The solution structure of the RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML.

Abstract

Acute promyelocytic leukaemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and the retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex (termed ND10, Kr bodies, nuclear bodies, PML oncogenic domains or PODs) which is disrupted in the APL disease state. PML contains a number of characterized motifs including a Zn2+ binding domain called the RING or C3HC4 finger. Here we describe the solution structure of the PML RING finger as solved by 1H NMR methods at physiological pH with r.m.s. deviations for backbone atoms of 0.88 and 1.39 A for all atoms. Additional biophysical studies including CD and optical spectroscopy, show that the PML RING finger requires Zn2+ for autonomous folding and that cysteines are used in metal ligation. A comparison of the structure with the previously solved equine herpes virus IE110 RING finger, shows significant differences suggesting that the RING motif is structurally diverse. The role of the RING domain in PML nuclear body formation was tested in vivo, by using site‐directed mutagenesis and immunofluorescence on transiently transfected NIH 3T3 cells. Independently mutating two pairs of cysteines in each of the Zn2+ binding sites prevents PML nuclear body formation, suggesting that a fully folded RING domain is necessary for this process. These results suggest that the PML RING domain is probably involved in protein‐protein interactions, a feature which may be common to other RING finger domains.

The promyelocytic leukemia protein PML interacts with the proline-rich homeodomain protein PRH: a RING may link hematopoiesis and growth control

“…For PML, disruption of other regions of the protein, including the leucine coiled coil and B-box motifs, did not a ect its ability to bind PRH indicating that these domains were not required for interaction. Western and immuno¯uorescence analysis con®rmed that PML, Z and PRH mutants used were produced to levels similar to respective wild-type proteins (Borden et al, 1995(Borden et al, , 1998a; data not shown).…”

“…Both colorometric and liquid culture assays were used to monitor results ( Figure 1a,b). For both PML and Z point mutations which caused misfolding of the RING domain (Borden et al, 1995(Borden et al, , 1998a, denoted PMLRING and ZRING, abrogated the ability of either protein to bind PRH. Other regions of PML were deleted to determine if they were necessary for the interaction.…”

“…RINGs are involved in protein interactions, probably do not bind nucleic acids directly and are often involved in macromolecular assemblages. Consistent with this possibility, PML nuclear bodies are disrupted if any of the zinc-binding residues in the RING or Bboxes are mutated (Borden et al, 1995Le et al, 1996). One 90 residue RING protein from an arenavirus, Z, associates with PML nuclear bodies, binds directly to PML, and can translocate bodies to the cytoplasm (Borden et al, 1998a).…”

“…Full-length human PML and Z cDNA were described (Borden et al, 1995(Borden et al, , 1998a and cloned into constructs with the GAL4 DNA binding domain (pAS-PML and pAS-Z) in order to carry out yeast 2-hybrid studies. Double point mutations in the RING domains of PML and Z which abrogate zinc binding and therefore folding of the RING were described (Borden et al, 1995(Borden et al, , 1998a.…”

Modulation of Fos-mediated AP-1 transcription by the promyelocytic leukemia protein

“…Conversely, another report indicated that both PML and the translocation fusion product PMLRARa are capable of inducing the transcriptional activity of steroid hormone receptors, and altering their subcellular distribution (GuiochonMantel et al, 1995). Because, PML does not bind DNA, it is postulated that its e ects on transcription are through protein-protein interactions with other factors involved in transcription regulation (Borden et al, 1995). Supporting the above hypothesis, recent analysis of the solution structure of the PML RING®nger suggests that this region may be involved in protein-porotein interactions (Borden et al, 1996).…”