Structural studies on a protein-binding zinc-finger domain of Eos reveal both similarities and differences to classical zinc fingers.
The oligomerization domain that is present at the C terminus of Ikaros-family proteins and the protein Trps-1 is important for the proper regulation of developmental processes such as hematopoiesis. Remarkably, this domain is predicted to contain two classical zinc fingers (ZnFs), domains normally associated with the recognition of nucleic acids. The preference for protein binding by these predicted ZnFs is not well-understood. We have used a range of methods to gain insight into the structure of this domain. Circular dichroism, UV-vis, and NMR experiments carried out on the C-terminal domain of Eos (EosC) revealed that the two putative ZnFs (C1 and C2) are separable, i.e., capable of folding independently in the presence of Zn(II). We next determined the structure of EosC2 using NMR spectroscopy, revealing that, although the overall fold of EosC2 is similar to other classical ZnFs, a number of differences exist. For example, the conformation of the C terminus of EosC2 appears to be flexible and may result in a major rearrangement of the zinc ligands. Finally, alanine-scanning mutagenesis was used to identify the residues that are involved in the homo- and hetero-oligomerization of Eos, and these results are discussed in the context of the structure of EosC. These studies provide the first structural insights into how EosC mediates protein-protein interactions and contributes to our understanding of why it does not exhibit high-affinity DNA binding.
Westman
Belinda J
BJ
School of Molecular and Microbial Biosciences, University of Sydney, Sydney, New South Wales 2006, Australia.
Perdomo
José
J
Matthews
Jacqueline M
JM
Crossley
Merlin
M
Mackay
Joel P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D051740:Ikaros Transcription Factor; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D055672:Static Electricity; D040681:Structural Homology, Protein; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers / Q000235:genetics
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C497239:IKZF1 protein, human; C418108:IKZF5 protein, human; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D014157:Transcription Factors; D029867:Xenopus Proteins; C497238:Zfpn1a1 protein, mouse; C118760:Zfpn1a4 protein, mouse; D051740:Ikaros Transcription Factor; D015032:Zinc