Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics

Abstract

Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function.

Identification and Characterization of an Inside-Out Folding Intermediate of T4 Phage Sliding Clamp

“…gp45 unfolds after a three-state transition with unfolding of NTD preceding CTD (52). We have previously shown that gp45 CTD undergoes MG-like intermediate formation during unfolding and refolding; here, CTD alone adopts MG conformation, whereas the NTD remains unfolded (52). In this article, we show that there occurs a huge conformational rearrangement in the gp45 CTD when it forms MG. Our data suggest that the domain swells and forms DMG wherein the surface residues flip inside the domain's hydrophobic core, causing burying of residues in a solventexcluded environment.…”

“…The cloning of various genes, protein expression and purification, and sitedirected mutagenesis were carried out as described previously (52), and are detailed in Supporting Material. Proteins were dialyzed against gp45 buffer (50 mM sodium phosphate pH 7.4, 50 mM NaCl, 1 mM EDTA, and 5% glycerol).…”

“…Unfolding studies in the presence of urea were performed as described previously (52,54); details are provided in Supporting Material. Eight micromoles of protein in specified urea concentrations were subjected to far-UV circular dichroism (CD), fluorescence emission, and anisotropy measurements and the recorded data were analyzed as detailed in Supporting Material.…”

“…gp45 forms a homotrimer with each subunit having two domains, i.e., N-terminal domain (NTD) and C-terminal domain (CTD) (51). gp45 unfolds after a three-state transition with unfolding of NTD preceding CTD (52). We have previously shown that gp45 CTD undergoes MG-like intermediate formation during unfolding and refolding; here, CTD alone adopts MG conformation, whereas the NTD remains unfolded (52).…”

Dissecting the structure–function relationship in lysozyme domain of mycobacteriophage D29‐encoded peptidoglycan hydrolase

“…S1. Molar ellipticity values at 222 nm were recorded and used to calculate fraction unfolded as described elsewhere . The unfolding transition of LD WT and its variants was also monitored using Differential scanning calorimetry on a Microcal VP‐DSC (GE Healthcare, Chicago, IL, USA) as detailed in Doc.…”