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Haspin kinase binds to a nucleosomal DNA supergroove

Nature Structural & Molecular Biology volume 32, pages 1030–1037 (2025)Cite this article

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Abstract

Phosphorylation of histone H3 threonine 3 (H3T3) by Haspin recruits the chromosomal passenger complex to the inner centromere and ensures proper cell cycle progression through mitosis. The mechanism by which Haspin binds to nucleosomes to phosphorylate H3T3 is not known. Here we report cryogenic electron microscopy structures of the human Haspin kinase domain bound to a nucleosome. In contrast with previous structures of histone-modifying enzymes, Haspin solely contacts the nucleosomal DNA, inserting into a supergroove formed by apposing major grooves of two DNA gyres. This binding mode provides a plausible mechanism by which Haspin can bind to nucleosomes in a condensed chromatin environment to phosphorylate H3T3. We identify key basic residues in the Haspin kinase domain that are essential for phosphorylation of nucleosomal histone H3 and binding to mitotic chromatin. Our structural data provide notable insight into a histone-modifying enzyme that binds to nucleosomes solely through DNA contacts.

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Fig. 1: Haspin binds to nucleosomal DNA.
Fig. 2: Electrostatic interactions stabilize Haspin binding to the nucleosomal DNA supergroove.
Fig. 3: Density corresponding the histone H3 tail and a small molecule in the Haspin active site.
Fig. 4: Haspin binds in an optimal position for engaging the H3 tail.
Fig. 5: Charge-reversal substitution of key Haspin residues disrupts Haspin binding to nucleosome and H3T3 phosphorylation activity.
Fig. 6: Positively charged Haspin kinase domain residues are important for recruiting full-length Haspin to chromatin and promoting H3T3 phosphorylation in cells.
Fig. 7: Haspin DNA supergroove binding surface is unique among structurally related proteins.

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Data availability

Models and cryo-EM maps were deposited in the PDB and Electron Microscopy Data Bank (EMDB) under the following accession codes: Haspin position 1 (PDB: 9B2S, EMDB: 44113), Haspin position 2 (PDB: 9B2T, EMDB: 44114) and Haspin local refinement (PDB: 9B2U, EMDB: 44115). Raw cryo-EM movies were deposited in the EMPIAR database under the accession code EMPIAR-11971. Source data are provided with this paper.

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Acknowledgements

We thank D. Sousa, D. Ding and K. Cai for their support with cryo-EM sample preparation and data collection at the Beckman Center for Cryo-EM at Johns Hopkins School of Medicine. We thank the Summer Academic Research Experience (SARE) Program at Johns Hopkins for providing support to host S.J.V. for a summer research experience in the Wolberger lab. We thank the Wolberger lab for their insights and discussions on the paper. This work was supported by National Institute of General Medical Sciences grants R35GM130393 (C.W.), R01GM133897 (A.J.H.) and R01GM114119 (A.J.H.) and National Cancer Institute grants F31CA261154 (C.W.H.), F31CA271743 (S.R.) and R01CA266199 (A.J.H.) of the National Institutes of Health, and by a National Science Foundation Graduate Research Fellowship (A.S.E.).

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Authors and Affiliations

  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Chad W. Hicks, Sanim Rahman, Xiangbin Zhang, Andrew S. Eneim, Stacy J. Vasquez & Cynthia Wolberger

  2. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Colin R. Gliech & Andrew J. Holland

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  1. Chad W. Hicks
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  2. Colin R. Gliech
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Contributions

C.W.H. performed cryo-EM data processing, structural modeling, map interpretation and in vitro experiments to assay Haspin binding and activity on canonical nucleosomes. C.R.G. performed cell-based experiments on Haspin localization. S.R. assayed Haspin phosphorylation of H3T3 on free histone H3 in vitro and on chromatin in cells. X.Z. prepared Haspin kinase domain mutant plasmid constructs. A.S.E. assayed Haspin binding to tailless nucleosomes. S.J.V. froze and clipped cryo-EM grids for data collection. A.J.H. oversaw execution and interpretation of cell-based experiments. C.W. oversaw all aspects of structure determination, biochemistry and data interpretation. C.W.H. and C.W. wrote the paper, with contributions and feedback from all other authors.

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Correspondence to Cynthia Wolberger.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editors: Sara Osman and Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology and team.

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Extended data

Extended Data Fig. 1 Cryo-EM data processing workflow for Haspin (465-798) bound to nucleosome.

General processing pipeline to obtain the final cryoEM maps of Haspin Position 1, Haspin Position 2, and Haspin Local Refinement.

Extended Data Fig. 2 Global and local resolution evaluation of both cryo-EM maps.

a, b) Fourier Shell Correlation (FSC) plots using gold-standard 0.143 cutoffs for both cryo-EM maps of Haspin bound to nucleosome, a) position 1, b) position 2. c, d) Local resolution estimation color depictions for both cryo-EM maps corresponding to Haspin bound to nucleosome, c) position 1, d) position 2. e, f) Cut-away slice view through the center of both Haspin cartoon models showing the fit to their respective cryo-EM maps, e) position 1, f) position 2.

Extended Data Fig. 3 Haspin structural alignments.

Structural alignments of cartoon model of Haspin (470-798) in position 1 over Haspin (468-798) (PDB: 2WB8)34, Haspin (470-798) (PDB: 3DLZ)27, and Haspin (470-798) (PDB: 4OUC)36.

Extended Data Fig. 4 Global and local resolution evaluation of Haspin local refinement cryo-EM map.

a) Fourier Shell Correlation (FSC) plot using fold-standard 0.143 cutoffs for the Haspin local refinement cryo-EM map of Haspin bound to nucleosome. b) Local resolution estimation color depictions for the Haspin local refinement cryo-EM map of Haspin bound to nucleosome. c) Cut-away slice view through the center of Haspin showing the fit of the model to the cryo-EM map.

Extended Data Fig. 5 Cryo-EM structure of H3 tail bound to Haspin is similar to crystal structure of H3 peptide bound to Haspin.

A previously reported crystal structure of Haspin (PDB: 4OUC)36 with bound H3 peptide (royal blue) was superimposed over our cryo-EM map of locally-refined Haspin (orange) containing bound H3 tail (light blue). Structures are depicted in cartoon and atom representation and show the similarity in the binding position of H3.

Extended Data Fig. 6 Haspin binding to canonical and tailless nucleosome.

Electrophoretic mobility shift assay (EMSA) showing binding of wild-type Haspin (465-798) to canonical unmodified nucleosome (199 bp) and tailless unmodified nucleosome (199 bp) at the indicated concentrations.

Source data

Supplementary information

Source data

Source Data Fig. 5

Unprocessed western blots and gels.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 6

Unprocessed blots.

Source Data Extended Data Fig. 6

Unprocessed gel.

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Hicks, C.W., Gliech, C.R., Rahman, S. et al. Haspin kinase binds to a nucleosomal DNA supergroove. Nat Struct Mol Biol 32, 1030–1037 (2025). https://doi.org/10.1038/s41594-025-01502-y

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