Thermal Characterization and Interaction of the Subunits from the Multimeric Bacteriophage Endolysin PlyC

Abstract

Bacteriophage endolysins degrade the bacterial peptidoglycan and are considered enzymatic alternatives to small-molecule antibiotics. In particular, the multimeric streptococcal endolysin PlyC has appealing antibacterial properties. However, a comprehensive thermal analysis of PlyC is lacking, which is necessary for evaluating its long-term stability and downstream therapeutic potential. Biochemical and kinetic-based methods were used in combination with differential scanning calorimetry to investigate the structural, kinetic, and thermodynamic stability of PlyC and its various subunits and domains. The PlyC holoenzyme structure is irreversibly compromised due to partial unfolding and aggregation at 46 °C. Unfolding of the catalytic subunit, PlyCA, instigates this event, resulting in the kinetic inactivation of the endolysin. In contrast to PlyCA, the PlyCB octamer (the cell wall-binding domain) is thermostable, denaturing at ~75 °C. The isolation of PlyCA or PlyCB alone altered their thermal properties. Contrary to the holoenzyme, PlyCA alone unfolds uncooperatively and is thermodynamically destabilized, whereas the PlyCB octamer reversibly dissociates into monomers and forms an intermediate state at 74 °C in phosphate-buffered saline with each subunit subsequently denaturing at 92 °C. Adding folded PlyCA to an intermediate state PlyCB, followed by cooling, allowed for in vitro reconstitution of the active holoenzyme.

Keywords: PlyC; antimicrobial; differential scanning calorimetry; endolysin; thermal stability; thermodynamic characterization.

Figure 1

Structure of the PlyC holoenzyme (PDB: 4F88). The PlyCA subunit contains the GyH catalytic domain (residues 1–205, blue), a central helical docking domain (residues 227–288, yellow), and a CHAP catalytic domain (residues 309–465, green). Linkers between domains (residues 206–226, 289–308) are depicted in red unless disordered (dashed lines or absent). The eight PlyCB subunits (A–H) are depicted in alternating cyan/magenta. The N-terminal residues of the PlyCB octamer docks to PlyCA via the helical docking domain.

Figure 2

Fifteen (15)-day kinetic stability analysis of PlyC. The kinetic stability of PlyC at 1 mg/mL in PBS, pH 7.2, was evaluated for 15 days at 4, 25 and 37 °C. At each temperature, an aliquot from three independent samples of PlyC was removed every 48 h and subsequently assayed for residual bacteriolytic activity toward S. pyogenes strain D471 using a turbidity reduction assay. Data were averaged and normalized to the bacteriolytic activity of each sample observed on day 1.

Figure 3

Thermal denaturation of PlyC. Top: The structural stability of the PlyC holoenzyme was assessed by incubating the endolysin at 1 mg/mL in PBS at temperatures ranging from 37 to 65 °C (left) or 42 to 48 °C (right) for 30 min. After heat treatment, the samples were cooled on ice and visualized via native PAGE. The absence of PlyC in wells corresponds to thermally induced protein unfolding and aggregation. Bottom: Lytic activity was normalized to unheated PlyC. +, ≥90% of unheated PlyC activity; −, ≤10% of unheated PlyC activity.

Figure 4

Purification of various PlyC-related constructs used in this study. The PlyC holoenzyme (114 kDa; PlyCA subunit = 50 kDa, PlyCB subunit = 8 kDa), the PlyCA catalytic subunit (50 kDa), the C-terminal CHAP domain of PlyCA (18 kDa), and the PlyCB CBD (64 kDa; PlyCB subunit = 8 kDa) were purified to near homogeneity as indicated by SDS-PAGE. Note that the PlyCB subunit routinely runs with the dye front and can appear lower than the 6.5 kDa marker despite its 8 kDa mass.

Figure 5

Thermodynamic analysis of PlyCA, PlyCB CBD, and the PlyC holoenzyme using DSC. The thermodynamic properties of (A) PlyCA, (B) PlyCB CBD, (C) PlyCB CBD (re-scanned), and (D) PlyC were experimentally determined using DSC. Each sample was heated in the calorimeter from 15 to 105 °C and subsequently cooled from 105 to 15 °C using scan rates of 15 or 60 K/h. The representative thermograms depicted were obtained from analyzing the protein samples in either PBS (PlyCA and PlyC) or phosphate buffer (PlyCB CBD).

Figure 6

Structural and kinetic stability of the PlyCB octamer. (A) Following a 10-min incubation in PBS at temperatures ranging from 65 to 90 °C, PlyCB at 1 mg/mL was chemically crosslinked with 1 mM BS³ and cooled on ice. The samples were analyzed using SDS-PAGE in order to evaluate the structural integrity of the PlyCB octamer. (B) AlexaFluor-labeled PlyCB CBD at 1 mg/mL in PBS was incubated at 4 to 100 °C for 30 min, cooled on ice, and then added to S. pyogenes. The kinetic stability of the heat-treated PlyCB samples (i.e., PlyCB that retains its ability to bind S. pyogenes following heat treatment) was analyzed by means of fluorescence microscopy. Scale bar: 5.0 µm.

Figure 7

Thermal reconstitution of the PlyC holoenzyme. To elucidate the folding dynamics of PlyC, an attempt was made to reassemble the holoenzyme structure using purified PlyCA and PlyCB subunits. In one scenario, PlyCA and the PlyCB CBD were incubated at room temperature. Alternatively, ice-cold PlyCA was mixed with the PlyCB CBD in PBS heated to 75 °C and then immediately placed on ice. A 1:1 molar ratio of PlyCA:PlyCB CBD was used in both instances. Used as an indirect measure of PlyC holoenzyme reconstitution, the bacteriolytic activity of each sample at equal molar concentrations was assayed against S. pyogenes using a turbidity reduction assay. Activity was normalized to that of PlyC. Isolated PlyCA (<1% activity of PlyC) and PlyCB CBD (no activity) were used as controls.