Journal of Molecular Biology
Crystal Structure of the Coiled-coil Dimerization Motif of Geminin: Structural and Functional Insights on DNA Replication Regulation
Introduction
Geminin, a polypeptide of about 25 kDa, occurs in the nuclei of higher eukaryotes and functions as both a negative regulator of genome replication and coordinator of differentiation. Geminin was discovered as a protein that is degraded when cells exit from mitosis, by the large ubiquitin–ligase complex known as the cyclosome or anaphase-promoting complex, APC. Geminin tightly interacts with CDT1,1., 2., 3., 4. a factor necessary for the recruitment of MCM helicase complex and inhibits the loading of this complex on chromatin. The destruction of geminin at mitotic exit releases CDT1, which can then serve to reload MCM proteins on chromatin. In Hela cells, geminin is synthesized throughout the cell-cycle, but the protein has a half-life of three to four hours during the S phase, becomes phosphorylated (at amino acid residues serine 45 and serine 49, an area closely adjacent to the destruction box motif) as S phase proceeds, and is degraded.2., 5., 6. The role of geminin in embryonic development has been investigated recently.7., 8. These studies demonstrated that murine geminin associates with members of the Hox-repressing polycomb complex, with the chromatin of Hox regulatory DNA elements and with Hox proteins.8 Geminin and Six3 transcription factor act antagonistically to control the balance between proliferation and differentiation, and probably Six3 competes with CDT1 binding to geminin.7
The analysis of deletion mutants of geminin6., 9. has defined three almost independent regions of the protein (Figure 1A). A destruction box for ubiquitin-mediated degradation during mitosis at the N terminus, followed by a neuralization domain and at the C terminus the DNA replication inhibition domain containing a conserved leucine zipper (LZ). Here, we have focused on the replication inhibition domain involving residues 87 to 168 in Xenopus geminin.6 This domain is highly conserved among vertebrates (Figure 1B). The corresponding region of human geminin (HsGem residues 76–160 in Figure 1B) has a predicted coiled-coil motif of five heptad repeats (amino acid residues 110–144) flanked by an N-terminal sequence rich in basic amino acid residues and a C terminus predicted to form a helix. Coiled-coil structural motifs are distributed widely in proteins, and genome database searches with coiled-coil prediction programs suggest that 3–5% of all protein residues exist as coiled coils.10 They are oligomerization motifs commonly occurring at the interface between separate protein chains. They are found in many cytoskeletal and contractile systems (e.g. intermediate filaments, nuclear lamins, and myosins), transcription regulators (e.g. Myc and Max, Fos and Jun, GCN4), viral envelope proteins (e.g. MoMLV, HIV, SIV, influenza).11 Less is known, however, about the structure of the geminin leucine zipper (LZ). The sequence of geminin-LZ (Figure 1B) shows the predominance of polar residues (22 of 35). This distribution of amino acids has been linked to “natively unfolded” proteins, which lack stable conformational order under physiological conditions.12 We report here the crystal structure of the coiled-coil region of human geminin from residues 110 to 145 (termed HsGem-LZ). We show that HsGem-LZ is natively unfolded at physiological pH and temperature but can be induced to form a coiled coil by decreasing the temperature. Sedimentation in density gradients provides the possibility of studying the degree of oligomerization of geminin and the low-resolution overall shape derived from small-angle, X-ray-scattering (SAXS) experiments fits with a tetrameric structure. Various geminin coiled coil-containing domains have been assayed for their ability to inhibit DNA replication. Our results show that the HsGem-LZ alone is not sufficient to inhibit DNA replication of sperm chromatin in Xenopus egg extracts. However, full inhibition can be obtained for HsGem-LZ containing an additional sequence at the N terminus. Taken together our structural, biophysical and functional results provide new insights into understanding how geminin may inhibit DNA replication initiation in eukaryotes.
Section snippets
Structure of the geminin-LZ
A peptide corresponding to the predicted coiled-coil fragment (residues 110–145) of human geminin capped by an N-terminal Thr residue (Figure 1A) was produced by standard peptide synthesis and crystallized as described.13 The crystal structure contains a dimer in the asymmetric unit and was determined by molecular replacement. We found a unique solution involving a parallel, two-stranded coiled coil. The structure refined at 1.47 Å resolution contains 74 residues and 125 water molecules, and has
Discussion
Here, we present the three-dimensional structure of the coiled-coil domain of human geminin expected to be involved in homodimerization of geminin. The structure of this leucine zipper (HsGem-LZ) consists of two α-helices wound tightly around each other in a parallel coiled-coil dimer. The structure of the HsGem-LZ peptide raises a number of questions about the function of the coiled-coil domain of geminin, as this region is required also for DNA replication inhibition6 and differentiation
Geminin constructs and bacterial protein expression
Deletion mutants of human geminin were made by PCR amplification (Master Mix Qiagen) and insertion into pET15(b) between the NdeI and BamHI sites. The sequences of the primers used to generate each construct were:
ggaattccatatgaaaaatcttggaggagtcacc
ggaattccatatgacccaggagtcatttgatctt
cgggatccttatgctacttctgccagttcttt
cgggatccttaaccattcagtctctctattag
Acknowledgements
This work was supported by grants from MENRT, FRM (to M.T.), and the Association pour la Recherche contre le Cancer. We acknowledge P. Vachette for assistance with the SAXS experiments, F. Hoh for help with diffraction data collection and J.M. Lhoste for helpful discussions.
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Cited by (28)
SIX3 and SIX6 interact with GEMININ via C-terminal regions
2019, Biochemistry and Biophysics ReportsCitation Excerpt :However, in SIX6 the 15 aa adjacent to the C- terminus of the homeodomain was shown to render a DNA binding specificity that is not present in SIX2, another member of the SIX class [15]. The human GEMININ is 209 aa-long and the 3D-structure of its protein interaction domain, the coiled-coil domain (aa 91–160), has been determined [16–18]. GEMININ homo-dimerises through the coiled-coil domain and exists as a tetramer under low salt condition (200 mM salt or lower).
Geminin a multi task protein involved in cancer pathophysiology and developmental process: A review
2016, BiochimieCitation Excerpt :It is a tetramer protein. The monomers of each dimer are known to interact with each other by coiled-coil domain interaction [18,19]. The peptide harbors leucine zipper domain consisting of internal polar residues and a negatively charged surface that interacts with the basic domain of interacting partners [19].
The Geminin and Idas coiled coils preferentially form a heterodimer that Inhibits Geminin function in DNA replication licensing
2013, Journal of Biological ChemistryCitation Excerpt :First, we analyzed the stability of dimerization of the coiled coil regions. The tGeminin homodimer had a Tm of 70.8 °C, significantly higher than the value of 35 °C previously reported for a smaller peptide comprising residues Leu-110–Ala-145 of Geminin (37). This suggests that the first and second heptads (Geminin 96–109) are very important for the stability of the coiled-coil.
Idas, a novel phylogenetically conserved geminin-related protein, binds to geminin and is required for cell cycle progression
2011, Journal of Biological ChemistryCitation Excerpt :A multiple sequence alignment of Geminin orthologues with Idas orthologues from human, mouse, and Xenopus is shown in Fig. 1A. Geminin and Idas exhibit low overall similarity but significant conservation in their central coiled-coil regions, where the human Idas and Geminin proteins share 53% identity (Fig. 1A, black box). The central region of Geminin, which is conserved in Idas, mediates Geminin homodimerization and binding to Cdt1 (20–23). The Geminin coiled-coil has unique properties, with three polar residues in a and d positions of the heptad repeats, which are also present in Idas (Fig. 1A, arrowheads), suggesting that Idas may both homodimerize and heterodimerize with Geminin.
Geminin is cleaved by caspase-3 during apoptosis in Xenopus egg extracts
2007, Biochemical and Biophysical Research CommunicationsCitation Excerpt :We have also shown that Geminin cleavage by caspase-3 is not relevant to its function as inhibitor of DNA synthesis. These findings are consistent with the observation that two cleavage sites by caspase-3 have been mapped to the C-terminus of Geminin in human cells [24], while the DNA replication inhibition domain of Geminin lies in the central and amino-terminal part of Geminin [1,4–6]. In this report, we have also shown that Geminin cleavage by caspase-3 can be observed in Xenopus egg extracts, both in solution and on chromatin, putting forward the first basis for the study of Geminin cleavage in in vitro systems that are amenable to biochemical manipulation.
Automated de novo phasing and model building of coiled-coil proteins
2015, Acta Crystallographica Section D: Biological Crystallography