Fig 1.
The sequence of D-HMG, a solubility enhanced variant of SOX9 incorporating the Group E specific dimerization region, a Gly-Ser rich linker in bold face, and the HMG domain. Two additional substitutions relative to the human SOX9 sequence (C72S, K82S) are also shown in bold face. Boxed sequences indicate the position of experimentally observed helices (α1/α2/α3) from a crystal structure of the SOX9-DNA complex (PDB: 4EUW) using the program, STRIDE [36]. Lines above the sequence indicate the position of helices predicted by PSIPRED [26]. Amino acids underneath the sequence indicate additional substitutions made to fine map the attributes of the dimerization region.
Fig 2.
Binding of SOX9 proteins to single and double sites.
(a) SOX9 protein fragments, either containing the HMG domain alone (HMG, open circles) or a high solubility variant containing the dimerization region and HMG domain (D-HMG, closed circles) were incubated at varying concentrations with 32P-labeled single site (S9WT) or double site (CC36) DNA and an EMSA was performed. Bands corresponding to single site and double site occupancy were measured, integrated and normalized. Each plot was fitted independently to a four parameter logistic curve.
Table 1.
Fitted parameters for the EMSA-based titrations in Fig 2.
Fig 3.
Nonradioactive EMSA assessment of substitution mutants in the dimerization region.
(a) The dimerization region sequence was placed on a helical wheel with hydrophobic amino acids marked black and hydrophilic amino acids marked white to show the amphipathic nature of the predicted helix. S78 and Q79 (underlined) were substituted with alanine in a previous study with no effect on dimerization [25]. (b) For each substitution mutant presented in Fig 1, an EMSA was performed with a CC36 double site DNA probe and a substoichiometric protein to DNA ratio. Mutants with a nonfunctional dimerization domain are observed as a mixture of one site and two-site occupancies while mutants that retain a functional dimerization domain are observed exclusively as a two site occupancy. Mutants that do not have a functional dimerization domain from this qualitative assay coincide with the hydrophobic amino acids on the helical wheel.
Fig 4.
A peptide from the dimerization region binds preassembled SOX9 DNA complexes.
(a) Sequence alignment of the HMG domains from three dimerizing Group E proteins (SOX8/9/10) and two non-dimerizing proteins, SRY and SOX18. (b) Four substitution mutants in the SOX9 HMG domain were chosen for analysis based upon sequence conservation among Group E family members and a high degree of side chain surface exposure. (b) Presentation of EMSA data is similar Fig 3. The observed single and double occupancy bands for the mutants A119E and L142Q demonstrated loss of dimerization (red). In contrast, the A118E and L145E mutants retain dimerization (green) (c) To solutions of stoichiometric, fully occupied complexes of protein (2 μM) and CC36 at (1 μM), an increasing amount of FITC-labeled peptide corresponding to amino acids 71–85 of the dimerization region (D-peptide) was added followed by an EMSA to resolve the complex. The presence of the peptide in the protein/DNA complex was determined by detection of FITC fluorescence (ex: 495 nm, em: 519 nm).
Fig 5.
Molecular docking of a potential dimerization region helix upon the SOX9 HMG domain.
Guided by substitution mutagenesis data, the dimerization region was modeled as one amphipathic helix (α0; blue), and docked onto a cleft formed by α1 and α2 of the HMG domain. Following the color scheme in Fig 4, amino acids that prevented and retained dimerization are red and green, respectively. Additional amino acids colored pink (W115, L123, L130, T138, L139, and T146) were include with A119 and L142 to form a contingous hydrophobic surface for peptide docking.
Fig 6.
A molecular model of the dimeric SOX9 complex.
(a) The sequence of the DNA duplex in the model. The two inverted sites in the palindrome represent canonical high affinity sequences for SOX family proteins. (b,c) Two views of the complex rotated 90° highlighting the bend in DNA and the distance in which the linker region can bridge the two proteins.