Alanine scanning mutagenesis

To investigate systematically which amino acids of Ikaros are critical for dimerization, the Ikaros DZF domain was studied using alanine-scanning mutagenesis (work performed by R.

Fang). This approach should reveal residue positions that contribute to dimerization.

To perform an alanine-scan of the Ikaros DZF domain a set of plasmids encoding mutant proteins was generated and assayed using the B1H system. PCR-mediated mutagenesis was used to introduce individual alanine substitutions at 47 different residue positions within the Ikaros DZF domain except at conserved positions that mediate zinc binding (cysteines and histidines). To analyze the effects of these mutations on homodimerization, plasmids were constructed encoding each of these 47 different mutants fused to λcI and introduced into the reporter strain to measure their β-galactosidase activity (R. Fang).

Figure 4.4 shows the results of the alanine-scan. The cutoff for defining a dimer-defective mutant was set to >1.5 fold the β-galactosidase activity obtained with wild-type Ikaros DZF domain. This cutoff was chosen to include the standard error of these experiments which is typically about 10%. Thus, mutants displaying β-galactosidase units 1.5-fold higher than wild-type were considered to possess a significant dimerization defect. Mutations in 21 of the 47 positions tested displayed β-galactosidase units comparable to that of the wild type (<1.5 fold activation), suggesting that those residues are not directly involved in dimerization. 10 of these alanine substitution mutants exhibited slightly higher dimerization activity. Alanine substitutions in the remaining 26 positions tested significantly affected dimerization. The nature and importance of the various residue positions are described in more detail below.

Predicted conserved hydrophobic residues:

Alanine substituted mutants Y07A, F16A, Y22A, F35A and F50A affected residues which are likely to form the hydrophobic core of the C2H2 ZF since these residue positions are defined as conserved in the typical C2H2 ZF motif (Figure 4.1, see also section 1.2.3). These mutants were all severely defective in dimerization. It is noteworthy that residue position 44 is also defined as highly conserved in the classical DNA binding C2H2 ZF and is typically a

phenylalanine. Interestingly, this residue is substituted by a serine in the Ikaros DZF domain and was not affected by an alanine mutation (see also section 4.7.3 below).

0 50 100 150 200 250 300

cI-NTD WT V06A Y07A K08A C09 E10A H11A C12 R13A V14A L15A F16A L17A D18A H19A V20A M21A Y22A T23A I24A H25 M26A G27A C28A H29 G30A F31A R32A D33A P34A F35A E36A C37 N38A M39A C40 G41A Y42A H43A S44A Q45A D46A R47A Y48A E49A F50A S51A S52A H53 I54A T55A R56A G57A E58A H59 R60A Dimerization Defective

N-terminal finger Linker C-terminal finger

VYKCEHCRVLFLDHVMYTIHMGCHGFRDPFECNMCGYHSQDRYEFSSHITRGEHR

Miller (β-Galactosidase) Units

A

β-sheets α-helix β-sheets α-helix

N-terminal finger Linker C-terminal finger

B

0 50 100 150 200 250 300

cI-NTD WT V06A Y07A K08A C09 E10A H11A C12 R13A V14A L15A F16A L17A D18A H19A V20A M21A Y22A T23A I24A H25 M26A G27A C28A H29 G30A F31A R32A D33A P34A F35A E36A C37 N38A M39A C40 G41A Y42A H43A S44A Q45A D46A R47A Y48A E49A F50A S51A S52A H53 I54A T55A R56A G57A E58A H59 R60A Dimerization Defective

N-terminal finger Linker C-terminal finger

VYKCEHCRVLFLDHVMYTIHMGCHGFRDPFECNMCGYHSQDRYEFSSHITRGEHR

Miller (β-Galactosidase) Units

A

β-sheets α-helix β-sheets α-helix

N-terminal finger Linker C-terminal finger

B

β-sheets α-helix β-sheets α-helix

β-sheets α-helix β-sheets α-helix

N-terminal finger Linker C-terminal finger

B

Figure 4.4 Alanine scan analysis of the Ikaros DZF domain in the B1H system. (A) Ikaros DZF mutants were tested for their ability to mediate homodimerization in the B1H system by performing galactosidase assays. The cutoff for defining dimer-defective mutants was set to >1.5-fold the β-galactosidase activity of wild-type Ikaros and is indicated by the red line. This cutoff was chosen to take account of the standard error of these experiments which is typically about 10%. Residues replaced by an alanine are shown below the graph and each substitution corresponds to the respective bar. Note that conserved cysteines and histidines are also shown but were not mutated. Residues were numbered according to McCarty et al., 2003 where the conserved tyrosine 2 residues before the first Zinc-ligating cysteine is defined as residue position 7. Controls expressing λcI and wild-type Ikaros are also shown.

Regions defining the N-terminal ZF, linker and C-terminal ZF are indicated on the bottom of the residue sequence. Values are presented as the mean calculated from three independent experiments. (B) Summary of residues that were affected by an Alanine-substitution. Zinc ligating cysteines and histidines are highlighted in blue and conserved residues are highlighted in yellow (Phe/Tyr) and pink (hydrophobic residues). Residues that abolished dimerization when mutated are boxed. Typical secondary structures are indicated above the amino-acid sequence. Figure 4.4A was kindly provided by K. Joung.

Predicted β-sheets:

Mutants E10A, H11A, V14A, L15A, L17A and D18A lie in the predicted ββ-sheet region of the first C2H2 ZF motif and were all dimerization defective, whereas mutants K08A and R13A did not affect dimerization. The ββ-sheet region in the second ZF contained five more residue positions that were affected by an alanine substitution (E36A, M39A, G41A, Y42A and D46A). Although we can not rule out that G41 participates in the interaction, we suspect that because of its features this residue is likely to play a structural role by for example providing flexibility in the β-sheet region of the second ZF. Mutations in the other residues in this region (N38A, H43A, S44A and Q45A) did not affect dimerization.

Predicted α-helices:

Residues 19-29 and 47-59 in fingers 1 and 2, respectively, are predicted to form the α-helix.

Mutants M21A, I24A, M26A, I54A and R56A in these regions significantly disrupt the dimerization while mutations in the other residues of the α-helices (H19A, V20A, T23A, G27A, C28A, R47A, S51A, S52A, T55A, G57A and E58A) did not affect dimerization.

Inter-finger linker:

The region between the two individual C2H2 zinc fingers is termed the linker region and contains a highly diverse sequence throughout the different DZF domains. Mutations in three of these residues G30A, D33A and P34A, disrupted dimerization whereas mutations F31A and R32A had no effect.

Overall, this preliminary study of the Ikaros DZF domain identified several residue positions that may contribute to dimerization. Theses residues are spread out throughout the whole DZF domain and could be directly involved in the interaction but could also affect the affinity indirectly, by for example changing the confirmation or stability of Ikaros. In fact, Western Blot analysis of the expression level of these mutated peptides in E. coli indicated that many of the mutants that are severely impaired in their ability to dimerize, are expressed at lower levels compared to the wild-type Ikaros DZF domain (R. Fang, personal communication).