Chapter 4. Genetic analysis of various DZF domains using mutagenesis
4.4 Analysis of the Hunchback DZF domain
4.4.1 Alanine scanning mutagenesis
An alanine scan was also performed with the DZF domain of Drosophila Hunchback PCR-mediated mutagenesis was applied to introduce individual alanine substitutions at 49 different residue positions within the DZF domain except at positions occupied by the conserved cysteines and histidines. This set of mutant proteins was then assayed using the B1H system.
As demonstrated in Figure 4.7, mutations in 18 of the 49 positions tested displayed β-galactosidase units comparable to that of wild-type, suggesting that those residues are not directly involved in dimerization. Three of these alanine substitution mutants exhibited slightly higher dimerization activity. Alanine substitutions in the remaining 31 positions tested impaired dimerization.
Predicted conserved hydrophobic residues:
Mutations in the conserved hydrophobic residues (Y07A, F16A, Y22A, F35A and L50A) all severely affected the dimerization as was the case for Ikaros. Interestingly, residue C44 was also impaired by an alanine. As mentioned above, this residue position is normally conserved in the C2H2 ZF motif (and is typically a phenylalanine) but no effect was detected for the Ikaros DZF domain when this residue was mutated.
Predicted β-sheets:
Seven mutations (K10A, Y11A, C12A, I14A, F16A, K17A and D18A) in the predicted ββ-sheet of the N-terminal finger and 5 mutations (K36A, C40A, G41A, D45A and G46A) in the ββ-sheet of the C-terminal finger disrupted dimerization whereas the remaining residue positions (E08, D13, N38, E42 and K43) in this region in both C2H2 ZFs seemed not to be impaired by alanine substitutions.
Predicted α-helices:
Affected residue positions in the predicted α-helices are L21, Y22, I24, M26, G27, Y28, V48, V52, M54, A55, R56 and A58 while the remaining positions (A19. V20, T23, S30, P47, G49, F51 and N57) in this secondary structure were not affected by alanine substitutions.
Inter-finger linker:
The linker region between the N-terminal and the C-terminal finger contained 2 additional mutations D32A and D33A that displayed severe dimerization defects.
Thus, this study identified several residue positions that may contribute to dimerization mediated by the Hunchback DZF domain. These residues could be directly involved in making contacts but could also be important for stability of the protein. Although some of
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cI-NTD WT I06A Y07A E08A C09 K10A Y11A C12A D13A I14A F15A F16A K17A D18A A19G V20A L21A Y22A T23A I24A H25 M26A G27A Y28A H29 S30A C31A D32A D33A V34A F35A K36A C37 N38A M39A C40A G41A E42A K43A C44A D45A G46A P47A V48A G49A L50A F51A V52A H53 M54A A55G R56A N57A A58G H59 S60A
Miller (β-Galactosidase) Units Dimerization Defective
N-terminal finger Linker C-terminal finger
β-sheets α-helix β-sheets α-helix
N-terminal finger Linker C-terminal finger
IYECKYCDIFFKDAVLYTIHMGYHSCDDVFKCNMCGEKCDGPVGLFVHMARNAHS
B A
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cI-NTD WT I06A Y07A E08A C09 K10A Y11A C12A D13A I14A F15A F16A K17A D18A A19G V20A L21A Y22A T23A I24A H25 M26A G27A Y28A H29 S30A C31A D32A D33A V34A F35A K36A C37 N38A M39A C40A G41A E42A K43A C44A D45A G46A P47A V48A G49A L50A F51A V52A H53 M54A A55G R56A N57A A58G H59 S60A
Miller (β-Galactosidase) Units Dimerization Defective
N-terminal finger Linker C-terminal finger N-terminal finger Linker C-terminal finger
β-sheets α-helix β-sheets α-helix
N-terminal finger Linker C-terminal finger
β-sheets α-helix β-sheets α-helix
β-sheets α-helix β-sheets α-helix
N-terminal finger Linker C-terminal finger
IYECKYCDIFFKDAVLYTIHMGYHSCDDVFKCNMCGEKCDGPVGLFVHMARNAHS
B A
Figure 4.7 Alanine scan analysis of the Hunchback DZF domain in the B1H system. (A) Hunchback DZF mutations were tested for their ability to abolish homodimerization in the B1H system by performing β-galactosidase assays. The cutoff is indicated by the red line and defined as in Figure 4.4. Residues replaced by an alanine are shown below the graph. Note that alanine residues in the original DZF domain were always replaced by a glycine. Zinc binding cysteines and histidines are also shown but were not mutated. Residues are numbered as in Figure 4.4. Controls expressing λcI and wild-type Hunchback are also shown. Values represent the mean calculated from three independent assays. (B) Summary of residues that were affected by an alanine-substitution. Conserved residues are highlighted in blue, yellow and pink as described in Figure 4.1. Residues that effected dimerization when mutated are boxed. Predicted secondary structures and individual ZF with linker region are also indicated.
these residues are adjacent to one another, overall they are spread out throughout the whole domain.
4.4.2 Residue “swap” scanning mutagenesis
To further investigate dimerization mediated by the DZF domain “swap” mutation analysis was also applied to the Drosophila Hunchback DZF.
To do this, 32 Hunchback “swap” mutants in which single residues were replaced with the corresponding residues from the Ikaros DZF domain were constructed. As described for the Ikaros DZF domain, we used PCR mutagenesis to individually introduce these mutations
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cI-NTD WT I06V E08K K10E Y11H D13R I14V F15L K17L A19H L21M Y28C S30G C31F D32R V34P K36E E42Y K43H C44S D45Q G46D P47R V48Y G49E L50F F51S V52S M54I A55T N57G A58E S60R Dimerization Defective
Miller (β-Galactosidase) Units
β-sheets α-helix β-sheets α-helix
N-terminal finger Linker C-terminal finger
IYECKYCDIFFKDAVLYTIHMGYHSCDDVFKCNMCGEKCDGPVGLFVHMARNAHS
B A
0 100 200 300 400 500 600
cI-NTD WT I06V E08K K10E Y11H D13R I14V F15L K17L A19H L21M Y28C S30G C31F D32R V34P K36E E42Y K43H C44S D45Q G46D P47R V48Y G49E L50F F51S V52S M54I A55T N57G A58E S60R Dimerization Defective
Miller (β-Galactosidase) Units
β-sheets α-helix β-sheets α-helix
N-terminal finger Linker C-terminal finger
β-sheets α-helix β-sheets α-helix
β-sheets α-helix β-sheets α-helix
N-terminal finger Linker C-terminal finger
IYECKYCDIFFKDAVLYTIHMGYHSCDDVFKCNMCGEKCDGPVGLFVHMARNAHS
B A
Figure 4.8 “Swap” scan analysis of the Hunchback DZF domain in the B1H system. (A) “Residue swap” mutants are defined as in Figure 4.5A and were tested for their ability to abolish dimerization by performing β-galactosidase assays. The cutoff for “dimerization defective” is indicated by a red line and defined in Figure 4.4. “Swap” mutations are indicated below the X-axis. Values represent the mean of three experiments. (B) Summary of residues that were affected by a “swap” mutation depicted on the amino acid sequence of the Hunchback DZF domain. Conserved residues are marked in blue, yellow and purple and residue positions that were impaired by a mutation are indicated by boxes. Predicted secondary structures and individual ZFs are also shown.
within the Hunchback DZF domain and subsequently assessed the ability of each of these swap mutants to homodimerize using the B1H system. It was found that 18 mutants yielded β-galactosidase activity <1.5 fold of the wild-type activity, suggesting that they did not affect dimerization of Hunchback (Figure 4.8). Mutations in the remaining 14 residue positions weakened or abolished the interaction (Figure 4.8). Some of the disruptive “swap”
substitutions occurred within the inter linker region (S30G, C31F and D32R) and at the predicted transition between the second β-sheet and the α-helix of the C-terminal finger (C44S, D45Q, P47R and V48Y). The remaining seven defective mutations (D13R, I14V, K17L, K36E, E42Y, F51S and V52S) are distributed throughout the DZF domain. Overall, these 14 residues may participate in contacts at the dimerization interface that are responsible for specificity of the Hunchback DZF domain.
4.4.3 Comparison of mutants identified by alanine scan and “swap” scan analysis
32 of the 49 residue positions differ in both Ikaros and Hunchback and were tested in both the alanine scan as well as in the residue swap experiments. Comparing the dimerization phenotype for these 32 pairs demonstrated that 18 pairs behaved the same in their ability to dimerize. Mutations in eight of the positions caused dimerization defects whereas mutations in the remaining ten positions did not affect dimerization. However, substitutions in 14 residue positions displayed different effects when replaced with alanine or swapped with the corresponding Ikaros amino acid. Eight of these residue positions were affected by an alanine but not by a swap substitution, whereas six positions were only impaired by the “swap”
substitution.
The remaining 17 alanine mutations are at residue positions that are the same in both the Hunchback and Ikaros DZF domains and were therefore not tested in the residue swap experiments. 11 of these mutations (Y7A, F16A, D18A, L21A, I24A, M26A, G27A, D33A, F35A, G41A and R56A) possessed significant dimerization defects and may provide additional information about residues involved in contacts at the dimer interface.