BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture

Abstract

The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.

Using cryo-EM to uncover mechanisms of bacterial transcriptional regulation

“…This is evidenced by the rapidly expanding structural repertoire of bacterial transcription complexes over the past two years, with >65 resolved by cryo-EM to date. As illustrated within this review, these structures have shed light on unique conformational changes not seen in previous crystallographic studies, which include: promoter remodelling to stabilise intermediate complexes of transcription initiation [ 22 , 23 , 37 ]; insights into the conformational plasticity during the transition from transcription initiation to elongation [ 22 , 28 ]; the cooperative assembly of the transcriptional repressor, NanR [ 26 ]; the effector-induced reconfiguration of BusR to bind a bipartite DNA motif [ 27 ]; and the interaction of Crl and WhiB7 with RNAP to tether the σ-factors that they regulate through protein–protein or protein–DNA interactions, respectively [ 32 , 34 ]. However, despite these advancements reviewed here, gaps in our knowledge remain.…”

“…Following 3D reconstruction, atomic models provide the basis to structurally and functionally interpret the cryo-EM density maps. Most commonly, this process involves the input of existing X-ray or NMR structures from the PDB [22,[26][27][28][29], however in their absence, the neural network-based, structure prediction programs, AlphaFold [109] or RoseTTA [110] now offer an alternative. To guide this initial structure into the target density map, various flexible fitting tools, such as cryo_fit within phenix [70], ISOLDE [71] or iMODFIT [72] in Chimera, Namdinator [73] among others [29,111] can be used to accommodate conformational heterogeneity by utilising molecular dynamics simulations or normal mode analysis [112].…”

“…The 70.5 kDa cryo-EM structure of dimeric NanR in complex with cognate DNA (PBD-6WFQ) [ 26 ]. ( F ) Streptococcus agalactiae ( Stag ) BusR binds palindromic promoter DNA as a tetramer to repress transcription (PDB-7OZ3) [ 27 ]. The 5′ and 3′ DNA strands have been annotated throughout.…”

“…BusR is a transcriptional repressor that binds the c-di-AMP molecule; a vital molecule in normal cellular growth conditions and a target for antibiotic development [ 61 ]. A recent cryo-EM structure of BusR revealed how it binds bipartite DNA motifs ( Figure 2F ) as a tetramer and proposes a new regulator family, as the protein architecture is unlike any other transcriptional regulator described [ 27 ].…”

“…Notably, bacterial RNAP transcription complexes suffer from severe preferential orientation [ 101 ], however, this is routinely combated by the addition of the zwitterionic detergent CHAPSO during sample preparation [ 22 , 23 , 39 , 40 ]. Other detergents, such as β-octyl glucoside have also been used to promote random particle distribution and discourage complex dissociation [ 27 , 102 , 103 ]. Recently, molecular goniometers have been constructed using DNA origami, which enable the DNA-binding protein to bind and be precisely oriented via a sequence-specific DNA stage [ 104 ].…”