Mutagenesis Study on the Zebra Fish SOX9 High-Mobility Group:  Comparison of Sequence and Non-Sequence Specific HMG Domains

Abstract

A unique class of proteins, containing high-mobility group (HMG) domain(s), recognizes unusual DNA structures and/or bends specific to AT-rich linear double-stranded DNA. The DNA binding feature of these proteins is exhibited in the HMG domain(s). Although the sequence specific and non-sequence specific HMG domains exhibit very high degrees of sequence similarity, the reasons for the difference between their DNA recognition mechanisms are unclear. A series of zebra fish SOX9 HMG domain mutants was prepared in an effort to elucidate the importance of various residues on protein stability and DNA binding. This study is the first of a comprehensive mutagenesis study on a sequence specific HMG domain. Comparing how various residues influence sequence specific and non-sequence specific HMG domains helps us to rationalize their mode of action. Positively charged amino acids concentrated at the surface of sequence specific HMG domains recognize specific, linear AT-rich DNA segments. After the negative charges at the surface of the DNA are neutralized, the hydrophobic residues of the protein may intercalate DNA. Phenylalanine at position 12 plays a crucial role in the sequence specific HMG domain. The differences in pI values, the instability index, and DNA contact regions between sequence and non-sequence specific HMG domains are associated with their functional modes.

DNA-binding properties of ARID family proteins

“…Some highly conserved residues have been identified as very important in sequence specificity of HMG domains, but these residues alone are not the sole determinant of sequence specificity [reviewed in (47)]. Rather, sequence-specificity appears to be a combination of effects of residues on the domain's positioning, affinity, its stability in complex with DNA, the number of interactions on the protein–DNA interface and the number of base-specific contacts (49). Studies of the HMG domain indicate the difference between sequence-specific and sequence-non-specific members of the same family is generally more complex than the simple substitution of contact residues for neutral residues.…”

The LEF-1 High-Mobility Group Domain Undergoes a Disorder-to-Order Transition upon Formation of a Complex with Cognate DNA

“…A recent alanine scanning mutagenesis study performed on the Sox-9 HMG domain from zebra fish revealed the importance of certain residues and their influence on protein stability and DNA binding (63). In addition, the authors calculated a theoretical "instability index" and reported the average instability indices to have contrasting values of 67 and 30 for the sequence-specific and non-sequencespecific subclasses, respectively (the higher the value, the greater the instability).…”

Functional Assessment of Residues in the Amino- and Carboxyl-Termini of Crustacean Hyperglycemic Hormone (CHH) in the Mud Crab Scylla olivacea Using Point-Mutated Peptides

“…The HPLC-purified glycine extended wild-type Sco-CHH (Sco-CHH-Gly), and glycine extended and alanine-substituted Sco-CHHs (I2A, F3A, D4A, D12A, R13A, Q51A, E54A, D60A, I69A, and V72A Sco-CHH-Gly) were individually dissolved in 10 mM PBS (150 mM NaCl, pH 6.8) at 20–30 μM; an aliquot (200 μl) of the samples was placed in a 1-mm cell and data collected from 260 nm to 200 nm in 0.5 nm increments at 25°C. Raw data were baseline corrected, smoothed, and transformed to obtain spectra in units of mean residue ellipticity and the ellipticity at 222 nm was used to estimate the α-helical content of the peptides [ 40 ].…”