Validation of the solution structure of dimerization domain of PRC1

Abstract

Cell-cycle dependent proteins are indispensible for the accurate division of cells, a group of proteins called Microtubule-associated proteins (MAPs) are important to cell division as it bind microtubules and participate with other co-factors to form the spindle midbody, which works as the workhorse of cell-division. PRC1 is a distinguishing member of MAPs, as it is a human MAP and works as the key in mediating daughter cell segregation in ana-phase and telo-phase. The physiological significance of PRC1 calls for a high resolution three-dimensional structure. The crystal structure of PRC1 was published but has low resolution (>3 Å) and incomplete sidechains, placing hurdles to understanding the structure-function relationships of PRC1, therefore, we determined the high-resolution solution structure of PRC1's dimerization domain using NMR spectroscopy. Significant differences between the crystal structure and the solution structure can be observed, the main differences center around the N terminus and the end of the alpha-Helix H2. Furthermore, detailed structure analyses revealed that the hydrophobic core packing of the solution and crystal structures are also different. To validate the solution structure, we used Hydrogen-deuterium exchange experiments that address the structural discrepancies between the crystal and solution structure; we also generated mutants that are key to the differences in the crystal and solution structures, measuring its structural or thermal stability by NMR spectroscopy and Fluorescence Thermal Shift Assays. These results suggest that N terminal residues are key to the integrity of the whole protein, and the solution structure of the dimerization domain better reflects the conformation PRC1 adopted in solution conditions.

Fig 1. Two-dimensional ¹H-¹⁵N HSQC spectrum and assignment of PRC1-DD.

Fig 2. Solution structures of PRC1-DD.

(a) Ribbon representation of the 20 lowest-energy structures. (b) Backbone chain trace of the 20 lowest-energy structures; helices H1 and H2 from the two monomeric units in the homodimer are colored separately in blue (H1, M1-E28), cyan (loop, I29-D32) and purple (H2, E34-L65); C and N tags signify C- and N-terminal ends, respectively. This and all other structural figures were generated using the program CHIMERA.

Fig 3. Comparison of the solution structure of PRC1-DD and the corresponding segments in crystal structure.

(a) Superimposition of ribbon representation of the PRC1-DD lowest-energy solution structure and crystal structure; H1 and H2 of the solution structure are colored yellow and orange, respectively, while H1 and H2 of the crystal structure are colored in cyan and blue, respectively; C and N tags signify C- and N-terminal ends, respectively. (c) Enlargement of the dotted box in (a). Figures were plotted using MATLAB, while the alignment was performed using CHIMERA.

Fig 4. Differences between solution and crystal structures.

(a)–(b) The hydrophobic-core composition of (a) solution and (b) crystal structures with residues having significant differences being tagged. (c) Relative solvent-accessible surface area (SASA) as a function of residue number (x-axis); each residue SASA value is represented as a black dot; green continuous line belongs to the SASA values of the crystal structure, while dotted red line represents solution-structure SASA values.

Fig 5. H/D exchange of PRC1-DD.

(a) Bar graph showing the protection factor of each amide proton in PRC1-DD, schematic representations of PRC1-DD structure were also provided on top of the figure. (b) Bar graph showing the unfolding free energy (ΔGex) of each residue. (c) Experimentally determined free energy of unfolding (ΔGex) mapped to PRC1-DD solution structure. The ΔG_ex ranges are: fast exchangers (purple) ΔGex ≥ 30000; medium exchangers (white) ΔG_ex = 15000–30000; slow exchangers (cyan) ΔG_ex ≤ 15000. Figures were drawn using CHIMERA.

Fig 6. Two-dimensional ¹H-¹⁵N HSQC spectra of Δ1R2M and R2E.

Ribbon representation of the (a) solution structure (gold) and (b) crystal structure (cyan) with the side chains M1 and R2 in orange and green, while the side chains of the residues interacting with M1 or R2 are shown in blue and mud yellow for solution and crystal structures, respectively. (c) Two-dimensional ¹H-¹⁵N HSQC spectra of Δ1R2M. (d) Two-dimensional ¹H-¹⁵N HSQC spectra of R2E, residues experiencing great chemical-shift changes (Δ comp ≥ 0.3 ppm) are labeled.

Fig 7. Fluorescence thermal shift assay of PRC1-DD and its mutants (bar graph representation of data in Table 2).

(a) Bar graph showing the mid-denaturation temperature (Tm) of wild-type PRC1-DD and its mutants. The name of each mutant is labeled on the x-axis of the figure, R2M refers to Δ1R2M. (b) Changes in Tm (ΔTm) of mutants compared with wild type. The respective mutants’s colors are as labeled. Graphs were produced in MATLAB, plotted from the mean and standard deviations (thin red T) of Tm from four parallel experiments; single star indicates statistical significance (p<0.05) and double star indicates strong significance (p<0.01).

Fig 8. Two-dimensional ¹H-¹⁵N HSQC spectrum of L50E and L51E.

Ribbon representation of the (a) solution structure (gold) and (b) crystal structure (cyan) with the side chains L50 and L51 in orange and green while the side chains of the residues interacting with L50 or L51 shown in blue and mud yellow for solution and crystal structures respectively. (c) Two-dimensional ¹H-¹⁵N HSQC spectra of L50E. (d) Two-dimensional ¹H-¹⁵N HSQC spectra of L51E, residues experiencing great chemical shift changes (Δ comp ≥ 0.3 ppm) were labeled.

Fig 9. Two-dimensional ¹H-¹⁵N HSQC spectra of E57A and E58A.

(a)-(b) Ribbon representation of the solution structure (gold) and crystal structure (cyan) with the side chains E57 and E58 in orange and green while the side chains of the residues interacting with E57 or E58 shown in blue and mud yellow for solution and crystal structures respectively. (c) Two-dimensional ¹H-¹⁵N HSQC spectra of E57A. (d) Two-dimensional ¹H-¹⁵N HSQC spectra of E58A, all residues with significant chemical shift changes (Δ comp ≥ 0.3 ppm) were labeled.

Fig 10. Results from fluorescence thermal shift assay (FTSA) of PRC1 1–486 and its mutants (bar graph representation of data in Table 3).

(a) Bar graph showing the mid-denaturation temperature (Tm) of wild-type PRC1 1–486 and its mutants. The name of each mutant is labeled on the x-axis of the figure, R2M refers to Δ1R2M. (b) Changes in Tm (ΔTm) of mutants compared with wild type. The mutants are color-coded according to the figure legend. Graphs were produced in MATLAB; the mean and standard deviation of Tm were calculated from four parallel runs of each experiment. single star indicates statistical significance (p<0.05) and double star indicates strong significance (p<0.01).

Fig 11. Schematic diagram of full-length PRC1 (a) crystal and (b) solution structure.

Fig 12. Two-dimensional ¹H-¹⁵N HSQC spectra of N terminal extension mutant (red) and wildtype (black).