nature structural & molecular biology: doi:10.1038/nsmb figure 3 sequence alignment of the...

Supplementary Figure 1

The DNA-binding domain of HsHSF1 (HsDBD).

(a-c) Superposition of the HsDBD structure in the HsDBD–HSE complex with the DBDs in the HsDBD–SatIII complex (a), with the NMR structure of the free HsDBD (PDB 2LDU, Liu, G. et al., 2011) (b) and with the KlDBD–HSE complex (PDB 3HTS, Littlefield, O. &

Nelson, H.C. Nat. Struct. Biol. 6, 464-470, 1999) (c). C traces are shown. (d) Interface between the C-terminal 20 residues and the main body of the HsDBD. The protein backbone of HsDBD from the HsDBD–SatIII complex is shown in ribbon representation; residues 13-100 are additionally represented as molecular surface. The hydrophobic interface residues in the C-terminal segment are highlighted

in stick representation. The hydrogen bond contact to helix 3 is shown as a dotted line.

Nature Structural & Molecular Biology: doi:10.1038/nsmb.3149


DBD-DBD interactions in HSF1–DNA complexes.

(a, b) The KlDBD–HSE complex crystal structure (pdb 3HTS) viewed along the two-fold symmetry axis (a), with the molecular contacts in the DBD–DBD interface highlighted in (b). Protein is shown in ribbon representation; DNA is shown in surface representation. Prominent sidechains are shown in stick representation; hydrogen bonds are represented by dotted lines. (c) Interactions between two asymmetric units along the DNA stacks in the crystal lattice of the HsDBD–SatIII complex. The duplexes are rotated out of register. Furthermore, two additional nucleotides were included into the DNA, shifting the DBDs ~6.8 Å further apart. (d, e) Potential interaction site of the wing domain in head-to-tail contacts. A view along the DNA is shown. A hydrophobic groove is situated between helices 2 and 3 (indicated by an arrow). Residues lining the groove are highlighted in stick representation. Panel e shows a surface representation with positive and negative charges is blue and red, respectively. Hydrophobic sidechains are shown in yellow.



Sequence alignment of the conserved segment of HsHSF1, CtHSF1 and CtSkn7.

Secondary structure elements are indicated above and below the sequences. Similar residues are shown in red and identical residues in white using bold lettering on red background. Blue frames indicate homologous regions. The downward arrowhead designates the residue critical for Skn7 DNA-binding properties. Asterisks denote acetylation sites in HsHSF1; red ovals designate putative hydrophobic layer residues in the HsHSF1 coiled-coil.



Interaction of CtSkn7 with DNA.

(a) Superposition of the DBDs from HsDBD–HSE and CtDBD–HSE complexes. (b) Superposition of the DBD pairs from CtDBD–HSE and CtDBD–SSRE structures. (c) Sequence alignment of representative Skn7 and HSF1 sequences, showing the systematic sequence divergence at residue 100 (CtSkn7 numbering). (d) Close-up of the interactions of CtDBD with the GGC triplet in SSRE. Sidechains of conserved residues are shown in stick representation. Hydrogen bonds are represented by dotted lines. The C-terminal tail of the DBD was omitted for clarity. (e, f) Omit density for the base-specific interactions in the CtDBD–SSRE complex. Panels e and f show the interactions of helix 3 with the GCC and GGC motifs, respectively (same orientation as in Fig. 4c). For calculating omit density, CtDBD residues 95-101 were deleted from the model, 0.2 Å random shifts applied to the coordinates with PDBSET and B-factors set to 20.00 Å2 to reduce model bias. These modified models were re-refined with Refmac5. Weighted 2Fo-Fc density maps at 1.0 sigma are shown for the missing segments, superposed on the complete model. Water atoms and the C-terminal tail residues were deleted for clarity. (g) Close-up of the interactions of CtDBD with the GAA triplet in HSE.



Biophysical characterization of CtSkn7 and the chimeric HsHSF1(CtHR-A/B) protein.

(a, b) Limited proteolysis of CtSkn7(40–220) in absence (a) and presence (b) of HSE DNA duplexes. CtSkn7(40–220) at 11 M was

treated with increasing amounts of proteinase K (PK) (0, 0.05, 0.15, 0.45, 1.35, 4.05, 12.15 and 36.45 g ml-1

) for 1 h on ice. The reaction was terminated by addition of 4 mM phenylmethylsulfonyl fluoride. The products were analyzed by SDS-PAGE and Coomassie

staining. (c, d) SEC-MALS analysis of CtSkn7(160–209) (c) and HsHSF1(CtHR-A/B) (d). Data show measurements of ~100 g protein. The calculated molar mass is indicated. The theoretical molar masses of CtSkn7(160–209) dimers and trimers are ~11.4 and ~17.1 kDa, respectively. The average observed mass of ~14.5 kDa is consistent with a ~1:1 mixture of dimer and trimer. The theoretical mass for HsHSF1(CtHR-A/B) trimer is ~171.8 kDa. (e, f) Cellular locations of HsHSF1-V (e) and HsHSF1(CtHR-A/B)-V (f) in HeLa cells in absence and presence of heat shock (HS) treatment. Nuclei are stained with DAPI.



Structural model for HsHSF1(13–182) bound to SatIII repeats.

Perpendicular views are shown. The C-termini point alternatingly in opposite directions; only every second HsHSF1 chain can contribute to a trimeric complex. The three HsHSF1 chains in one complex are indicated in blue, yellow and beige. The DNA is shown in stick representation. The linkers were modelled with Modeller as implemented in the ModLoop server. The helical bundle is a homology model based on the CtSkn7(160–209) trimer structure.


SUPPLEMENTARY TABLES

Supplementary Table 1. Sequences of DNA oligonucleotide duplexes. fl, fluorescently labelled; Ctrl, control.

Name 5´–3´ DNA sequence

HSE ggTTCtaGAAcc

SatIII cGGAATGGAATg

SSRE ATTTGGCTGGGCC

fl-HSE 6FAM-cgacagTTCtaGAAacagacg

fl-SSRE 6FAM-cgacagGGCtaGCCacagacg

fl-Ctrl 6FAM-cgacagatacagatacagacg


nature structural & molecular biology: doi:10.1038/nsmb figure 3 sequence alignment of the...

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