Crystal structure of an atypical cobalamin riboswitch reveals RNA structural adaptability as basis for promiscuous ligand binding

Abstract

Cobalamin riboswitches encompass a structurally diverse group of cis-acting, gene regulatory elements found mostly in bacterial messenger RNA and are classified into subtypes based on secondary and tertiary characteristics. An unusual variant of the cobalamin riboswitch with predicted structural features was identified in Bacillus subtilis over a decade ago, but its structure and mechanisms of cobalamin selectivity and translational control have remained unsolved. We present the crystal structure of the aptamer domain of this atypical cobalamin riboswitch and a model for the complete riboswitch, including its expression platform domain. We demonstrate that this riboswitch binds to multiple cobalamin derivatives and correlate its promiscuous behavior to its structure and unique arrangement of peripheral elements. Comparative structural analyses between conventional cobalamin riboswitches and the B. subtilis cobalamin riboswitch reveal that the likely basis for this promiscuous ligand binding is intrinsic structural adaptability encoded in the RNA structure. It suggests that cobalamin selectivity might ultimately be viewed as existing on a spectrum of affinity for each derivative rather than as belonging to distinct types based on ligand specificities. Our work provides an interesting and notable example of functional coupling of ligand-sensing and adaptive folding by a structured RNA molecule.

Cobalamin riboswitches are broadly sensitive to corrinoid cofactors to enable an efficient gene regulatory strategy

“…Notably, deletion of btuR, which encodes the adensosyltransferase that installs the Ado upper ligand group, rendered the B. subtilis btuF Cbl-riboswitch reporter insensitive to exogenously supplied CNCbl, MeCbl, and OHCbl, while retaining dose-dependent repression in response to AdoCbl (Figure S1). This strongly suggests that the B. subtilis btuF Cbl-riboswitch only responds to Cbl containing the 5'-deoxyadenosine upper ligand, in contrast with a report suggesting that this riboswitch aptamer can also bind MeCbl and OHCbl (Chan and Mondragón, 2020).…”

“…These structural models show that the base-on tails are contained within the binding site, whereas the tails of the base-off Cbl structures protrude away from the binding site and clash with the L5-L13 kissing loop. Although the B. subtilis btuF (Figure S8C-D) and Symbiobacterium thermophilum cblT (Figure S8E-F) riboswitch models do not contain the L13 structure of the kissing loop, some of the modeled base-off tails clash with L5 of the aptamer in these structures (Chan and Mondragón, 2020; Peselis and Serganov, 2013). The kissing loop has been shown to play a key mechanistic role of sensing the corrinoid-binding state of the aptamer domain to influence downstream regulatory structures in the expression platform (Lussier et al, 2015; Polaski et al, 2016).…”

“…We next sought to identify how structural differences in the corrinoid tail affect the response of semi-selective Cbl-riboswitches to corrinoids. There are no predicted hydrogen bond interactions between the lower ligand and the RNA in the X-ray crystal structures of Cbl-bound riboswitches, making it difficult to surmise how a Cbl-riboswitch might distinguish between corrinoids (Chan and Mondragón, 2020; Johnson et al, 2012; Peselis and Serganov, 2013). Could the overall structural conformation of the corrinoid, rather than specific interactions between the RNA and the corrinoid tail, influence Cbl-riboswitch activity?…”

Modelling the impact of magnesium ions concentration on the folding of the SAM-II riboswitch

“…Crystal structures of riboswitches often show one or more magnesium ions bound to the RNA (Huang and Lilley 2018;C. Lu et al 2010;Chan and Mondragón 2020;Furukawa et al 2015) though in some cases these ions can be found in unexpected locations or even absent (Gilbert et al 2008;Garst et al 2008) despite the clear evidence that these riboswitches undergo structural changes in the presence of these ions (Chen et al 2012;Garst et al 2008).…”

“…The number of magnesium ions that interact with the RNA and the affinity of the RNA to these ions are variable [5,11]. Crystal structures of riboswitches often show one or more magnesium ions bound to the RNA [2,[11][12][13] though in some cases these ions can be found in unexpected locations or even absent [14,15] despite the clear evidence that these riboswitches undergo structural changes in the presence of these ions [7,15].…”