Crystal Structure of the Coiled-coil Dimerization Motif of Geminin: Structural and Functional Insights on DNA Replication Regulation

We have determined the crystal structure of the coiled-coil domain of human geminin, a DNA synthesis inhibitor in higher eukaryotes. We show that a peptide encompassing the five heptad repeats of the geminin leucine zipper (LZ) domain is a dimeric parallel coiled coil characterized by a unique pattern of internal polar residues and a negatively charged surface that may target the basic domain of interacting partners. We show that the LZ domain itself is not sufficient to inhibit DNA synthesis but upstream and downstream residues are required. Analysis of a functional form of geminin by density sedimentation indicates an oligomeric structure. X-ray solution scattering experiments performed on a non-functional form of geminin having upstream basic residues and the LZ domain show a tetramer structure. Altogether, these results give a consistent identification and mapping of geminin interacting regions onto structurally important domains. They also suggest that oligomerization properties of geminin may be implicated in its inhibitory activity of DNA synthesis.

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.