Identification of the Tolfenamic Acid Binding Pocket in PrbP from Liberibacter asiaticus.

Lei Pan,Fernando A Pagliai,Claudio F Gonzalez,Graciela L Lorca,Christopher L Gardner

doi:10.3389/fmicb.2017.01591

Abstract

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that was found to regulate gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. It was found that inactivation of PrbP, using the inhibitor tolfenamic acid, resulted in a significant decrease in the overall transcriptional activity of L. asiaticus, and the suppression of L. asiaticus infection in HLB symptomatic citrus seedlings. The molecular interactions between PrbP and tolfenamic acid, however, were yet to be elucidated. In this study, we modeled the structure of PrbP and identified a ligand binding pocket, TaP, located at the interface of the predicted RNA polymerase interaction domain (N-terminus) and the DNA binding domain (C-terminus). The molecular interactions of PrbP with tolfenamic acid were predicted using in silico docking. Site-directed mutagenesis of specific amino acids was followed by electrophoresis mobility shift assays and in vitro transcription assays, where residues N107, G109, and E148 were identified as the primary amino acids involved in interactions with tolfenamic acid. These results provide insight into the binding mechanism of PrbP to a small inhibitory molecule, and a starting scaffold for the identification and development of therapeutics targeting PrbP and other homologs in the CarD_CdnL_TRCF family.

Highlights

Huanglongbing (HLB) or citrus greening, is a devastating disease threatening the citrus industry in Asia, Africa, and America (Graça, 1991; da Graça and Korsten, 2004; Bové, 2006; Gottwald, 2010; Wang and Trivedi, 2013)
We focused on the identification and characterization of the tolfenamic acid binding pocket (TaP, Tolfenamic acid Pocket) in Predicted RNA polymerase binding protein (PrbP)
Additional studies in Myxococcus, Mycobacterium, and Thermus species later revealed that members of this family are important RNA polymerase interacting proteins, that regulate transcription initiation (Stallings et al, 2009; GarciaMoreno et al, 2010; Bae et al, 2015; Rammohan et al, 2015)

Summary

Introduction

Huanglongbing (HLB) or citrus greening, is a devastating disease threatening the citrus industry in Asia, Africa, and America (Graça, 1991; da Graça and Korsten, 2004; Bové, 2006; Gottwald, 2010; Wang and Trivedi, 2013). The causative agent is the phloem-limited bacteria Liberibacter asiaticus, L. africanus, and L. americanus (Bové, 2006; Gottwald, 2010; Wang and Trivedi, 2013) It was first documented in Asia over a century ago (Graça, 1991; da Graça and Korsten, 2004; Bové, 2006), HLB was not widely recognized until its emergence in the United States between 2004 and 2005 (Bové, 2006). Tolfenamic Acid Binding Site in PrbP culture conditions of L. asiaticus remain elusive (Davis et al, 2008; Sechler et al, 2009; Parker et al, 2014), which has severely impeded the understanding of the physiology of L. asiaticus, and the development of an effective treatment for HLB

Methods

Results

Conclusion