Structural basis for homeodomain recognition by the cell-cycle regulator Geminin Bo Zhou, Changdong Liu, Zhiwen Xu, and Guang Zhu1 Division of Life Science and State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Kowloon, Hong Kong Edited by Barry Honig, Columbia University, Howard Hughes Medical Institute, New York, NY, and approved April 20, 2012 (received for review January 21, 2012) Homeodomain-containing transcription factors play a fundamental regulatory activities through protein–protein interactions remain role in the regulation of numerous developmental and cellular largely unexplored. processes. Their multiple regulatory functions are accomplished The mutual regulation between Geminin and Hox or Six3 through context-dependent inputs of target DNA sequences and homeodomain proteins have been identified to coordinate cell collaborating protein partners. Previous studies have well estab- proliferation and differentiation processes (12, 14). Geminin was fi lished the sequence-specific DNA binding to homeodomains; initially identi ed as a cell-cycle regulator critical for maintaining however, little is known about how protein partners regulate genome stability and euploidy (15, 16). During G1 phase, Cdc6 their functions through targeting homeodomains. Here we report and Cdt1 are recruited by origin recognition complex to the the solution structure of the Hox homeodomain in complex with replication origins and in turn required for the loading of mini- chromosome maintenance complex onto DNA to form the pre- the cell-cycle regulator, Geminin, which inhibits Hox transcrip- replication complexes (pre-RC) (17, 18). Geminin physically tional activity and enrolls Hox in cell proliferative control. Side- interacts with Cdt1, and sequesters Cdt1 from its role in the pre- chain carboxylates of glutamates and aspartates in the C terminus RC assembly, thus preventing reinitiation of DNA replication of Geminin generate an overall charge pattern resembling the (19, 20). The interaction of Hox with Geminin could promote DNA phosphate backbone. These residues provide electrostatic cell proliferation and mediate Hox-induced enhancement of interactions with homeodomain, which combine with the van hematopoietic stem cell activity (21). Meanwhile, this binding der Waals contacts to form the stereospecific complex. We further also characterizes Geminin as an inhibitor of the transcriptional showed that the interaction with Geminin is homeodomain sub- activity of Hox proteins. This mutual regulation was suggested class-selective and Hox paralog-specific, which relies on the sta- to result from the competitive binding between Hox and Cdt1 to pling role of residues R43 and M54 in helix III and the basic amino the coiled-coil domain of Geminin (12). acid cluster in the N terminus. Interestingly, we found that the In the present study, we first delineated the specific homeo- C-terminal residue Ser184 of Geminin could be phosphorylated domain binding region (HBR) on Geminin through pull-down by Casein kinase II, resulting in the enhanced binding to Hox and assay, isothermal titration calorimetric (ITC) measurement, and more potent inhibitory effect on Hox transcriptional activity, indicat- NMR titrations. Surprisingly, the HBR is located at the C ter- ing an additional layer of regulation. This structure provides minus of Geminin and separated from the Cdt1-binding coiled- insight into the molecular mechanism underlying homeodomain- coil domain. Using solution NMR techniques, we solved the protein recognition and may serve as a paradigm for interactions structure of Gem-HBR in complex with the homeodomain of fi between homeodomains and DNA-competitive peptide inhibitors. Hoxc9 (Hoxc9-HD). The de ned molecular mechanism for the regulation between Hox and Geminin was verified by mutagen- esis, and biochemical and cellular assays. We also found the he homeodomain transcription factors play fundamental C-terminal residue Serine 184 of Geminin at the complex in- roles in genetic control of development, including body plan T fi – terface could be phosphorylated by Casein kinase II (CK2), which speci cation, pattern formation, and cell fate determination (1 increased the binding affinity to Hox, and the mutant that mimics 4). This protein family includes Hox, extended Hox, NK, LIM, S184 phosphorylation showed enhanced inhibitory effect on Hox BIOCHEMISTRY POU, paired, and atypical subclasses based on evolutionary fi transcriptional activity. Furthermore, we showed key residues classi cations (5). These proteins are all characterized by a 60-aa determining the selectivity of Geminin to various homeodomain DNA binding domain, the homeodomain, which is encoded by subclasses and Hox paralogs. a 180-bp DNA sequence known as the homeobox. The homeo- domain folds into three α-helices and the latter two form a helix- Results turn-helix motif typical for DNA binding (6). The molecular Delineation of Homeodomain-Binding Site on Geminin. We first mechanisms of how homeodomains recognize target DNA performed an extensive search for the Hox-binding domain on sequences have been extensively studied. For Hox proteins, their Geminin by pull-down assay using Hoxc9-HD and deletion frag- homeodomains contain identical DNA base-contacting residues ments of Geminin based on the report that Geminin interacts and have very similar DNA-sequence specificity. Cooperative with Hox homeodomains (12). Our results suggest that direct DNA binding with other cofactors is thought to enhance the physical interaction occurs between Hoxc9-HD and the C ter- transcriptional specificity of Hox proteins (7). minus of Geminin, spanning residues 138–209 (Fig. 1A). To In addition to the role in DNA recognition, homeodomains narrow down the binding region, we used smaller fragments of have been found to serve as protein interaction targets to regu- Geminin and found that Geminin(151–170) weakly interacts with late the functions of homeodomain transcription factors or other proteins. For example, the Hox homeodomains are involved in the interactions with proteins, such as Smad1 (8), Smad4 (9), CBP (10), HMG1 (11), and Geminin (12). These protein inter- Author contributions: B.Z. and G.Z. designed research; B.Z. and C.L. performed research; actions are mostly described to influence regulatory activities B.Z., C.L., and Z.X. analyzed data; and B.Z., Z.X., and G.Z. wrote the paper. that define the level of target-gene activation or repression, and The authors declare no conflict of interest. also link homeodomain proteins function to a variety of de- This article is a PNAS Direct Submission. velopmental pathways, such as chromatin remodeling, cell sig- Data deposition: The NMR, atomic coordinates, chemical shifts, and restraints have been naling, and cell cycle regulation (13). Despite the accumulating deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2LP0). knowledge on the functional importance of the homeodomain– 1To whom correspondence should be addressed. E-mail: [email protected]. protein interactions, the molecular mechanism and sequence This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. signatures of homeodomains in modulating DNA binding or 1073/pnas.1200874109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1200874109 PNAS | June 5, 2012 | vol. 109 | no. 23 | 8931–8936 Downloaded by guest on September 29, 2021 Fig. 1. C terminus of Geminin specifically interacts with Hoxc9. (A) Hoxc9-HD was pulled down by GST-tagged Gem-FL and its deletion mutants, and vi- sualized by Coomassie blue staining. (B) The defined region of Geminin required for Hoxc9-HD interaction. Hoxc9-HD was pulled down by GST-tagged fragments of Geminin C terminus and visualized by Coomassie blue staining. (C) Part of the 1H-15N HSQC spectra of 15N-labeled Hoxc9-HD in free form (red) overlaid with that titrated with nonlabeled Gem-HBR at a molar ratio of 1:2 (blue). (D) Part of the 1H-15NHSQCspectraof15N-labeled Gem-HBR in free form (red) overlaid with that titrated with nonlabeled Hoxc9-HD at a molar ratio of 1:2 (blue). (E) ITC measurement of Hoxc9-HD binding to Gem-HBR. (F) Schematic illustration of reported and currently identified protein binding motifs on Geminin. Hoxc9-HD and Geminin(171–190) (Gem-HBR) strongly inter- modest binding affinity is in agreement with the fast exchange acts with Hoxc9-HD (Fig. 1B). state of the complex formation demonstrated by NMR titrations. The specificity of the interaction between Hoxc9-HD and We also determined the binding affinity between Hoxc9-HD and Gem-HBR was further validated through NMR titration ex- Geminin full-length protein (Gem-FL), which was calculated to periments. In heteronuclear single quantum coherence (HSQC) be 3.3 ± 0.3 μM based on ITC measurement (Fig. S2E), indi- spectra of 15N-labeled Hoxc9-HD titrated with Gem-HBR and cating that although Gem-HBR provides the specific binding site vice versa, the chemical-shift perturbations of amide proton and for Hoxc9-HD, amino acids in other regions of Geminin could nitrogen resonances of a defined set of residues indicate a spe- further enhance the binding affinity through nonspecific contacts. cific binding (Fig. 1 C and D and Fig. S1). In contrast, titration of Our results, obtained by multiple approaches, consistently Hoxc9-HD with Geminin(151–170) showed no obvious changes demonstrate that the specific Hox-binding region of Geminin is in chemical shift, suggesting that the weak binding observed situated at the C terminus, encompassing residues 171–190, which in the GST pull-down assay was nonspecific, or the interaction is different from the Cdt1-binding domain (22, 23) and partially was too weak to be detected by NMR (Fig. S2A). Titration of overlaps with the Brg1-binding region (24) (Fig. 1F). 15N-labeled Hoxc9-HD with Geminin(151–190) and vice versa showed similar chemical-shift pattern as the titration with Gem- Structure Determination of Hoxc9-HD/Gem-HBR Complex by NMR. HBR, except that the peaks broadened more rapidly, indicating Using the solution NMR techniques, we determined the 3D a higher affinity (Fig. S2 B and C). These results demonstrate structure of the Hoxc9-HD/Gem-HBR complex.