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The Geminin and Idas coiled coils preferentially form a heterodimer that inhibits Geminin function in DNA replication licensing.

Caillat C, Pefani DE, Gillespie PJ, Taraviras S, Blow JJ, Lygerou Z, Perrakis A - J. Biol. Chem. (2013)

Bottom Line: Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer.In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression.The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas-Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions. The crystal structure of the homology regions of Idas in complex with Geminin showed a tight head-to-head heterodimeric coiled-coil. This Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin, still with high affinity (∼30 nm), but with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression. The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.

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The structure of the Idas-Geminin complex.A, a diagram of the domain structure for Geminin and Idas·coiled-coil domain (CC), destruction box (DB), homeodomain binding region (HD). The limits of the crystallization construct (tIdas-tGeminin) and those of the coiled coil are numbered and denoted as thicker boxes. B, a sequence alignment of the seven heptads involved in the coiled coil. C, a schematic representation of the crystal structure of the tGeminin-tIdas structure. Differences in the a and d register positions are in bold. D–F, the structures of the Geminin homodimer, alone (D) and from the complex with Cdt1 (E and F).
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Figure 5: The structure of the Idas-Geminin complex.A, a diagram of the domain structure for Geminin and Idas·coiled-coil domain (CC), destruction box (DB), homeodomain binding region (HD). The limits of the crystallization construct (tIdas-tGeminin) and those of the coiled coil are numbered and denoted as thicker boxes. B, a sequence alignment of the seven heptads involved in the coiled coil. C, a schematic representation of the crystal structure of the tGeminin-tIdas structure. Differences in the a and d register positions are in bold. D–F, the structures of the Geminin homodimer, alone (D) and from the complex with Cdt1 (E and F).

Mentions: The Idas and Geminin coiled-coil regions form a head to head (parallel) heterodimer (Fig. 5). The secondary structure is α-helical from residues Pro-94 to Asn-159 for Geminin and from Pro-178 to Leu-241 for Idas. The overall structure is similar to the coiled-coil domain of the Geminin-Geminin homodimer (Fig. 5, D–F). The root mean square deviation (r.m.s.d.) of the Idas to the Geminin monomers is 4.2 Å when the two monomers are compared as single rigid bodies but has an r.m.s.d.f of 1.4 Å when compared as two rigid bodies each, as optimized by the RAPIDO algorithm (39). Idas has an r.m.s.d.f of 0.8 and of 1.2 Å when compared with each of the two Geminin monomers of the Geminin homodimer structure (19) (PDB code 1UII). Geminin from the Idas-Geminin complex had an r.m.s.d.f of 1.5 and 1.1 Å compared with the same two Geminin monomers of the homodimer. This suggests that when our heterodimer structure is compared to the Geminin homodimer structure, Geminin in the heterodimer resembles the second Geminin monomer of the homodimer (PDB code 1UII, chain B), and Idas resembles the first Geminin monomer (PDB code 1UII, chain A). That information has been used in all subsequent superpositions presented here, where Geminin is always superposed to the second monomer of the 1UII homodimer. This is also reflected in our preference to refer to an Idas-Geminin heterodimer in the context of this study.


The Geminin and Idas coiled coils preferentially form a heterodimer that inhibits Geminin function in DNA replication licensing.

Caillat C, Pefani DE, Gillespie PJ, Taraviras S, Blow JJ, Lygerou Z, Perrakis A - J. Biol. Chem. (2013)

The structure of the Idas-Geminin complex.A, a diagram of the domain structure for Geminin and Idas·coiled-coil domain (CC), destruction box (DB), homeodomain binding region (HD). The limits of the crystallization construct (tIdas-tGeminin) and those of the coiled coil are numbered and denoted as thicker boxes. B, a sequence alignment of the seven heptads involved in the coiled coil. C, a schematic representation of the crystal structure of the tGeminin-tIdas structure. Differences in the a and d register positions are in bold. D–F, the structures of the Geminin homodimer, alone (D) and from the complex with Cdt1 (E and F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814758&req=5

Figure 5: The structure of the Idas-Geminin complex.A, a diagram of the domain structure for Geminin and Idas·coiled-coil domain (CC), destruction box (DB), homeodomain binding region (HD). The limits of the crystallization construct (tIdas-tGeminin) and those of the coiled coil are numbered and denoted as thicker boxes. B, a sequence alignment of the seven heptads involved in the coiled coil. C, a schematic representation of the crystal structure of the tGeminin-tIdas structure. Differences in the a and d register positions are in bold. D–F, the structures of the Geminin homodimer, alone (D) and from the complex with Cdt1 (E and F).
Mentions: The Idas and Geminin coiled-coil regions form a head to head (parallel) heterodimer (Fig. 5). The secondary structure is α-helical from residues Pro-94 to Asn-159 for Geminin and from Pro-178 to Leu-241 for Idas. The overall structure is similar to the coiled-coil domain of the Geminin-Geminin homodimer (Fig. 5, D–F). The root mean square deviation (r.m.s.d.) of the Idas to the Geminin monomers is 4.2 Å when the two monomers are compared as single rigid bodies but has an r.m.s.d.f of 1.4 Å when compared as two rigid bodies each, as optimized by the RAPIDO algorithm (39). Idas has an r.m.s.d.f of 0.8 and of 1.2 Å when compared with each of the two Geminin monomers of the Geminin homodimer structure (19) (PDB code 1UII). Geminin from the Idas-Geminin complex had an r.m.s.d.f of 1.5 and 1.1 Å compared with the same two Geminin monomers of the homodimer. This suggests that when our heterodimer structure is compared to the Geminin homodimer structure, Geminin in the heterodimer resembles the second Geminin monomer of the homodimer (PDB code 1UII, chain B), and Idas resembles the first Geminin monomer (PDB code 1UII, chain A). That information has been used in all subsequent superpositions presented here, where Geminin is always superposed to the second monomer of the 1UII homodimer. This is also reflected in our preference to refer to an Idas-Geminin heterodimer in the context of this study.

Bottom Line: Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer.In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression.The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas-Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions. The crystal structure of the homology regions of Idas in complex with Geminin showed a tight head-to-head heterodimeric coiled-coil. This Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin, still with high affinity (∼30 nm), but with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression. The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.

Show MeSH
Related in: MedlinePlus