring N atoms within a radius of