The crystal structure and dimerization interface of GADD45gamma

Abstract

Gadd45 proteins are recognized as tumor and autoimmune suppressors whose expression can be induced by genotoxic stresses. These proteins are involved in cell cycle control, growth arrest, and apoptosis through interactions with a wide variety of binding partners. We report here the crystal structure of Gadd45gamma, which reveals a fold comprising an alphabetaalpha sandwich with a central five-stranded mixed beta-sheet with alpha-helices packed on either side. Based on crystallographic symmetry we identified the dimer interface of Gadd45gamma dimers by generating point mutants that compromised dimerization while leaving the tertiary structure of the monomer intact. The dimer interface comprises a four-helix bundle involving residues that are the most highly conserved among Gadd45 isoforms. Cell-based assays using these point mutants demonstrate that dimerization is essential for growth inhibition. This structural information provides a new context for evaluation of the plethora of protein-protein interactions that govern the many functions of the Gadd45 family of proteins.

Fig. 1.

Monomeric structure and sequence conservation of Gadd45 isoforms. (A) Ribbon representation of the Gadd45γ monomer. Secondary structural elements are marked, and residues that are identical among all Gadd45 isoforms are colored yellow. This figure was created by using PyMOL (53). (B) Amino acid sequence alignment of Gadd45 family members. Identical residues are boxed in gray. Secondary structural elements observed in Gadd45γ crystals are marked above the sequences. This figure was created by using ESPript (54, 55).

Fig. 2.

Ribbon representation of four possible dimer interfaces. In each case interacting regions marked in dark blue for one monomer interact with regions colored yellow in the monomer colored green. Secondary structural elements are marked as in Fig. 1A.

Fig. 3.

Ribbon representations of interactions 1 and 2. The color coding is the same as in Fig. 1A. (A) Dimer generated by interaction 1. (B) Dimer generated by interaction 2. (C) Close-up view of the interactions of helices α3 in dimer 1. (D) Close-up view of the interactions of helices α3 in dimer 2.

Fig. 4.

Growth inhibition of HepG2 cells by wild-type and mutant Gadd45γ. Growth inhibition was assayed by using alamarBlue staining and by manual counts of colonies after crystal violet staining. Results shown are an average of three to four experiments with associated standard errors of the mean.

Fig. 5.

Potential interaction of Gadd45γ with PCNA. Portions of PCNA proposed to be involved in Gadd45 interactions are marked in yellow. Proposed PCNA-binding regions of Gadd45 isoforms are mapped in dark blue onto a Gadd45γ dimer. The potential binding surfaces on PCNA and on the Gadd45γ dimer are spaced ≈45 Å apart. The regions of Gadd45 reported to bind p21 are shown in pink, and p21^WAF1/CIF1 is shown as a light blue ribbon. PCNA-p21^WAF1/CIF1 coordinates are PDB entry 1AXC (2, 43).