Molecular mechanism of MLL PHD3 and RNA recognition by the Cyp33 RRM domain

Abstract

The nuclear protein cyclophilin 33 (Cyp33) is a peptidyl-prolyl cis-trans isomerase that catalyzes cis-trans isomerization of the peptide bond preceding a proline and promotes folding and conformational changes in folded and unfolded proteins. The N-terminal RNA-recognition motif (RRM) domain of Cyp33 has been found to associate with the third plant homeodomain (PHD3) finger of the mixed lineage leukemia (MLL) proto-oncoprotein and a poly(A) RNA sequence. Here, we report a 1.9 A resolution crystal structure of the RRM domain of Cyp33 and describe the molecular mechanism of PHD3 and RNA recognition. The Cyp33 RRM domain folds into a five-stranded antiparallel beta-sheet and two alpha-helices. The RRM domain, but not the catalytic module of Cyp33, binds strongly to PHD3, exhibiting a 2 muM affinity as measured by isothermal titration calorimetry. NMR chemical shift perturbation (CSP) analysis and dynamics data reveal that the beta strands and the beta2-beta3 loop of the RRM domain are involved in the interaction with PHD3. Mutations in the PHD3-binding site or deletions in the beta2-beta3 loop lead to a significantly reduced affinity or abrogation of the interaction. The RNA-binding pocket of the Cyp33 RRM domain, mapped on the basis of NMR CSP and mutagenesis, partially overlaps with the PHD3-binding site, and RNA association is abolished in the presence of MLL PHD3. Full-length Cyp33 acts as a negative regulator of MLL-induced transcription and reduces the expression levels of MLL target genes MEIS1 and HOXA9. Together, these in vitro and in vivo data provide insight into the multiple functions of Cyp33 RRM and suggest a Cyp33-dependent mechanism for regulating the transcriptional activity of MLL.

Figure 1

The crystal structure of the RRM domain of Cyp33 determined at 1.85 Å resolution. (a) Architecture of Cyp33: the amino-terminal RRM domain and the catalytic CYP domain. (b) Ribbon diagram of the RRM structure.

Figure 2

Binding of the RRM domain of Cyp33 to the PHD3 finger of MLL. (a) Schematic of MLL. The PHD3 finger is shown as a white oval. (b) Superimposed ¹H,¹⁵N HSQC spectra of the ¹⁵N-labeled Cyp33 RRM domain, collected in the absence and presence of a two-fold excess of unlabeled MLL PHD3. (c) Superimposed ¹H,¹⁵N HSQC spectra of the ¹⁵N-labeled MLL PHD3 finger, collected in the absence and presence of a two-fold excess of unlabeled Cyp33 RRM. (c) Representative ITC curves used to calculate the binding affinity for the interaction between the Cyp33 RRM domain and the MLL PHD3 finger.

Figure 3

The MLL PHD3-binding site of the Cyp33 RRM domain. (a) A histogram shows normalized ¹H,¹⁵N chemical shift changes in backbone amides of ¹⁵N-labeled RRM upon addition of unlabeled PHD3 at a protein ratio of 1:1. (b, d) Residues that exhibit significant PHD3-induced resonance perturbations in (a) are mapped on the ribbon diagram (b) and the surface (d) of the Cyp33 RRM domain. Colored bars indicate significant change being greater than an average plus one standard deviation. (c) The binding affinities of wild type Cyp33 RRM and mutants for MLL PHD3, as measured by ITC. Interaction of the RRMΔ40–45 mutant was examined by NMR. Other mutants generated, including G48A, F54A, RRMΔ41–42, RRMΔ41–43 and RRMΔ40–43, precipitated during dialysis for ITC experiments.

Figure 4

Dynamics of the Cyp33 RRM domain bound and unbound. (a–c) Relaxation parameters of the RRM domain in the absence (blue) and the presence (red) of a 1.5-fold excess of MLL PHD3. R₁, R₂ and NOE values were determined for backbone amide groups and are plotted for each residue of the Cyp33 RRM domain. The RRM secondary structure is shown above the graphs. (d) The most affected (due to the interaction with PHD3) residues of Cyp33 RRM are colored in shades of purple in the ribbon diagram of the RRM structure.

Figure 5

The RNA-binding site of the Cyp33 RRM domain. (a) Eight superimposed ¹H,¹⁵N HSQC spectra of 0.1 mM Cyp33 RRM, collected as the AAUAAA RNA construct was gradually added. The spectra are color-coded according to the molar protein-RNA ratio (inset). (b) A histogram shows normalized ¹H,¹⁵N chemical shift changes in backbone amides of ¹⁵N-labeled RRM (aa 1–90) upon addition of a six-fold excess of AAUAAA. (c, d) Residues that exhibit significant RNA-induced resonance perturbations in (b) are labeled and colored in shades of green on the ribbon diagram of the structure (c) and on the surface (d) of the Cyp33 RRM domain (aa 1–83). Colored bars indicate significant change being greater than an average plus one and a half standard deviation. (e) Representative binding curves used to determine the K_d values of the Cyp33 RRM-RNA interaction by NMR spectroscopy. (f) The RNA binding affinities of the wild type and mutant RRM domain measured by NMR. (g) Alignment of the RRM domain sequences of human proteins: absolutely, moderately and weakly conserved residues are colored brown, green and yellow respectively. The RNP2 and RNP1 motifs required for the interaction with RNA are outlined by red rectangles. The secondary structure of the Cyp33 RRM domain is shown below the sequences.

Figure 6

The MLL PHD3-binding site and the RNA-binding site of the Cyp33 RRM domain partially overlap. (a) The Cyp33 RRM domain is shown as a ribbon diagram. Residues of RRM that are perturbed by either PHD3, RNA or both ligands are colored red, green and yellow, respectively. The MLL PHD3-binding site and the RNA-binding site of the Cyp33 RRM domain are indicated by red and green circles, respectively. (b) Superimposed ¹H,¹⁵N HSQC spectra of the Cyp33 RRM domain (0.1 mM) in the ligand-free form (black), after addition of 0.6 mM RNA (green), and after subsequent addition of 0.25 mM MLL PHD3 (red).

Figure 7

Cyp33 decreases MLL-dependent gene transcription. Gene expression levels were quantified in transfected 293T cells using quantitative real-time RT-PCR (a). (b) A model of the Cyp33-MLL association.