BIOPHYSICAL STUDIES ON STRUCTURAL TRANSITIONS OF T7 BACTERIOPHAGE ENDOLYSIN

Abstract

Bacteriophages are extremely diversified prokaryotic viruses that act on bacteria. Certain classes of bacteriophage known as lytic phages cause complete lysis of susceptible bacterial culture with the aid of endolysins. Endolysins hydrolyze specific bonds in the peptidoglycan, which triggers bursting and release of mature phage particles from bacterial host. Endolysins have been categorized into four classes that include: glycosidase (muramidase), endopeptidase, amidohydrolase (amidase) and lytic transglycosylase. Endolysins infecting Gram-positive bacteria contain an enzymatic catalytic domain (ECD) and a cell binding domain (CBD), whereas most endolysins from a Gram-negative background possess only an enzymatic catalytic domain. Endolysins from T7L, T4L,  phage, K11, and KP32 etc., are the major representatives of a single-domain family. Among these endolysins, amidases (also called as an N-acetylmuramoyl-L-alanine amidases) are known for their rapid lysis activity. They destabilize the peptidoglycan by separating the glycan polymer from the stem peptide by cleaving a critical amide bond between the glycan moiety (MurNAc) and the peptide moiety (L-alanine). Bacteriophages such as T7, T3, K11, Thermus scotoductus phage vB_Tsc2631, and Bacillus anthracis prophage etc., comprise the single domain endolysin amidase family. These proteins have a molecular weight around 16-20 kDa and share the conserved α-β fold. T7 endolysin (T7 lysozyme-T7L) is produced by T7 bacteriophage from Podoviridae family. It is a 151 amino acid long (17 kDa) globular bi-functional protein containing Zn+2 as a co-factor. T7L facilitates the rapid lysis of Gram-negative bacteria and inhibit T7 RNA polymerase at the end of their lytic multiplication cycle. Zinc is required for its amidase activity but not for RNAP inhibition. T7 endolysin contains five β-strands and three α-helices, and has a prominent cleft. Zinc atom is located in the catalytic cleft and anchored to the protein by three side-chain ligands, His17, His122, and Cys130, and connected to the hydroxyl group of Tyr46 through a water molecule. The current thesis unravels the molecular insights on pH dependent structure-folding-dynamics-function relationship of T7 endolysin by employing various biophysical techniques. Chapter 1 includes an overview of the origin of endolysins, classification, domain organization, structural diversity, mechanism of action, and comparative structural analysis of different phage endolysins with mammalian peptidoglycan recognition proteins (PGRPs) /bacterial VIII endolysins with a special emphasis on the structural-enzymatic relationship of phage endolysins. The chapter also presents the concepts and applications of engineered endolysins, which includes optimization and development of endolysin into potential therapeutic compounds by means of rational design, recent applications and patents in the field of medicine, food safety, agriculture and biotechnology. Further, a brief overview of biophysical techniques used to achieve the proposed research work was presented. Chapter 2 unravels the characterization of T7 endolysin structural features, and influence of pH on its structural stability and enzymatic activity. T7L was successfully expressed and purified by transforming E. coli BL 21 (DE3) cells with plasmid (pAR4593) containing T7L gene. Purification of T7L was done by using combination of chromatographic techniques and confirmed its monomeric state. The mechanism of pH-dependent differential activity profiles and structural features were investigated by employing cell lysis assay and various biophysical techniques including CD, fluorescence, size-exclusion chromatography and NMR techniques etc. Studies established that pH plays a major role in maintaining the structural integrity of the T7L structure and lowering the pH results in the loss of lysis activity. T7L exhibited a pH dependent reversible structural transition from native state to partially folded (PF) conformation below pH 6. Structural studies indicated a partial loss of secondary/tertiary structures in PF states (pH range 5-3) compared to its native state. Moreover, T7L PF states displayed differential binding characteristics of 8-anilino-1- naphthalenesulfonic acid (ANS) dye. Although zinc is essential for T7L amidase activity, CD experiments indicated that zinc does not influence this pH-induced structural transition. Structural analysis of T7L suggested that a network of buried His residues are the source for the observed pH induced structural transition Chapter 3 provides a detailed study of structure, stability and dynamic features of T7L native conformation at pH 8, 7 and 6. Temperature dependent far-UV CD measurments indicate thermal melting of secondary structure above 50 ⁰C and formation of insoluble aggregates or precipitates at ~ 70 ⁰C. Urea dependent unfolding experiments based on CD and fluorescence evidenced for differential structural stablities and unfolding characterstics of pH 6 conformation compared to pH 8 and 7. Backbone resonance assignment of T7L at pH 7 was obtained using a set of conventional triple-resonance NMR experiments. Heterogeneous intensities of the NH cross peaks and lack of several sequential connections in the 3D experiments indicating an inherent dynamic nature of T7L IX native state. The structural heterogeneity of the native state is further confirmed by assigning the multiple conformations of T7L segments. The dynamic behavior and the heterogeneity are regulated by the pH, and the low pH state (pH 6) is comparatively more dynamic than the higher pH (8, 7) native state conformations. Dynamics measurements using NMR relaxation experiments provided detailed sequence specific information along the polypeptide chain at various time scales. It is observed that the T7L shows significant chain dependent conformational dynamics in its R1 and R2 values. Steady state NOEs show constant values (~ 0.82) over the polypeptide chain, evidencing for a rigid T7L backbone across fast (nano to picosecond) time scale motions. Residues at pH 6 experienced more fluctuations in R1, R2 and NOE values as compared to pH 8 and 7. Further, hydrogen exchange (HX) experiment reveals that majority of protected residues belong to the structural elements and account for the stability of the native state. Chapter 4 delineates the structure-stability-dynamics relationship of T7L low pH partially folded conformations. Detailed biophysical and NMR studies revealed that these PF states differ in their structural and stability characteristics and exhibit differential dynamics. Urea dependent unfolding features of PF state at pH 5 and 4 evidenced for a collapsed conformation at intermediate urea concentrations (i.e. 2 M and 4 M), although pH 3 conformation resulted in uniform melting of structural elements. The pH 3 PF conformation is less stable as compared to pH 5 and 4 conformations as evidenced by free energy (ΔG) calculations. Thermal denaturation experiments delineated that the pH 5 conformation is thermally irreversible in contrast to pH 3 due to formation of insoluble aggregates. Temperature dependent amidase activity measurements show that thermally challenged pH 3 conformation achieve 100 % of its activity when the pH is adjusted back to 7 although the PF conformations do not possess lysis activity. In order to unravel the structural characteristics of T7L low pH PF states, backbone resonance assignments of pH 3 conformation was obtained by using triple resonance NMR experiments. A total of 37 residues were assigned out of ~ 42 observable peaks. Amide proton temperature coefficient analysis indicated that these assigned residues were devoid of H-bonding and located in the unstructured elements. Few residues such as S68, H69 (loop 34) and S148 (C-terminal) are notable as their temperature coefficients are < -4 ppb/K at pH 5 and are > -5 ppb/K at pH 4 and 3 suggesting that these residues might be involved in some local H-bonding interactions at pH 5 that melted upon lowering the pH. Further, 15N transverse relaxation rate measurements (R2) were used to probe their dynamics and conformational exchange behavior in s to ms time scale. It was observed that residues having enhanced R2 values are mainly X concentrated in the loop 34 and C-terminal region. Residue level analysis of the NMR based urea and temperature dependent HSQC spectra established that the structural elements 1-helix and 3- 4 segment of the N-terminal half of T7L is the major source for differential unfolding characteristics of PF states. All these results established that the low pH PF states of T7L are heterogeneous and exhibit differential structural, unfolding, thermal reversibility, and dynamic features. In summary, the current thesis investigated the structure-function relationship of T7L at residue level. Detailed insights were obtained on the stability and dynamic features of various T7L conformations such as native state and partially folded. Unraveling these biophysical features of a single domain bacteriophage endolysins serve as guiding attributes in assessing the structural and functional constraints of a single domain endolysins that are specifically designed as next generation enzybiotics by engineering them into chimeolysins and artilysins.