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Structural basis for heteromeric assembly and perinuclear organization of keratin filaments

Nature Structural & Molecular Biology volume 19, pages 707–715 (2012)Cite this article

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Abstract

There is as yet no high-resolution data regarding the structure and organization of keratin intermediate filaments, which are obligate heteropolymers providing vital mechanical support in epithelia. We report the crystal structure of interacting 2B regions from the central coiled-coil domains of keratins 5 and 14 (K5 and K14), expressed in progenitor keratinocytes of epidermis. The interface of the K5–K14 coiled-coil heterodimer has asymmetric salt bridges, hydrogen bonds and hydrophobic contacts, and its surface exhibits a notable charge polarization. A trans-dimer homotypic disulfide bond involving Cys367 in K14's stutter region occurs in the crystal and in skin keratinocytes, where it is concentrated in a keratin filament cage enveloping the nucleus. We show that K14-Cys367 impacts nuclear shape in cultured keratinocytes and that mouse epidermal keratinocytes lacking K14 show aberrations in nuclear structure, highlighting a new function for keratin filaments.

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Figure 1: K5 and K14 domain structure, and crystal structure of the K5–K14 2B heterocomplex.
Figure 2: Structure-function relationship of EBS.
Figure 3: A trans-dimer, K14 Cys367–mediated homotypic disulfide bond in the K5–K14 structure.
Figure 4: Calcium-dependent regulation and localization of the keratin-associated disulfide bond.
Figure 5: Localization of the keratin-associated disulfide bond in newborn-mouse epidermis, and impact of K14 loss on nuclear size and shape.
Figure 6: Direct linkage of K14 Cys367 and the maintenance of the round nuclear shape under high calcium medium.
Figure 7

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Acknowledgements

We thank members of the Coulombe and Leahy laboratories for guidance and support, M. Becker and N. Venugopalan for beamline assistance, and S. Bailey for comments. General Medicine and Cancer Institutes Collaborative Access Team receives support from the US National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Sciences (Y1-GM-1104). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract DE-AC02-06CH11357. These studies were otherwise supported by US National Institutes of Health grants AR42047 (to P.A.C.) and HD055545 (to D.J.L.).

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  1. Chang-Hun Lee

    Present address: Present address: College of Convergence, School of Basic Science, Daegu Gyeongbuk Institute of Science and Technology, Daegu, Korea.,

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA

    Chang-Hun Lee, Byung Min Chung & Pierre A Coulombe

  2. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Min-Sung Kim & Daniel J Leahy

  3. Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Pierre A Coulombe

  4. Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Pierre A Coulombe

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Contributions

C.-H.L. purified and crystallized the protein, analyzed the crystal structure, and performed in silico, in vitro and in vivo experiments. M.-S.K. collected, processed and refined the crystallographic data. B.M.C. performed the reconstitution experiment in A431 keratinocytes. D.J.L. aided in experimental design and provided guidance for several aspects of crystallography. P.A.C. designed the study, analyzed the data and wrote the manuscript in collaboration with C.-H.L., M.-S.K., B.M.C. and D.J.L.

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Correspondence to Pierre A Coulombe.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–2 and Supplementary Note (PDF 1937 kb)

Supplementary Movie 1

Three-dimensional reconstruction of z-stack images (0.05 μm increment, depth = 5 μm; 101 images) captured by confocal microscopy of mouse skin keratinocytes cultured in low-calcium medium. Red, K14; blue, nucleus. (MOV 4477 kb)

Supplementary Movie 2

Three-dimensional reconstruction of z-stack images (0.05 μm increment, depth = 5 μm; 101 images) captured by confocal microscopy of mouse skin keratinocytes cultured in high-calcium medium. Red, K14; blue, nucleus. (MOV 4482 kb)

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Lee, CH., Kim, MS., Chung, B. et al. Structural basis for heteromeric assembly and perinuclear organization of keratin filaments. Nat Struct Mol Biol 19, 707–715 (2012). https://doi.org/10.1038/nsmb.2330

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