Download Ternary complex structure of Aristaless and Clawless bound to DNA

Supplementary results
Optimization of Al and Cll constructs for structure determination
To identify the minimal regions of Al and Cll where each homeodomain
protein binds cooperatively to DNA, we overexpressed constructs of Al and Cll based
on the degree of conservation of the amino acid sequence of each protein. Al and its
homologues share high sequence similarity in two regions: the homeodomain (85 - 144
of Al) (Figure 1A) and the OAR domain (376 - 391 of Al). The OAR domain is thought
to negatively regulate the DNA-binding affinity of Al in vivo and in vitro (Brouwer et
al., 2003). Because these two domains are connected by a sequence that is predicted to
be a disordered region of the protein, we overexpressed the highly conserved
homeodomain region of Al (Al-HD: -5 - 63 of the Al homeodomain) and Cart1
(Cart1-HD: -1 - 61 of the Cart1 homeodomain) for the structural analysis. When the
amino acid sequence of Cll was compared with its homologues, the highly conserved
region was found to be extended from its homeodomain to neighboring residues (Figure
1B). To verify the importance of this extension, we overexpressed 6 Cll constructs
(Cll-N3C3 (N3 - C3 in Figure 1B), Cll-N3C2 (N3 - C2), Cll-N2C3 (N2 - C2), Cll-N2C2
(N2 - C2), Cll-N1C2 (N1 - C2), and Cll-N2C1 (N2 - C1)) and 2 Hox11L1 constructs
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(Hox11L1-N3C2 (N3 - C2 in Figure 1B) and Hox11L1-N1C2 (N1 - C2)), and analyzed
their cooperative DNA-binding ability with Al-HD and Cart1-HD by electrophoretic
mobility shift assay (EMSA). The EMSA results on the cooperative DNA binding of
Hox11L1 with Al and Cart1 are shown in Supplementary Figure 1. When N3-N1 of
Hox11L1 was deleted, Hox11L1 lost its cooperative DNA-binding ability with Al.
Hox11L1-N3C2 also binds DNA cooperatively with Cart1, though its binding affinity
and DNA-binding cooperativity are weaker than those of All-Cll and All-Hox11L1.
For the structure determination of the Al and Hox11L1 homeodomains, we
used shortened Al and Hox11L1 constructs (residues 91 - 147 and 162 - 216,
respectively) which include three  helical regions of the homeodomain. For the
structure determination of the binary complex of Al-DNA, we used Al-HD and 14 bp of
blunt-end double-stranded DNA,
5’-GGGTTTAATTAGGG-3’ (the recognition
sequence of Al is underlined). For the structure determination of the ternary complex of
Al-Cll-DNA, we used Al-HD and Cll-N2C3, which include the minimal region for the
cooperative DNA binding (Cll-N2C2), because this pair of proteins yielded the best
quality crystals. We obtained crystals of the ternary complex using 17 bp of a
blunt-ended double-stranded DNA, 5’-GGCTTAATTAATTGCGG-3’ (the recognition
sequences of Al and Cll are underlined) (Figure 1C).
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Structure determinations of the Al homeodomain, Hox11L1 homeodomain, the
binary complex of Al-DNA, and the ternary complex of Al-Cll-DNA
The crystal structures of the Al and Hox11L1 homeodomains were determined
at 1.00 Å and 1.54 Å, respectively, with good stereochemistry (Supplementary Figure
2AB). These structures were refined to R/Rfree values of 17.3/18.4% and 20.1/22.3%,
respectively. For the structures of the Al and Hox11L1 homeodomains, we built
structural models of 7 - 57 residues and 8 - 60 residues of the homeodomain,
respectively. The outer regions of the proteins were disordered in the crystals. The
structures of three  helices of each homeodomain were highly similar to the
preexisting homeodomain structures. The three  helices of the Al homeodomain fit
closely to those of the Pax homeodomain (Wilson et al., 1995), which is a paired class
homeodomain like Al, with a root mean square deviation (r.m.s.d.) in 51 aligned C
atom positions of 0.6 Å (Supplementary Figure 3A). When the structure of the Hox11L1
homeodomain was compared with those of the Hox homeodomains found in the ternary
complexes of Hox-Pbx/Exd-DNA (Passner et al., 1999; Piper et al., 1999), Hox11L1
showed a high degree of similarity among the three  helical regions. The r.m.s.d. in the
aligned 53 C atom positions between the structure of the Hox11L1 homeodomain and
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those of other Hox homeodomains (HoxB1 and Ubx) were 0.6 Å and 0.8 Å,
respectively (Supplementary Figure 3B).
The binary complex structure of Al-DNA was determined at a resolution of
2.25 Å and refined to R and Rfree values of 22.8% and 25.1%, respectively
(Supplementary Figure 2C). The final model contained one Al homeodomain (residue
range of 3 - 60), one 14 bp of DNA duplex, one acetic acid molecule, and 29 water
molecules in the asymmetric unit. The structure of the Al homeodomain is similar to
that of the apo form of the Al homeodomain, with an r.m.s.d. value of 0.6 Å for 51
aligned
C atoms.
The Al
homeodomain
recognizes
the DNA sequence
(5’-T5T6A7A8T9N10A11-3’) by N-terminal arm residues (Arg3 and Arg5) inserted into
the minor groove and Val47, Gln50, and Asn51 of the 3 helix bound to the major
groove (Supplementary Figure 2D). Arg3 and Arg5 recognize the first three bases of
sequence (5’-T5T6A7-3’) by direct hydrogen bonds. Val47, Gln50, and Asn51 form
direct or water-mediated hydrogen bonds and/or van der Waals contacts with bases from
the major groove of the DNA to recognize the 5’- A7A8T9N10A11-3’ sequence. The DNA
recognition mechanism of the Al homeodomain of the Al-DNA complex is essentially
identical to that of the Al-Cll-DNA complex, though Gln50 and Asn51 form additional
water-mediated hydrogen bonds to Thy9. By the binding of Al-HD, the DNA bends 14 o
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toward Al-HD.
The ternary complex structure of the Al-Cll-DNA complex was determined at a
resolution of 2.70 Å and refined to R and Rfree values of 23.4% and 28.2%, respectively.
The final model contained two Al-Cll-DNA complexes in the asymmetric unit (chains
A(Al)B(Cll)CD(dsDNA) and E(Al)F(Cll)GH(dsDNA), respectively). Though the two
complexes are essentially identical, the first complex appeared to be better refined and
built than the other complex when the B factors of the models and the range of the
determined residue structures were compared. We therefore used the coordinates of the
complex comprised of chains ABCD in the Results and Discussion sections of this
report unless otherwise stated.
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Supplementary figure legends
Supplementary Figure 1. Cooperative DNA binding of Hox11L1 with Al and Cart1
(A) Cooperative DNA binding of Al and Hox11L1. The following Al and Hox11L1
constructs were added to each binding reaction: lane 1, no protein: lanes 2, 3, and 4,
17.5, 35.0, and 70.0 pmol of Hox11L1-N3C2, respectively; lanes 5, 6, and 7, 20 pmol of
Al-HD and 17.5, 35.0, and 70.0 pmol of Hox11L1-N3C2, respectively; lanes 8, 9, and
10, 35.0, 70.0, and 140 pmol of Hox11L1-N1C2, respectively; lanes 11, 12, and 13, 20
pmol of Al-HD and 35.0, 70.0, and 140 pmol of Hox11L1-N1C2, respectively. Each
binding reaction contains 2 pmol of FITC-labeled dsDNA. (B) Cooperative DNA
binding of Cart1 and Hox11L1. The following Cart1 and Hox11L1 constructs were
added to each binding reaction: lane 1, no protein: lanes 2, 3, and 4, 17.5, 35.0, and 70.0
pmol of Hox11L1-N3C2, respectively; lanes 5, 6, and 7, 20 pmol of Cart1-HD and 17.5,
35.0, and 70.0 pmol of Hox11L1-N3C2, respectively; lanes 8, 9, and 10, 35.0, 70.0, and
140 pmol of Hox11L1-N1C2, respectively; lanes 11, 12, and 13, 20 pmol of Cart1-HD
and 35.0, 70.0, and 140 pmol of Hox11L1-N1C2, respectively. Each binding reaction
contains 2 pmol of FITC-labeled dsDNA.
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Supplementary Figure 2. Structure determinations of the Al homeodomain, the
Hox11L1 homeodomain, and the binary complex structure of Al-DNA
(A) Homeodomain structure of Al. The color-coding runs from blue in the N-terminal
region to red in the C-terminal region. Water molecules, a cadmium ion, and a chloride
ion are shown by spheres colored red, yellow, and green, respectively. (B) The
homeodomain structure of Hox11L1. Water molecules and the sodium ion are shown by
spheres colored red and purple, respectively. Sulfate ions are shown by a stick model.
(C) The binary complex structure of Al-DNA. The color-coding of Al-HD runs from
blue in the N-terminal region to red in the C-terminal region. Water molecules and
acetate ion were shown by spheres colored red and sticks colored orange, respectively.
(D) Base recognition mode of Al in the Al-DNA complex. Residues of Al are colored
blue. The bases recognized by Al are colored cyan. Hydrogen bonds are indicated with
arrows, and van der Waals contacts are shown as lines terminating in a circle.
Water-mediated hydrogen bonds are indicated by the letter w. Residues interacting with
DNA from a minor groove are underlined.
Supplementary Figure 3. Superposition of the homeodomain structures
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(A) Stereo diagram showing the superposition of the DNA-free structure of the Al
homeodomain (green), the homeodomain structure of Al in the Al-Cll-DNA complex
(cyan), and the homeodomain structure of Pax (magenta). (B) Stereo diagram showing
the superposition of the homeodomain structure of Hox11L1 (blue), the homeodomain
structure of Cll in the Al-Cll-DNA complex (red), and the homeodomain structure of
HoxB1 (gray).
Supplementary Figure 4. Temperature factor of the Al-Cll-DNA complex
The relative temperature factor is indicated by a color gradient from blue (low) to red
(high).
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Supplementary Figure 1. Miyazono et al.
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Supplementary Figure 2. Miyazono et al.
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Supplementary Figure 3. Miyazono et al.
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Supplementary Figure 4. Miyazono et al.
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