Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.


Suppl. Figure 1: Time-dependent changes in anisotropy upon binding of RNA to Hera
A 3'-fluorescein-labeled 9mer RNA (500 nM) exhibits a low fluorescence anisotropy of r = 0.025 due to rapid rotational motion. The anisotropy increases to r = 0.085 when addition of 1 µM 32mer RNA allows for duplex formation, leading to slower rotational tumbling. Addition of 1 µM Hera and formation of the Hera-32/9mer complex caused an instantaneous increase in anisotropy to r = 0.135, justifying a 2 min equilibration time in anisotropy titrations. Measurements were performed in 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl 2 at 25°C. Fluorescence was excited at 495 nm and detected at 530 nm.
Suppl. Figure 2: Binding of RNA to Hera and Hera domains (A) Comparison of K d values of 32mer RNA complexes of Hera_RBD, Hera_370-510 (DD, RBD), Hera_core and Hera_208-419 (RecA_C, DD) with full-length Hera. The values are consistent with the RBD as the major RNA binding platform in the absence of ATP. The core contributes to RNA binding, but the DD does not. Measurements were performed in 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl 2 at 25°C with 50 nM 5´-fluorescein-labeled 32mer RNA. Fluorescence was excited at 495 nm and detected at 530 nm. (B) Stoichiometric titrations of the 5´-fluorescein-labeled 32mer (5 µM) with Hera and Hera_RBD. The broken lines indicate the lower and upper limit of the equivalence points. The data support a 1:1 stoichiometry of RNA binding to the RBD and to Hera (one RNA molecule per Hera protomer). Measurements were performed in 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl 2 at 25°C. Fluorescence was excited at 495 nm and detected at 530 nm. (C) Electrophoretic mobility shift assay of 32mer and 21mer binding to Hera_RBD, and quantification of the RNA fraction bound. Samples were incubated in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl and 12% (v/v) glycerol at 25°C for 30 min prior to gel electrophoresis. The numbers below the lanes indicate the protein concentration in µM. The RNA concentration was 6 µM. The K d values can be estimated to ~7 µM (32mer) and ~23 µM (21mer). (D) Isothermal calorimetric titration of the 21mer RNA with Hera in 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl 2 at 25 °C. 20 µM 21mer RNA in the cell was titrated with Hera_RBD (400 µM). The injection time was 20 s, with 300 s equilibration time between injections. The lack of an initial baseline precludes quantification of the K d value, but it can be estimated to ~ 40 µM, consistent with the value determined by EMSA (panel C) and NMR (see main text).

Suppl. Figure 3: Complete assignment of 1 H, 15 N-HSQC spectrum, and changes in chemical shifts upon binding of different RNAs.
(A) Complete assignments for the backbone amide groups of the Hera-RBD in its free form. Shown is the 1 H, 15 N-HSQC spectrum recorded at 37°C with all assignments indicated. The two signals for the single side chain amide group of Q457 are connected by a horizontal line and labeled as Q457sc. The signal of the side chain amide group of W435 is labeled as W435sc. Unassigned arginine side chain signals which are folded into the spectrum are labeled as sc. (B) Overlay of the 1 H, 15 N-HSQC spectra of the free RBD (black) and spectra in the presence of 5mer (dark blue), 19mer (red), 21mer (light blue), and 21mer_mod RNA (green). The directions of the chemical shift changes are indicated by the arrows. Spectra were measured in 25 mM Bis-Tris/HCl, pH 6.0, 50 mM NaCl, 10% (v/v) D 2 O at 37°C. The Hera_RBD concentration was 100 µM.
Suppl. Figure 4: NMR and CD analysis of RBD secondary structure (A) Influence of 21mer RNA binding on secondary structure propensities in the Hera-RBD. Shown are the results of a PECAN-analysis of the backbone chemical shifts (H N ,N H , CO, CA, CB) for the free protein (black) and protein bound to the 21mer RNA (red). Light blue lines indicate the positions of secondary structure elements as observed in the X-ray structure. Spectra were measured in 25 mM Bis-Tris/HCl, pH 6.0, 50 mM NaCl, 10% (v/v) D 2 O at 37°C. (B) Far-UV CD spectra of Hera_RBD (black), 32mer RNA (red), and the RBD/32mer RNA complex (blue). Spectra were measured with 10 µM protein in 50 mM potassium phosphate buffer, pH 7.5 at 25°C. (C) The spectrum of the RBD/RNA complex (blue) is identical to the sum of the individual spectra (purple), indicating that RNA binding does not induce secondary structure formation.
Suppl. Figure 5 : The affinity of Hera_RBD for the GGGC-RNA is independent of the C-tail Fluorescence anisotropy titration of a 5'-fluorescein-labeled GGGC-RNA with (A) Hera_RBD and (B) Hera_RBD_∆C-tail. The K d values for the RNA/RBD complexes are 8 µM in the absence and 11 µM in the presence of the C-tail, indicating that the C-tail does not contribute to binding of this RNA. The K d value for the RBD/GGGC complexes are virtually identical to the K d value of the Hera_RBD_∆C-tail/32mer complex (13 µM, cp. Figure 2, main text). These data are in-line with the core of Hera_RNA binding the GGGU sequence in the single-stranded region of the 32mer RNA, and the ∆C-tail contacting the hairpin. Measurements were performed with 50 nM RNA in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl with GST-Hera_RBD at 25°C. Fluorescence was excited at 495 nm and detected at 530 nm.
Suppl. Figure 6: Structural features of RBD-bound RNA (A) Imino proton region of a 1D-1 H-spectrum of the free 21mer RNA. 5 signals with chemical shifts typical for imino protons of Watson-Crick base pairs are visible and their NOE-based assignment is given. (B) Overlay of the imino group region of the 1 H, 15 N-HSQC of 15 Nguanine-labelled 21mer RNA bound to unlabelled Hera-RBD with a 15 N-decoupled 1D-1 H-NMR-spectrum of the RNA/protein-complex and a 15 N-selected-1D-1H-NMR-spectrum of 21mer RNA bound to 15 N-labelled Hera-RDB (B) Comparison of the three spectra shows that of the 10 signals observed in this spectral range three belong to protein backbone amide groups (red spectrum). Two are not correlated to 15 N-nuclei when either 15 N-labelled RNA or 15 N-labelled protein is used and therefore most likely belong to protein OH-groups. NOESY-spectra suggest that these signals belong to two protein tyrosine residues. Five signals represent guanine imino groups of the bound RNA. Four of these signals have chemical shifts typical of guanine imino groups in Watson-Crick-base pairs and belong to the helical stem of the 21mer RNA which is still present when the RBD is bound. The fifth signal (red arrow) belongs to a hydrogen-bonded guanine not in a canonical base pairing interactions. Moreover, this signal is not observable in the free RNA (not shown). Thus, this signal corresponds most likely to a guanine interacting directly with the protein via hydrogen bonding in agreement with the binding mode for Gua2 in the X-ray structure of the Hera-RBD in complex with 5'-GGGC-3'. (C) Comparison of 1D-and 15 N-filtered 1D-spectra of the free RBD show that the two signals belonging to OH-groups are already observable in the free protein and that their protection from exchange is not caused by RNA-binding. All spectra were measured in 25 mM Bis-Tris/HCl, pH 6.0, 50 mM NaCl, 10% (v/v) D 2 O at 37°C.
Suppl. Figure 7: RNA unwinding by Hera (A) Unwinding of the 32/9mer RNA substrate (0.5 µM, containing a 3'-fluorescein-9mer) by Hera (5 µM) in the presence of 5 mM ATP. The unwinding reaction was stopped at different time-points, and double-stranded substrate and single-strand released were separated by native PAGE. Quantification of the fraction of RNA unwound (bottom panel) reveals that Hera unwinds the 32/9mer RNA substrate with a rate constant of 0.012 s -1 . (B) Unwinding of a donor/acceptor-(Cy3/Cy5)-labeled 32/9mer RNA substrate (0.5 µM), followed by a reduction in acceptor fluorescence due to a loss of FRET upon release of the single strand. Cy3 fluorescence was excited at 554 nm and Cy5 fluorescence detected at 666 nm. Hera (5 µM) unwinds the 32/9mer RNA substrate with a rate constant of 0.017 s -1 (plus a slower phase with k = 0.003 s -1 ) in the presence of 1 mM ATP (black, arrow). No unwinding (above background) is detected in the presence of 1 mM ADPNP (cyan). (C) Unwinding of the 32/9mer RNA substrate (50 nM 9mer, 1 µM 32mer) followed by fluorescence anisotropy of a fluorescein attached to the 3´-end of the 9mer. Fluorescence was excited at 495 nm and detected at 530 nm. In the 32/9mer RNA the anisotropy is 0.103. Addition of 500 nM trap RNA (unlabeled 9mer) leads to a slight decrease in anisotropy. Formation of the Hera-32/9mer -complex results in an anisotropy of r = 0.115. After ATP addition (4 mM, arrow), the anisotropy decreases due to release of the 9mer upon duplex unwinding. ADPNP (4 mM) does not support unwinding (cyan). The half-life of the unwinding reaction is ca. 45 s, corresponding to a rate constant of unwinding of ca. 0.015 s -1 .
All reactions were performed in 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl 2 at 25°C in the presence of a 10-fold excess of non-labeled trap strand to ensure single turnover conditions. The similar rate constants of unwinding and the lack of unwinding in the absence of ATP (not shown) and in the presence of ADPNP in all three assays confirm that these methods report on duplex unwinding.