Figure 1.
The definition of WD40 blade and binding faces of WD40-repeat protein.
(a) In sequence order, every WD40 repeat protein is composed of strand d–a–b–c. Structurally, the first strand d and the last strand a–b–c form a four-strand anti-paralleled β-sheet, a WD40 blade, which encloses β-propeller structure. The other WD40 blades are formed by sequential strand a–b–c–d. (b) WD40 proteins have top, side and bottom faces. Top face: loops connecting strand b–c and strand d–a; Bottom face: loops connecting strand a–b and strand c–d; Side face: the grooves between two neighbored WD40 blades. (c) A majority of interactions are focused on the top faces.
Figure 2.
Empirical methods for identifying hotspots on the top faces of WD40 proteins.
(a). Identifying hotspots by two sequential steps: 1. Determining loops composed of top faces; 2. Choosing the functional residues on two loops. (b). The 16th, 18th and 34th residues are repeatedly involved in the protein-protein interaction. Here, strand d–a–b–c was presented in the sequence order. (c). Three hotspots proposed by Stirnimann et al.3 (marked by red-stars) are incorrectly mapped on the common sequence pattern.
Figure 3.
The hydrogen bonds and structural features in the general WD40 blade by strand a–b–c–d.
Although the enclosed WD40 blade has the different order in sequence, their hydrogen bonds, β-bulges and DHSW tetrad are in common. the DHSW tetrad: Asp-His-Ser/Thr-Trp (in red). WDb–a β-bulge: formed in strand b and a; WDc–d β-bulges: formed in strand c and d. X, R1, R2 (in blue). Three residues, R1-2, R1 and D-1 colored in pink are highlighted here for the following discussion of interaction on the top face. R1 as a binding residue is “R1 residue” of WDb–a bulge. R1-2 locates on two residues before R1 of WDb–a. D-1 locates before Asp of DHSW tetrad.
Figure 4.
The summation of WDb–a and WDc–d β–bulges.
(a) The schematic view and residue dominances of WDb–a and WDc–d bulges. (b) Two-side view of β-bulge. X, R1:α-conformation; R2: β-conformation.
Table 1.The.
percentages of the 20 natural amino acid residues in the X, R1 and R2 positions of WDb–a and WDc–d β-bulges.
Figure 5.
The interaction involved in WDb–a β-bulges between two neighbored WD40 blades.
(a) and (b) show the hydrogen-bonding interactions provided by two WDb–a of neighboring WD40 blades in 3D and 2D, respectively. Those residues composed of WDb–a bulges are shown in stick as numbered in BUB3 (1YFQ). The hydrogen bonds, HB1 and HB2, are formed inter-blade by Ser213 backbone (X+1 position) and Ser212 (X position) side chain with Val254 (R1-1) at the other WD40 blade. (c) χ2 dihedral angles of R1 residues. As χ2 is at gauche-conformation, Arg points upward. If χ2 is at anti-conformation, Arg point to WD40-1, which is unacceptable due to the crowdedness.
Figure 6.
The summation of crystallographic and mutagenesis studies of hotspots on the top faces of 19 representative WD proteins.
As depicted on the top left corner, every WD40 blade is composed of four-strand anti-paralleled β-sheet implicitly. R1-2, R1 and D-1 residues are colored by blue, red and green, respectively. All listed residues are supposed to be important for protein-protein interaction on the top face. Some of them highlighted by the underlines were further convinced by mutagenesis studies.
Figure 7.
Three representative binding modes provided by WD40 proteins.
Binding negatively-charged group: R465 and R505 of two neighboring WD40 blades at FBW7 interact with phosphorylated Thr80 of Cyclin E. Binding positively-charged group: EED applies two R1, F97 and Y147, as well as Y365 to selectively interact with trimethyl-H3K27. Interacting hydrophobic group: Hydrophobic ring formed by a series of residues in TLE1 accommodates WRPW tetrapeptide. R1-2, R1 and D-1 residues are colored by blue, red and green, respectively.
Figure 8.
R1-2, R1 and D-1 are predicted from WD40 blade primary sequence with the use of structural features.
The residues of WD40 blade are in the order of frequencies along the vertical line. The blue-colored residues are those in the DHSW tetrad. R1, R2 and X of β-bulges are highlighted by green color except for R1 of WDb–a. The red-colored residues, R1-2, R1 and D-1, are potential hotspots. Fa and Fb are two fingerprint sequences. η represents bulky residues: Ile, Leu and Val. χ represents Trp, Phe, Tyr, Ile, Leu and Val. ψ represents residues with small side chains: Gly, Ala, Ser, Cys, Thr. φ are potential hotspots.
Figure 9.
The correlation of R1 propensity between x-ray crystal structure and WD40 blade primary sequences.
The most dominant residues, Ser, Thr, Asn, Arg, Leu, Trp, Phe, Tyr and Lys are qualitatively consistent.
Figure 10.
The consistency of predicted R1, R1-2 and D-1 in WD40 blades of Met30p and MDV1p with the mutagenesis studies.
The blue-colored residues are in the predicted DHSW tetrad. The green-colored residues are in the predicted WDb–a and WDc–d bulges. The red-colored residues were studied by the mutagenesis. The last WD40 blade is composed of strand d at the beginning of WD40 domain and the last strand a, b and c.
Figure 11.
The potential “hotspots” on the top face of Tup11.
The predicted R1-2, R1 and D-1 residues in WD40 blades marked by red-color. The underlined residues besides Cys337 and Thr467 are substituted by Ala or Ser. The last WD40 blade is composed of strand d at the beginning of WD40 domain and the last strand a, b and c.
Figure 12.
ITC measurements of Fep1 and Tup11 wild type as well as its Ala substitutions.
(a). The ITC profile of the wild type Tup11-Fep1 interaction. (b). The measured binding affinities of the interactions between Tup11 as well as its mutants and Fep1 by ITC. (c). The critical residues on top face of Tup11.