N-Terminus-Mediated Solution Structure of Dimerization Domain of PRC1

Abstract

Microtubule-associated proteins (MAPs) are essential for the accurate division of a cell into two daughter cells. These proteins target specific microtubules to be incorporated into the spindle midzone, which comprises a special array of microtubules that initiate cytokinesis during anaphase. A representative member of the MAPs is Protein Regulator of Cytokinesis 1 (PRC1), which self-multimerizes to cross-link microtubules, the malfunction of which might result in cancerous cells. The importance of PRC1 multimerization makes it a popular target for structural studies. The available crystal structure of PRC1 has low resolution (>3 Å) and accuracy, limiting a better understanding of the structure-related functions of PRC1. Therefore, we used NMR spectroscopy to better determine the structure of the dimerization domain of PRC1. The NMR structure shows that the PRC1 N terminus is crucial to the overall structure integrity, but the crystal structure bespeaks otherwise. We systematically addressed the role of the N terminus by generating a series of mutants in which N-terminal residues methionine (Met1) and arginine (Arg2) were either deleted, extended or substituted with other rationally selected amino acids. Each mutant was subsequently analyzed by NMR spectroscopy or fluorescence thermal shift assays for its structural or thermal stability; we found that N-terminal perturbations indeed affected the overall protein structure and that the solution structure better reflects the conformation of PRC1 under solution conditions. These results reveal that the structure of PRC1 is governed by its N terminus through hydrophobic interactions with other core residues, such hitherto unidentified N-terminal conformations might shed light on the structure−function relationships of PRC1 or other proteins. Therefore, our study is of major importance in terms of identifying a novel structural feature and can further the progress of protein folding and protein engineering.

Keywords: N-terminal domain of PRC1; N-terminus-mediated core packing; homodimerization; hydrophobic core packing; protein regulator of cytokinesis; solution structure.

Figure 1

Two-dimensional ${}^{1}H{-}^{15}N$ HSQC spectra and assignment of PRC1-DD.

Figure 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.

Figure 3

Comparison of the solution structure of PRC1-DD and the corresponding segments in the 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 cyan and blue, respectively. C and N tags signify C- and N-terminal ends, respectively. (b) A different orientation of (a). (c) Enlargement of the dotted box in (a,b). (d) Bar graph showing the RMSD values of residues between solution structure and the corresponding segment in the crystal structure for a single monomeric unit when aligning all backbone heavy atoms. Figures were plotted using MATLAB; the alignments were performed using CHIMERA.

Figure 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’s SASA value is represented as a black dot, the green continuous line belongs to the SASA values of the crystal structure, while the dotted red line represents solution-structure SASA values.

Figure 5

Schematic diagram of PRC1-DD (a): crystal and (b) solution structure.

Figure 6

Two-dimensional ${}^{1}H{-}^{15}N$ HSQC spectra of N-terminal extension mutant (red) and wild type (black).

Figure 7

Two-dimensional ${}^{1}H{-}^{15}N$ HSQC spectra of $\Delta$1R2M 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 ${}^{1}H{-}^{15}N$ HSQC spectra of $\Delta$1R2M. (d) Two-dimensional ${}^{1}H{-}^{15}N$ HSQC spectra of R2E; residues experiencing great chemical shift changes ($\Delta$ comp ≥ 0.3 ppm) are labeled.

Figure 8

Fluorescence thermal shift assay of PRC1-DD, PRC1-486 and its mutants (bar graph representation of data in Table 2 and Table 3). (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. (b) 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 $\Delta$1R2M. Graphs were produced in MATLAB, plotted from the mean and standard deviations (thin red T) of Tm from four parallel experiments; single star indicates statistical significance ($p<0.05$) and double star indicates strong significance ($p<0.01$).