supplementary information€¦ · supplementary figure 1 the k5-k14 l2–2b heterocomplex impacts...

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Supplementary Information

Ms. Title: “Structural basis for heteromeric assembly and perinuclear organization

of keratin filaments” Authors: Chang-Hun Lee, Ming-Sun Kim, Byung Min Chung, Daniel J. Leahy,

and Pierre A. Coulombe Bloomberg School of Public Health & School of Medicine Johns Hopkins University, Baltimore, MD, USA

Content: Supplementary Figures 1–4

Description of Supplementary Movies 1 and 2 Supplementary Tables 1–2 Supplementary Note References

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Nature Structural & Molecular Biology: doi:10.1038/nsmb.2330

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� Supplementary Figure 1 The K5-K14 L2–2B heterocomplex impacts keratin assembly in vitro and in vivo. (a) High-speed sedimentation assay was applied to address whether the purified K5-K14 L2–2B heterocomplex impacts the polymerization of full-length K5 and K14. ‘S’ denotes the supernatant or non-filamentous proteins, and ‘P’ denotes the pelleted fraction or filamentous proteins. The L2–2B heterocomplex cannot sediment on its own, but disrupts K5-K14 filament assembly when added prior to, but not after, initiation of polymerization; (b) Visualization of K5-K14 assemblies using negative staining and electron microscopy. Some of the 10 nm filaments


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appear broken or discontinued in the sample in which full-length K5-K14 is polymerized in the presence of the K5-K14 L2–2B heterocomplex. Filament width is not significantly altered. Bar = 100 nm; (c) Myc-K14 L2–2B and/or His-GFP-K5 L2–2B were transiently transfected into newborn mouse skin keratinocytes in primary culture. In doubly-transfected cells, K14 L2–2B (red channel) co-localizes with K5 L2–2B (data not shown) and is associated with a collapse of the endogenous keratin filament network. In cells singly transfected with Myc-K14 L2–2B, no significant change is observed in keratin filament organization. Bar = 10 μm. Collectively, these findings suggest that the K5-K14 heterotypic complex used for crystallization mimics a functional assembly intermediate. (d) Sequence alignment for the 2B domains of human type I keratins (top) and type II keratins (bottom). Amino acids are listed using the single letter code, and color code refers to the chemical properties of their side-chain. The heptad repeat (abcdefg) is given underneath the aligned sequences; the conserved four-residue stutter sequence is denoted with �x� (see arrowhead bar). The blue and red bars respectively denote the regions of K5 and K14 included in the crystal structure of the K5-K14 heteromeric complex. This sequence alignment was generated by ClustalW using publicly availably human keratin sequences.


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Supplemental Figure 2 Interaction map at the interface between participating α-helices in IF coiled-coils. (a) Distribution of contacts at the interface of select coiled-coil forming proteins. Source of the atomic coordinates are as follow: K5-K14 (this study; PDB: 3TNU), K5-K5 and K14-K14 (homology modeling performed using 3TNU as template), vimentin (PDB: 1GK4), lamin A/C (PDB: 1X8Y), cortexillin I (PDB: 1D7M). Note that the crystal structure of lamin A/C has a monomer in the asymmetric unit. Therefore, the coiled coil dimer was generated with a symmetry operator, and serves as a control possessing perfectly symmetric dimer. Interactions were assessed with LigPlot program. For K5-K14, the K5 and K14 coils are represented on the Y-and X-axes, respectively. Dots located on the diagonal line reflect direct “a-a” of “d-d” contacts (see single arrow for an example); opposing pairs of dots along the diagonal line indicate symmetric (bidirectional) interaction (see double arrows); and single dots away from the diagonal line reflect asymmetric (unidirectional) contacts (see triple arrows) at the interface between the two monomeric α-helices. Black dots denote hydrophobic interactions and red dots denote electrostatic interactions excluding water-mediated interactions; (b) Quantitation of the number of hydrophobic, electrostatic, and hydrogen bond contacts per heptad repeat along the coiled-coil interface for various dimers (see a). The K5-K14 structure stands out in that it has the most asymmetric salt bridges and the least “symmetric contact clusters” (the latter are “islands” composed of a “diagonal dot” with at least one pair of “symmetric dots”). (c) Correlation between the phenotype of previously characterized, biochemically-defined K5 and K14 mutants and the interaction map derived from the K5-K14 crystal structure. Point mutations studied by other researchers1,2 are denoted by arrows in the K5-K14 interaction map, and the associated assembly defect is given in-between parentheses. Point mutations causing EBS are also shown in red letter. This analysis conveys that mutations altering contacts made at the coiled coil interface alter dimer structure and/or introduces defects in filament assembly and/or structure. Comment on the significance of this comparative analysis There are differences in the type of bonds occurring in the coiled-coil interface of the 2B domains of K5-K14 and lamin A which, respectively, typify IF proteins that form obligate heterodimers or homodimers on their way to producing IFs 3,4. A predominant distinction is that all contacts within the coiled-coil interface of the homodimeric lamin A 2B crystal, whether hydrophobic-, electrostatic-, or hydrogen-based, are symmetric and bidirectional in nature. By contrast, the coiled-coil interface of the K5-K14 2B crystal shows many asymmetric (i.e., unidirectional), in addition to symmetric, contacts of all three types. Interestingly, the coiled-coil interface of the vimentin 2B homodimer has a hybrid character with key features from each of the keratin and lamin dimers. Such features are consistent with the observation that vimentin undergoes facultative heteromeric assembly in vitro and in vivo. In vivo, vimentin preferentially heterodimerizes when in the presence of compatible assembly partners in vivo, e.g., desmin, nestin, GFAP, and alpha-internexin5–8. Whether such principles hold true awaits the availability of additional IF subunit crystals along with site-directed mutagenesis studies altering key interface contacts in the IF coiled-coil interface.


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Supplementary Figure 3 Additional findings about K14-C367 and disulfide bond formation in vitro and in vivo. (a) 2Fo-Fc Electron density map of the region immediately surrounding the disulfide bond; (b) Phenanthroline/copper-mediated oxidation of the K5-K14 L2–2B–T (Tail) heteromeric complex in vitro. The left panel shows that the disulfide bonds in K14 are only forming under oxidative condition for all three K14 L2–2B variants tested, and that each are fully reversible to the reduced form after >1 h incubation with TCEP. The right panel shows that the K14 WT and C389A variant are able to maintain disulfide-bonded homodimers under partial reduction condition (i.e, treatment with TCEP for only 30 min). The outcome is different for the C367A variant, which shows near complete reduction under such conditions. (c) Non-reducing SDS-PAGE coupled with Western blotting of proteins isolated from mouse skin keratinocytes in vivo. (d) K14 variants with cysteine-to-alanine substitutions are filament assembly-competent. In the upper panels, full-length, untagged K14 WT and the two Cys-to-Ala variants were individually co-transfected with full length K5 into NIH3T3 fibroblasts, which are keratin-negative. All three forms of K14 were able to form a keratin filament network de novo in this setting. In the lower panels, GFP-fused versions of full length K14 WT, and the two Cys-to-Ala variants were individually transfected into mouse 308 skin keratinocytes. Again, all three forms of K14 were able to incorporate into the pre-existing keratin filament networks, which otherwise show identical morphologies. These findings support the view that neither Cys367 nor Cys389 is required for filament assembly or the formation of a cytoplasmic network of filaments in vivo. Bar = 10 μm.


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Supplementary Figure 4 Calcium promotes changes in keratin filament organization and in nuclear morphology. (a-c) Mouse 308 keratinocytes in culture were adapted to low calcium medium for 24 h, then subjected to either low calcium medium for a further 24 h (a) or switched to high calcium medium for 24 h (b, c). Under low calcium, the shape of the nuclei is more ellipsoidal, and keratin filaments are organized into long and large bundles that run parallel to the main axis of elongated cells (see a). Under high calcium, the nucleus adopts a rounder shape and keratin filaments intersect one another at various angles around the nucleus. Moreover, there is


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co-localization of the signals for disulfide bonds and keratin in the immediate perinuclear space (see arrow in inset for panel b). In c (specificity control), the labeling dye for disulfide bonds was omitted but otherwise the preparation was processed exactly as for frames a, b. Bar = 10 μm. (d) Frequency of nuclei showing a larger aspect ratio (> 2.0), a indirect measure of their shape, in mouse 308 keratinocytes cultured in low or high calcium media for 24 hr as described for a–c. Low calcium culture condition promotes higher aspect ratios, reflecting a more ovoid shape (see a). By comparison, high calcium promotes lower aspect ratios, reflecting a rounder shape (see b and c); (e) The overall size of nuclei is higher in high calcium compared to low calcium medium. Together, the findings reported in frames d and e show that exposure to high calcium medium causes many skin keratinocyte nuclei to become larger in size, and rounder in shape. This data complements the information reported in Figure 4.


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Description of Supplementary Movies

Supplementary Movie 1 Supplementary Movie 2

Supplementary Movies 1 and 2 were generated from the three-dimensional reconstruction of z-stack images (0.05 μm increment, depth = 5 μm; 101 images contributed to each movie) captured by confocal microscopy of mouse 308 skin keratinocytes cultured under low calcium medium (left column) and high calcium medium (right column), respectively, and stained for K14 antigens (red chromophore) and DNA (depicting the nucleus; blue chromophore). Representative individual still images are shown here. Bars = 10 μm.


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�� Inter-strand and intra-strand interactions in the K5-K14 2B coiled-coil�

Inter-strand interaction

K5 residue atom K14 residue atom Distance (�) Interaction

Glu385 OE1 Arg335 NH2 3.8 Salt bridge

Arg397 HO1 h, d H2O a - 1.9 Glu344

Lys404 H13 h, d H2O a - 1.9 Glu344

H2O - Glu344 OE2 3.8 Arg397, Lys404

H2O - Gln348 OE1 2.8 Cys407

Cys407 SG a H2O h, d - 2.1 Gln348

Glu422 OE2 a Tyr366 HO1 h, d 2.4 H-bond

Lys431 NZ Glu381 OE2 3.5 Salt bridge

Glu466 OE2 Arg416 NH2 3.6 Salt bridge

Tyr470 OH a Arg416 HO7 h, d 2.5 H-bond

Intra-strand interaction


Lys460 NZ Asp464 OD2 3.0 Salt bridge


Arg407 NH1 Glu411 OE2 3.5 Salt bridge

Lys405 NZ Glu409 OE2 3.7 Salt bridge

Structure analysis was done using LigPlot and PyMol v.1.2. Distance criteria were set in default in LigPlot. Distance measurement was done with hydrogen atoms added to the structure by LigPlot. h added hydrogen atoms by the program a hydrogen bond acceptor d hydrogen bond donor

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Supplementary Table 2 Map of Epidermolysis Bullosa Simplex Mutations in the K5-K14 Crystal Amino Acid

Substitution in EBS patients

Type of EBS 9 Color in Figure 2

Putative Interaction partner

Reference

K5 K404E localized Green K14-Glu344 10 A428V general. Cyan K14-Ile373 11 A438D localized Green Surface exposed 10 L463P general Cyan K14-Glu409, Ile412 12 E466D general Cyan K14-Arg416 13, 14 I467L localized K14-Ile408, Leu412, 11 I467T DM Blue Tyr415 15, 16 I467M general 17 T469P DM Blue Surface exposed 18 R471C general Cyan Surface exposed 16 E475K DM Blue Surface exposed 10, 19 E475G DM 20, 21 G476D localized Green Surface exposed 22 K14 I377N localized

general Magenta

K5-Leu435 16, 23

I377T localized 11 E381K localized Yellow K5-Lys431, Leu435 16 L384P general Magenta K5-Ala422 18, 24, 25 R388C localized Surface exposed 11, 14, 23, 26

R388G general Magenta 16 R388H localized 22 R388P general 16 L401P localized Yellow K5-Tyr453, Leu456,

Met457 14

L408M localized Yellow K5-Lys460, Ile467 10 E411K DM Red Surface exposed 26 I412N DM Red K5-Leu463, Ile467 26 A413T general Magenta Surface exposed 27 Y415H general K5-Ile467, Tyr470, 14, 28 Y415H DM Red Leu474 16, 22, 29, 30

Y415C localized 31 R416P DM Red K5-Glu466, Tyr470 32 R417P DM Red Surface exposed 16, 18 L418V general Magenta Surface exposed 11 L419Q DM Red K5-Leu474 10, 28

Note: One mutation in K14 (K14-E422K) and three mutations in K5 (K5-E477K, K5-E477X and K5-E477G) are not included here because the corresponding amino acids are missing in the crystal structure of the K5-K14 2B coiled-coil.


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Supplementary Note Reconstitution of full length K5-K14 filaments in vitro pET-K5 and pET-K14 were transformed into the E. coli strain BL21 (DE3) pLysS and BL21(DE3), respectively, to produce the corresponding recombinant human proteins as inclusion bodies33,34. The K5 and K14 proteins were purified in multiple steps consisting of isolation of the keratin inclusion bodies, urea-based solubilization of keratin proteins, and ion exchange chromatography including Hi-TrapQ and MonoQ columns (GE Healthcare) as described previously35,36. Heterotypic complexes containing purified K5 and K14 in an equimolar ratio were prepared and purified using the MonoQ column. Suitable fractions were identified by SDS-PAGE, their concentration adjusted to 1 mg/ml, and used for filament reconstitution by serial dialysis using the following three buffers at room temperature: (a) 9 M urea and 25 mM Tris-HCl, pH 7.4, with 25 mM β-ME for 4 h; (b) 2 M urea and 5 mM Tris-HCl, pH 7.4, with 5 mM β-ME, for 1 h; and (c) 5 mM Tris-HCl, pH 7.5, with 5 mM β-ME overnight. The NaCl concentration of the last buffer was 0 mM (standard assembly condition). In some experiments, filament bundling was promoted by dialyzing reconstituted filaments in standard assembly buffer supplemented with 10 mM NaCl (ref. 1 and refs. therein). To test K5-K14 filament assembly in the presence of the L2–2B heterocomplex, the latter was added before initiation of serial dialysis with K5-K14. The polymerization efficiencies of K5-K14 or K5-K14 L2–2B complex were assessed by high-speed sedimentation (150,000 g; 30 min) as described3. Filament structure was examined by negative staining (1% uranyl acetate) and transmission electron microscopy using a Hitachi 7600 (Hitachi) instrument. Forced Oxidation of [K5 2B - K14 2B] or [K5 2B - K14 2B-Tail] heterocomplexes in vitro To perform forced oxidation in vitro, the 2B domains of these K14 variants were subcloned into the pHMT vector37 for bacterial expression, then purified as described in the Methods section.

Transfection of full-length K14 variants or L2–2B fragments Skin keratinocytes were isolated from P2 neonatal mice, seeded for primary culture38, and used to assess the function of L2–2B fragments in an in vivo setting. Transient transfection of K5 L2–2B and/or K14 L2–2B, subcloned in the mammalian expression vector pBK-CMV, was performed using lipofectamine 2000 according to the manufacturer's instructions. At 48 hour after transfection, cells were fixed with 3.3% paraformaldehyde for 15 min, extracted with 0.1% Triton X-100 in PBS buffer for 5 min, and processed for indirect immunofluorescence using a monoclonal antiserum against c-myc (9E10 [Cell Signaling]; 1:1,000 dilution) or a rabbit polyclonal anti-K5 (AF-138 [Covance]; 1:1,000 dilution), followed by a rhodamine-conjugated goat anti–mouse secondary antibody (1:2,000) and a FITC-conjugated goat anti-rabbit secondary antibody (1:2,000; from [Kirkegaard and Perry Laboratories, Inc.]). The NIH3T3 fibroblast cell line (American Type Culture Collection) was used to analyze the de novo assembly properties of keratin proteins in a keratin-free setting. Co-transfection of the human K5 (WT) and K14 variants (WT, K14-C367A, K14-C389A) were performed as described above. At 72 hr after transfection, cells were fixed and immunostained as described above. Image recordings of transfected cells were obtained at room temperature using a confocal microscope (Axiovert 200 microscope with a 510 Meta module; Carl Zeiss, Inc.) fitted with a 40× 1.4 NA Plan-Apochromat oil objective and a 100× 1.4 NA Plan-Apochromat oil objective lenses. Microscope operation and image acquisition was done using the LSM 510 software (Carl Zeiss, Inc.). When indicated, pixel information from the confocal images was analyzed using ImageJ software.


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supplementary information€¦ · supplementary figure 1 the k5-k14 l2–2b heterocomplex impacts...

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