Reconsidering the czcD (NiCo) Riboswitch as an Iron Riboswitch

Abstract

Recent work has proposed a new mechanism of bacterial iron regulation: riboswitches that undergo a conformational change in response to Fe^II. The czcD (NiCo) riboswitch was initially proposed to be specific for Ni^II and Co^II, but we recently showed via a czcD-based fluorescent sensor that Fe^II is also a plausible physiological ligand for this riboswitch class. Here, we provide direct evidence that this riboswitch class responds to Fe^II. Isothermal titration calorimetry studies of the native czcD riboswitches from three organisms show no response to Mn^II, a weak response to Zn^II, and similar dissociation constants (∼1 μM) and conformational responses for Fe^II, Co^II, and Ni^II. Only the iron response is in the physiological concentration regime; the riboswitches' responses to Co^II, Ni^II, and Zn^II require 10³-, 10⁵-, and 10⁶-fold higher "free" metal ion concentrations, respectively, than the typical availability of those metal ions in cells. By contrast, the "Sensei" RNA, recently claimed to be an iron-specific riboswitch, exhibits no response to Fe^II. Our results demonstrate that iron responsiveness is a conserved property of czcD riboswitches and clarify that this is the only family of iron-responsive riboswitch identified to date, setting the stage for characterization of their physiological function.

Figure 1

(A) Sequence alignment of the Eba, Lmo, and Cce riboswitch constructs used in this study. Metal ligands are indicated with a red inverse caret (∨). (B) X-ray crystal structure of the Eba riboswitch with Co^II ions bound (PDB code 4RUM). Metal-binding nucleotides are numbered according to Figure 1A, and Co^II ions are shown as salmon spheres. The occupancy of C4 (anomalous signal 5σ) was significantly lower than that of C1, C2, and C3 (anomalous signals ∼12σ). Reprinted with permission from Xu and Cotruvo, copyright 2020 American Chemical Society. (C) Fluorescence response of Lmo-1 to first-row transition metal ions, with free metal concentrations buffered with 1 mM citrate. Full details and parameters from fits to the Hill equation (one set of sites) are given in Table 1.

Figure 2

Representative thermograms from ITC studies of the Eba czcD riboswitch (8.4–9.2 μM RNA) with Co^II (A) or Fe^II (B). The data are fitted to a model with one set of equivalent binding sites. Fitted parameters are provided in Table 2. Conditions: 30 mM MOPS, 100 mM KCl, 3 mM MgCl₂, pH 7.2, 20 °C.

Figure 3

Sensei RNA does not respond to iron. (A) Sequence alignment of the Eba czcD riboswitch and the Hdu Sensei RNA constructs used for ITC. The Sensei construct is the same one described by Ramesh and co-workers. (B) ITC thermograms of Hdu Sensei (15 μM) titrated aerobically with 225 μM Co^II (left) and anaerobically with 225 μM Fe^II (right). The thermograms do not show evidence of significant metal binding and conformational change, beyond nonspecific interactions; compare Figures 2 and 4 for czcD riboswitches. Conditions: 30 mM MOPS, 100 mM KCl, 3 mM MgCl₂, pH 7.2, 20 °C.

Figure 4

Representative thermograms from ITC studies of (A) Lmo and (B) Cce czcD riboswitches (8.2–9.9 μM RNA) with Co^II (left) or Fe^II (right). The data are fitted to a model with one set of equivalent binding sites. Fitted parameters are provided in Table 2. Conditions: 30 mM MOPS, 100 mM KCl, 3 mM MgCl₂, pH 7.2, 20 °C.

Figure 5

Comparison of K_d,app values for response of the Eba czcD riboswitch to transition metal ions (open red box for Mn^II, open red circles for Fe^II, Co^II, Ni^II, and Zn^II) with the calculated ranges of intracellular labile metal ion concentrations (black lines) determined for the Salmonella model system. The latter ranges consist of the free metal concentrations at which the relevant metal-sensing transcription factor gives 10, 50 (center), or 90% of its transcriptional response. Concept adapted from Young, Robinson, and co-workers, with our data added. See http://creativecommons.org/licenses/by/4.0/ for license information. In the case of Mn^II, the red box indicates that the K_d,app for the riboswitch is estimated to be 100–1000 μM (Figure S4). Because our experiments were carried out at 20 °C, but the prior ones were at 25 °C, our K_d values are slight underestimates relative to the intracellular labile metal ion concentrations. We also note that labile metal concentrations may differ somewhat from the Salmonella model, depending on the organism and aerobic versus anaerobic conditions.