Fig 1.
Atomic resolution structure of MsCI4•thalidomide.
(A) The overall structure of MsCI4•thalidomide from the trigonal crystal form. (B) Superposition of the latter (in green) with the three MsCI4•thalidomide monomers from the orthorhombic crystal form (4V2Y, in yellow) and the two MsCI4•thalidomide monomers from the hexagonal crystal form (this work, in red). While the conformation of the protein and the glutarimide moiety is virtually identical, the orientation of the phthaloyl moieties differ by up to 13° between the structures. (C) Stereo close-up of the thalidomide molecule and the three tryptophan residues of the aromatic cage of the atomic resolution structure, together with an omit electron density map at 1.2 Å resolution. The omit map was calculated for thalidomide and the three indoles and is contoured at 6.5 sigma. Whereas individual atoms of the indoles and of the glutarimide ring are sharply localized in the density, the phthaloyl moiety is poorly resolved due to thermal disorder. (D) Only thalidomide with omit map. (E) Only the tryptophans with omit map.
Table 1.
Crystallization conditions and cryo protection / washing procedure.
Table 2.
Data collection and refinement statistics.
Fig 2.
Partial unfolding of MsCI4 upon loss of thalidomide.
Crystals of the orthorhombic crystal from two crystallization conditions were washed for 40 h in a solution without thalidomide. For the initial structure (grey) and both experiments, “Wash I” (brown) and “Wash II” (black), the monomer that had the ligand washed out (chain C) is depicted in two perspectives. Loss of ligand was accompanied by the unfolding of three regions. The structures after washing are overlaid with the initial structure in transparent. On the bottom, the sequence alignment of MsCI4 with human cereblon details the unfolded regions and indicates secondary structure elements. The unfolded regions are shaded. In “Wash I”, the unfolding of the first region is incomplete, so the hairpin formed by the 3rd and 4th β-strand is still folded, albeit shifted in position. The C-terminal segment deleted in the R419X mutant of human cereblon and the corresponding segment in MsCI4 are underlined. Key-residues are highlighted red.
Fig 3.
MsCI4 in different conformations in the hexagonal crystal form.
Top: The 4 monomers in the ASU are found in three different conformations. The yellow and green monomer are in the known thalidomide-bound conformation. The blue monomer is in a conformation similar to the thalidomide-bound one, but its binding site is occupied by a DMSO molecule, which is accompanied with a displacement of one tryptophan of the aromatic cage (red). The pink monomer is in an overall distorted conformation and does not have the aromatic cage formed: the β3-β4 hairpin has another strand register, one of the tryptophans is flipped, and another tryptophan is displaced by 30Å (all in red). A second conformation of the 3rd flexible region is displayed in another shade of red. Bottom: The blue and the pink monomers form an intertwined dimer, in which the displaced tryptophan of the pink monomer completes the aromatic cage of the blue one, trapping a DMSO molecule in a binding site with a modified architecture. Here, the second conformation of the 3rd flexible region of the pink monomer is in orange.
Fig 4.
An ensemble of the available MsCI4 conformations.
(A) Superposition of the thalidomide-bound conformation (yellow), the blue and pink monomers from Fig 3, the “Wash I” structure (thin dark grey), and the “Wash II” structure (thick dark grey). One alternate conformation of the pink monomer in the 3rd flexible region is depicted in purple. (B) same as (A), but from the other perspective and in stereo. (C) The consensus (Wash II) structure together with the individual ensembles of the 1st, 2nd and 3rd flexible regions.
Fig 5.
Biophysical characterization of MsCI4 and mutants.
(A) Far-UV CD spectra of wild-type (WT) MsCI4 and mutants MsCI4WW/FF and MsCI4WWK/FFX, and (B) corresponding melting curves, monitored at 222 nm. (C) Thermal stability of MsCI4 and binding deficient MsCI4YW/AA in presence of thalidomide, uridine, thymidine and cytidine, as determined in the thermal shift assay. Only thalidomide and uridine lead to a shift in melting temperature with (two-tail) p-values of 1.5e-3 and 4.9e-2 as determined in a two sample equal variance t-test. (D) Tryptophan fluorescence spectra of MsCI4WW/FF in presence of uridine, thymidine and cytidine. The emission maximum is shifted to shorter wavelength only in presence of the binder uridine, which can be indicative for the folding of the binding site. Note also the pronounced quenching effect indicative for binding. (E) same as (C) for the MsCI4WWK/FFX mutant. As for the WT, thalidomide and uridine lead to a thermal shift, with p-values of 4.0e-4 and 1.2e-3. (F) same as (D) for the MsCI4WWK/FFX mutant. The presence of uridine has the same effect as on the WT.
Fig 6.
The thalidomide binding domain of mouse cereblon exhibits flexibility comparable to MsCI4.
The mouse domain in complex with (blue, sand) and without thalidomide (yellow, shades of brown) is compared to MsCI4•thalidomide (transparent white). (A) Two thalidomide-bound domains from 4TZC arranged as an intertwined dimer. (B) Superposition of one monomer to MsCI4•thalidomide, illustrating the unfolded nature of the first flexible region. (C) Same as (B) but from another perspective, in stereo. (D) Apo mouse domains in 3WX2 are found in two conformations, “yellow” and “brown”, forming an endless array of interactions in the crystal lattice. (E) Superposition of both apo conformations onto MsCI4•thalidomide, showing the first flexible region in different conformations. (F) Same as (E) but from another perspective, in stereo. Note that the conformation of the tryptophans on the left is reminiscent of the flipped tryptophan in the intertwined MsCI4 dimer.
Fig 7.
The flexible regions and mental retardation mutation in the context of human full length cereblon.
At the top, interaction partner DDB1 is represented by a sphere. The thalidomide binding domain is in green (rigid core), yellow (flexible regions) and red (deleted C-terminal part in the MsCI4WWK/FFX mutant). The exact boundaries of the segments are defined in Fig 2. The N-terminal extension of cereblon is in brown, the remainder of the protein grey.