Understanding disease mutations from the crystal structure of SOX9 with DNA

SRY(Sex determining Region Y)-box or SOX transcription factors are important in early development and maintenance of different cell pools after birth. Of the ~20 SOX proteins (SRY, SOX1-SOX15, SOX17, SOX18, SOX21 and SOX30), SOX2, SOX9 and SOX10 mutations are primarily disease-associated: SOX2 with Combined Pituitary Hormone Deficiency, Microphthalmia, Septo-optic dysplasia and anophthalmic syndrome; SOX9 with Campomelic Dysplasia (affects development of the reproductive and skeletal system); and SOX10 (~94% sequence identity to SOX9) with Waardenburg Syndrome (affects audition and pigmentation in hair, eyes and skin; and specifically with WS types 2 and 4). As part of our Protein Structure Initiative (PSI)-Biology partnership, we performed structural and mutational analyses including x-ray crystallography and surface plasmon resonance assays, on the DNA-binding HMG domain of SOX9 with duplex DNA. Crystals were obtained in C222 space group and the structure was determined by molecular replacement to 2.77 Å resolution with final Rcryst/Rfree of 24.8/27.8%. The overall structure of the SOX9-DNA complex is similar to other SOX/SRY protein complexes. The SOX9-DNA protein-DNA interactions suggested a panel of mutations to assay for biochemical activity, which allowed us to understand the molecular basis of five mutations identified in Campomelic Dysplasia. These mutated residues have direct contact with DNA as well as indirect contacts, i.e., these mutations lead to allosteric secondary structure changes in the protein, which affect residues in direct contact with DNA. Due to the very high sequence identity between SOX9 and SOX10, our crystal structure also helps to rationalize the effect of SOX10 mutations in Waardenburg Syndrome. This work is supported by NIH grants U54 GM094586 and U01 GM094614. SSRL operations are funded by DOE BES, and the SSRL SMB program by DOE BER, NIH NCRR BTP and NIH NIGMS.

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative

The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins—including proteins for which reliable homology models can be generated—on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long.