Structural plasticity of T4 transcription co-activator gp33 revealed by a protease-resistant unfolded state Radhakrishnan Mahalakshmi a, * , Svetlana Rajkumar Maurya a, 1 , Bhawna Burdak a, 1 , Parini Surti a, 1 , Manoj S. Patel a , Vikas Jain b, ** a Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India b Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India article info Article history: Received 30 July 2017 Accepted 10 August 2017 Available online xxx Keywords: gp33 Protease resistance Protein-urea interaction Structural plasticity abstract Gene 33 protein (gp33) is a transcriptional coactivator for late genes of the T4 bacteriophage. gp33 possesses a 5-helix bundle core, with unstructured N- and C-terminal regions that account for >50% of the protein sequence. It plays a unique role of interacting with host RNA polymerase, couples tran- scription with DNA replication, and plays the dual function as repressor and co-activator in phage transcription. Here, we identify protein structural plasticity as the molecular basis of the dual nature in gp33. We find that gp33 has the peculiar property of remaining protease insensitive in its urea-unfolded state. Using NMR studies with spectroscopic measurements, we propose that intra-protein interactions are replaced by protein-urea interactions in gp33. This process not only unfolds gp33 but also renders it protease-resistant. Our studies shed new light on the unique structural malleability of gp33 that might be important in its transition from a repressor to a late transcription co-activator. © 2017 Elsevier Inc. All rights reserved. 1. Introduction The T4 bacteriophage late gene transcription requires the T4 gene 55 (gp55) that acts as a sigma factor and recognizes the T4 late gene promoters [1,2]. T4 phage also produces a late gene tran- scription co-activator, gp33. The gp55-mediated transcription is generally repressed by gp33. However, in the presence of another phage encoded protein, gp45 (sliding clamp protein), the late gene transcription is enhanced several fold [3,4]. The C-terminal tails of both gp55 and gp33 interact with the sliding clamp and bring about late gene transcription. While the C-terminal region of gp55 is dispensable and allows RNA polymerase to achieve more than basal transcription, the gp33 C-terminal region is essential to achieve any transcription in the presence of gp45 [1,3]. In other words, when the gp33 C-terminal region is absent, transcription remains repressed. Thus gp33 has a dual function as repressor and co- activator in the transcription biology of T4 phage. It is thus of great interest to understand how one protein performs two con- trasting functions. The crystal structure of T4 gp33 bound to the flap domain of Escherichia coli RNA polymerase is available [5]. The structure of the ordered region of gp33 is made up of five helices, and forms the protein core (Fig. 1A). This core binds to the b-flap domain of RNA polymerase b-subunit. Thirty one residues at the N-terminus and 10 residues at the C-terminus were not visible in the electron density map, and were assumed to be disordered [5]. Other ex- periments also showed that ~55% of the gp33 protein remains disordered in solution [6]. Whether these unstructured regions of gp33 are relevant for its contrasting function is unclear, and calls for detailed analysis of the protein. Using biophysical studies, we now report that gp33 exhibits structural plasticity as evident from protease resistance upon denaturation with urea. Such a behavior is unique for a soluble protein, and to the best of our knowledge, has Abbreviations: <l>, average wavelength; BSA, bovine serum albumin; CD, cir- cular dichroism; C m , mid-point of chemical denaturation; f U , unfolded fraction; GdnHCl, guanidine hydrochloride; gp33, gene 33 protein; HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; PK, proteinase K; PMSF, phenylmethane sulfonyl fluoride; SDS-PAGE, sodium dodecylsulfate e polyacrylamide gel electrophoresis. * Corresponding author. Molecular Biophysics Laboratory, Department of Bio- logical Sciences, Room #324, III Floor, Block C, Academic Building 3, Indian Institute of Science Education and Research, Bhauri, Bhopal, 462066, India. ** Corresponding author. Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Room #325, III Floor, Block C, Academic Building 3, Indian Institute of Science Education and Research, Bhauri, Bhopal, 462066, India. E-mail addresses: maha@iiserb.ac.in (R. Mahalakshmi), vikas@iiserb.ac.in (V. Jain). 1 These authors contributed equally. Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2017.08.038 0006-291X/© 2017 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (2017) 1e6 Please cite this article in press as: R. Mahalakshmi, et al., Structural plasticity of T4 transcription co-activator gp33 revealed by a protease- resistant unfolded state, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.08.038