Cryo-electron microscopy structure of a human PRMT5:MEP50 complex

Abstract

Protein arginine methyl transferase 5 (PRMT5) is a signaling protein and histone modifying enzyme that is important in many cellular processes, including regulation of eukaryotic gene transcription. Reported here is a 3.7 Å structure of PRMT5, solved in complex with regulatory binding subunit MEP50 (methylosome associated protein 50, WDR77, p44), by single particle (SP) cryo-Electron Microscopy (cryo-EM) using micrographs of particles that are visibly crowded and aggregated. Despite suboptimal micrograph appearance, this cryo-EM structure is in good agreement with previously reported crystal structures of the complex, which revealed a 450 kDa hetero-octameric assembly having internal D2 symmetry. The catalytic PRMT5 subunits form a core tetramer and the MEP50 subunits are arranged peripherally in complex with the PRMT5 N-terminal domain. The cryo-EM reconstruction shows good side chain definition and shows a well-resolved peak for a bound dehydrosinefungin inhibitor molecule. These results demonstrate the applicability of cryo-EM in determining structures of human protein complexes of biomedical significance and suggests cryo-EM could be further utilized to understand PRMT5 interactions with other biologically important binding proteins and ligands.

Fig 1. Chemical structures of SAM, SAH, sinefungin and dehydrosinefungin.

The amino acid moieties of sinefungin and dehydrosinefungin are joined to the ribose ring through a saturated single bond and a desaturated double bond, respectively.

Fig 2. Raw micrographs and 2D class averages.

(A) A section of a negative stained grid imaged with an actual magnification of 50,607x is shown. The inset in the top left corner shows the 2D class average corresponding to over 50% of the observed particles. The diamond-like shape of the PRMT5 core is well-resolved, but the MEP50 is poorly resolved. The bottom right scale bar is 90 nm. (B) A section of PRMT5:MEP50 particles embedded in vitreous ice is shown. The image is a motion corrected summation of 40 individual movie frames at an actual magnification of 50,000x. A low contrast, central ‘donut hole’ is visible in individual particles (circled). Particles of similar orientation to the (A) inset appear like ‘headless turtles’. Ice and/or ethane contamination and aggregated protein are indicated by * and **, respectively. The bottom right scale bar is 30 nm. (C) Representative 2D class averages calculated from cryo-images are shown with inverted contrast. The maximal PRMT5:MEP50 particle dimension is approximately 15 nm. Black arrows highlight small filamentous aggregates in (A) and (B), and pore loop density for residues 488–493 in (C).

Fig 3. Structures of PRMT5 and the PRMT5:MEP50 complex.

(A) An individual PRMT5 subunit from Protein Databank Entry 4GQB is represented as a ribbon diagram with dehydrosinefungin (white C atoms) and a histone H4 derived peptide (yellow C atoms) depicted as CPK spheres. The TIM barrel domain, Rossmann fold and β-barrel domains are colored blue, red and green, respectively. (B) The 3D reconstruction of the PRMT5:MEP50 complex, viewed along a dyad axis, is represented as a transparent gray map contoured at 3 rmsd (0.036). The PRMT5 subunits (colored as in Fig 3A) and the MEP50 subunits (colored magenta) were rigid body fit within the map as heterodimeric units. Black arrows highlight weak or missing density for PRMT5 residues 173–179. Density for loop residues 488–493 is highlighted by stars. Views of the 3D reconstruction along the other two central dyad axes are available in S3 Fig. (C) A resolution of 3.7 Å for the reconstruction is indicated at FSC = 0.143.

Fig 4. Details of the cryo-EM map in blue mesh.

(A) A section of β-sheet present in the PRMT5 subunit shows good strand separation and side chain densities (B). (C) A section of α-helix in PRMT5 is shown with good helical pitch and bulky side chain density. (D) The seventh β-propeller in MEP50 is shown with good strand separation and bulky side chain density. (E) A peak in the Cryo-EM map, corresponding with binding of the dehydrosinefungin inhibitor (yellow C atoms), is well-resolved from the surrounding PRMT5 groups (cyan C atoms and ribbon). Carbon, nitrogen and oxygen atoms are colored yellow, blue and red, respectively, excepting where noted for panel E. The map is contoured at 4 rmsd (0.048) in panels (A), (B) and (C), and at 3 rmsd (0.036) in panels (D) and (E).