FIR and FBP are nucleic acid binding proteins involved in c-myc transcription regulation. (a) Top – Model of FUSE-dependent regulation of c-myc proto-oncogene. Bottom – FUSE DNA sequences (non-coding strand) used in this study. (b) Domain organization of FIR, FBP and FBP3 with the protein constructs used in this study (indicated by arrows). Amino acid sequence of FBP and FBP3 Nbox are given.

DNA binding and protein dimerization by FIR RRM1-RRM2. (a) X-ray structure of FIR RRM1-RRM2 bound to ssFUSE DNA (2qfj). The FIR RRM1-RRM2 dimer is represented with a grey ribbon and residues whose H^N protons are less than 10 Å from the dimer interface are displayed in blue. The nucleotide visible in the X-ray structure is displayed in orange. This structural representation was generated using the program PYMOL (www.pymol.org). (b) Isotherms for binding of the ssFUSE29 DNA to FIR RRM1-RRM2 (NMR). Ser268 and Ser269 are two residues of RRM2 ~5 Å away from the proposed dimer interface which do not change their chemical shift in response to DNA binding. (c) CSP of FIR RRM1-RRM2 resonances upon addition of ssFUSE29 or TGTGT pentamer. CSP by the ssFUSE29 (blue) largely overlap with CSP by the TGTGT pentamer (yellow) defining a common binding surface (green). Small differences are observed in the protein N-terminal helix, that is opposite to the dimerization area which can be attributed to transient contact with additional nucleotides in the longer DNA. FIR RRM1-RRM2 does not dimerize on ssFUSE29 DNA. These structural representations were generated using the program PYMOL (www.pymol.org).

FIR–FBP interaction represents a novel recognition mode in the RRM family. Ribbon representations of the structures of protein–protein complexes between RRM domains (grey) and their binding partners (blue and green): Y14–Mago–PYM (1rk8), UPF3b–UPF2 (1uw4), p14–SF3b155 (2f9d), Raver1–Vinculin (3h2u), U2AF35–U2AF65 (1jmt), U2AF65–SF1 (1o0p), SPF45–SF3b155 (2peh), U2B″–U2A′ (1a9n) and FIR–FBP (this study). In the PTB–Raver1 complex the Raver1 peptide position is reported (blue) on the structure of PTB RRM2 in the complex with RNA (2adb), according to the published model 22. U2AF35, U2AF65 and SPF45 RRM domains belong to the UHM subfamily, that bind a conserved Trp residue in the ligand peptides and includes the third RRM of PUF60, an isoform of FIR protein 25. The cartoon representation of the different structures were generated using the program MOLMOL 26.

FBP Nbox recruits FIR to ssFUSE DNA. (a) BLI binding assays show that the presence of the Nbox in FBP construct increases the affinity of FIR for the DNA. The BLI biosensors were derivatized with ssFUSE40 and exposed to different combinations of protein constructs (indicated on the right), as reported in the figure. RU changes in three parallel experiments are displayed. (b) FBP3 Nbox does not mediate recruitment of FIR to the DNA. BLI biosensors were derivatized with ssFUSE40 and exposed to different combinations of protein constructs (indicated on the right), as reported in the figure. The RU changes observed in three parallel experiments are displayed. (c) Isotherms for FBP and FBP3 Nbox binding to FIR RRM1-RRM2 (NMR). K_d are reported ± 2 × s.d. (d) Three different modes of FBP–FIR coupling and their effect on FIR binding and c-myc transcription. Left – In the absence of a coupling between FBP and FIR the affinity of FIR for the ssFUSE (K_d ~7 μM) would be too low for FIR to bind the ssFUSE. FBP activation of c-myc transcription (bottom) would therefore continue unperturbed. Middle – Boxed, the physiological scenario. The weak binding of the Nbox to FIR RRM2 and the 50-amino acid linker between the Nbox and KH1 are responsible for the physiological weak coupling between FBP and FIR. This coupling is necessary to create the required peak in c-myc expression (bottom) and regulate the cell cycle. Right – A stronger coupling between FBP and FIR created by, for example, a small molecular weight compound binding at the interface between the two molecules that would increase two orders of magnitude the apparent affinity of FIR for the FBP–ssFUSE complex, would speed FIR recruitment and lead to a shorter peak in c-myc expression (bottom).

FIR and FBP bind to ssFUSE DNA with different affinities. (a) Ribbon representation of FIR RRM1-RRM2. RRM1 is in light blue, RRM2 in dark blue and the amino terminal and linker alpha helices (α_N and α_L) are colored in white-to-light blue and a light blue-to-dark blue color gradients, respectively. (b) DNA binding surface of FIR RRM1. Residues showing substantial chemical shift perturbation (CSP) upon titration with the ssFUSE29 are in blue, residues whose amide resonance is not assigned are in dark grey. The position of the single nucleotide visible in the X-ray structure (orange) has been reported on the FIR RRM1-RRM2. The side chains of the aromatic residues located in the conserved RNP2 and RNP1 nucleic acids binding motifs (Tyr115, Phe155, Phe157) are displayed. The DNA molecule binds the canonical nucleic acid binding surface of the RRM1 domain. The structural representations in Figures 2a and 2b were generated using the program PYMOL (www.pymol.org). (c) Top – Graphic representation of FIR RRM1-RRM2 DNA sequence preference. The picture was generated by plotting our SIA data with the Weblogo server (http://weblogo.berkeley.edu/logo.cgi). Bottom – NMR binding isotherms of four ssDNA pentamers interacting with FIR RRM1-RRM2. The experiments were performed in 50 mM NaCl at 37 °C. RRM1-RRM2 has a moderate sequence specificity for Ts or Gs in all nucleobase positions. K_d are reported ± 2 × s.d. (d) Binding of increasing concentrations of FBP Nbox-KH1-KH4 (0.5 nM, 1 nM, 2 nM, 4 nM, 8 nM) (left), FBP KH1-KH4 (0.5 nM, 1 nM, 2 nM, 4 nM, 8 nM) (middle) and FIR RRM1-RRM2 (0.31 μM, 0.63 μM, 1.25 μM, 2.5 μM, 5 μM and 10 μM) (right) to ssFUSE40, as recorded by BLI. The experiments were performed in 150 mM NaCl at 25 °C. ssFUSE binding to FBP is three orders of magnitude tighter than FUSE binding to FIR. K_d are reported ± s.d.

FBP Nbox interacts with FIR RRM2 using a sparsely packed hydrophobic surface. (a) Left – Isotherm for FBP Nbox binding to FIR RRM1-RRM2 (NMR). K_d is reported ± 2 × s.d. Right – Superimposed ¹⁵N–¹H correlation spectra recorded during a titration of FIR RRM1-RRM2 with FBP Nbox. The spectra are color-coded according to the peptide:protein ratio: from red (0:1) to purple (6:1). (b) Left – Weighted chemical shift changes of FIR RRM1-RRM2 upon addition of FBP Nbox versus protein sequence. Right – Weighted chemical shift changes of FBP Nbox upon addition of FIR RRM1-RRM2 versus peptide sequence. NH peaks that disappear during the titration are assigned a Δδ_avg value of 0.28. (c) Solution structure of FIR RRM1-RRM2 (surface representation) in complex with FBP Nbox (ribbon representation – blue). FIR RRM1-RRM2 residues showing substantial chemical shift perturbation upon binding to FBP Nbox are colored in red. The hydrophobic residues of FBP Nbox involved in the interaction (left) and the four alanine residues of FBP Nbox in the central part of the interface (right) are labeled. The FIR RRM2 secondary structure elements involved in peptide binding are indicated. This structural representation was generated using the program PYMOL (www.pymol.org). (d) Surface representations of FIR RRM2 (left), of the solvent exposed surface of FBP Nbox (middle), and of the FIR-interacting surface of FBP Nbox (right). Hydrophobic residues are shown in yellow, residues with positive and negative charges are shown in blue and red respectively. This structural representation was generated using the program PYMOL (www.pymol.org).

FIR RRM1-RRM2 independently binds FBP Nbox and ssFUSE on two physically separated sites located on opposite sides of the molecule. (a) Surface representation of FIR RRM1-RRM2. Residues showing substantial CSP upon addition of ssFUSE29 or FBP Nbox peptide are colored in blue and red respectively. This structural representation was generated using the program PYMOL (www.pymol.org). (b) Superimposed ¹⁵N-¹H correlation spectra show that FIR RRM1-RRM2 interacts independently with a ssFUSE29 and the FBP Nbox peptide. The spectra of RRM1-RRM2+ssFUSE DNA, RRM1-RRM2+FBP Nbox and RRM1-RRM2+ssFUSE DNA+FBP Nbox are in blue, red and green respectively. A representative region containing one RRM1 resonance perturbed by DNA binding (Val114) and one RRM2 resonance perturbed by peptide binding (Asp222) is displayed. The chemical shift changes of RRM1-RRM2 peaks in the protein–DNA and protein–peptide complexes are additive in the three molecule complex, indicating independent binding.