A multitude of physiological and non-physiological signals induce members of the mammalian transcription factor NF-kB leading to immune and inflammatory responses, regulation of programmed cell death, cell growth and differentiation. The NF-kB family is comprised of five different subunits that share extensive sequence and structural homology within the rel homology region. This region is responsible for subunit association and DNA binding. We have done extensive structural and biochemical work to understand the detailed mechanisms of NF-kB dimer formation and NF-kB/DNA complex formation. The major findings are that residues in the dimerization domain that are not a part of the dimer interface also play roles in affecting the stability of the dimers in addition to the residues at the interface and the NF-kB/DNA complexes exhibit multiple conformations. The focus of the current proposal is multifaceted. We will investigate how dimerization of NF-kB subunits is fine-tuned by residues outside of the dimer interface. We will also investigate how conformational variations in NF-kB/kB DNA complexes affect enhancer specific multiprotein assembly and transcription using the HIV-LTR proximal promoter as a model system. NF-kB dimers are a sought after molecular target for therapeutic intervention. This study also seeks to understand molecular interactions between specific small molecule drugs and NF-kB. These drugs specifically inhibit the formation of NF- kB/DNA complexes and structural knowledge in this area might be useful in developing more potent drugs in the future. We want to extend our study towards understanding the structural features and specificity determinants of the activation domain of p65. We use the combined approaches of structural biology and biochemistry to achieve our goals. Our ability to produce large quantities of proteins and the knowledge to generate crystals will enable us to successfully complete our proposed work. We hope that the proposed biochemical work will be helpful for better biological understanding of NF-kB as well as in developing useful small molecule drugs for anti inflammatory reactions.
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