Understanding the basis of substrate recognition by the RiPP biosynthetic enzyme SuiB Open Access

Abstract

Finding new ways to combat antibiotic resistant bacterial infections has become a serious concern in the scientific community. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a newly classified group of natural products which have vast structural and chemical diversity correlated with antifungal, antibacterial, allelopathic, and antiviral activity. The modular and intrinsically tolerant biosynthetic pathways of RiPPs makes them a highly promising venture for bioengineering and downstream drug development. The RiPP recognition element (RRE) of RiPP biosynthetic enzymes has been implicated in the identification and transfer of ribosomally synthesized peptides to the active site for post-translational modification in a leader-dependent fashion. However, new questions about the role of the RRE domain in modifying enzymes and the function of the leader sequence in substrate peptides arose from the crystal structure of the RiPP peptide/enzyme pair of SuiA/SuiB which had the leader region of SuiA bound outside the RRE domain of SuiB. This study aims to analyze the interactions between the leader region of SuiA and the RRE domain of SuiB both to gain structural insights into the basis for precursor peptide recognition and clarify the role of the RRE. Molecular docking simulations were used in an attempt to build models of potential interactions between the SuiA-leader and the SuiB-RRE, however, the crystallographically-derived SuiA-leader structure failed to dock. Molecular dynamics simulations on the precursor peptide showed the a-helix of the leader region denaturing in solution, which suggested the binding conformation of the peptide leader sequence is non-helical. This result was verified by circular dichroism spectroscopy, indicating that and interactions between the SuiA leader and the RRE domain of SuiB, occur with SuiA as a random coil as opposed to the a-helical conformation observed in the active site. X-ray crystallography was attempted to identify the specific interactions between the SuiA-leader and the SuiB-RRE. Crystals were obtained, but these experiments are ongoing. We are currently in the process of optimization to improve diffraction quality. 

Table of Contents

1. INTRODUCTION………………………………………………………………………………………………………………….1

1.1 Introduction to Ribosomally Synthesized and Post-translationally Modified Peptides………………………………………………………………………………………………………………………1

1.2 RiPP Biosynthesis…………………………………………………………………………………………………..2

1.3 SuiA and SuiB: RiPP class of precursor peptide and radical SAM modifying enzyme……..………………………………………………………………………………………………………………..3

1.4 Research Overview………………………………………………………………………………………………..7

2. METHODS…………………………………………………………………………………………………………………………..8

2.1 Computational Methods………………………………………………………………………………………..8

2.2 Mass Bacterial Expression of NusA-RRE…………………………………………………………………10

2.3 Circular Dichroism Spectroscopy…………………………………………………………………………….15

2.4 Crystallography……………………………………………………………………………………………………..16

3. RESULTS & ANALYSIS………………………………………………………………………………………………………….17

3.1 Molecular Docking of SuiA-leader with SuiB-RRE and Comparison with Analogous Homologs…………………………………………………………………………………………………………………….19

3.2 Molecular Docking of a different class of RiPP peptide/modifying-enzyme pair………………………………………………………………………………………………………………………………27

3.3 Circular Dichroism Spectroscopy of SuiA-leader and NusA-RRE………………………………30

3.4 X-ray Protein Crystallography of SuiA-leader and NusA-RRE…………………………………..32

4. DISCUSSION………………………………………………………………………………………………………………………..36

5. CONCLUSION………………………………………………………………………………………………………………………38

6. REFERENCES………………………………………………………………………………………………………………..……..41

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Chemistry, Physical Chemistry, Molecular
Keyword	Modifying enzymes Post-translational modifications RiPPs SuiA Crystallography SuiB RRE Molecular Docking Biosynthesis
Committee Chair / Thesis Advisor	Katherine M. Davis, Emory University
Committee Members	Khalid Salaita, Emory University Deepika Das, Emory University

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