Structural and biophysical characterization of the mitochondrial fission protein Fis1, and, The transcription factor Sp1 and its cognate binding site

Part I. Fis1 is an integral membrane protein that controls the assembly, membrane distribution, and function of the mitochondrial fission machinery in Saccharomyces cervesiae. Fis1 is conserved from yeast to humans, implying a common role for Fis1 in mitochondrial fission. Fis1 from both yeast and human have been structurally and biophysically characterized by various biochemical and spectroscopic methods and X-ray crystallography. The 2.0 A resolution crystal structure of the cytosolic domain of human Fis1 (hFis1) has been determined. The structure reveals an anti-parallel array of alpha-helices homologous to tetratricopeptide repeat (TPR) proteins. Structure-based sequence alignments of hFis1 uncovered two divergent TPR motifs; the first TPR motif differs from the TPR consensus sequence by a 3-residue insertion in a turn that may be important for function. These TPR helices create an amphiphilic, concave surface that can accommodate a helix or, possibly, an extended segment. Indeed, this putative binding surface mediates homodimer formation of hFis1 in the crystal. The physiological relevance of the homodimerization is questioned by evidence for only the monomer in solution, but is substantiated by yeast two hybrid assays from another laboratory. Structural and biochemical evidence presented here alludes to the oligomerization of Fis1 potentially being triggered by a membrane-like environment. The structure of hFis1 and biophysical studies of both yFis1 and hFis1 provide insight into the proposed binding interactions that mediate mitochondrial fission.; Part II. Sp1 is a general transcription factor that binds GC-rich sequences to regulate the expression of a large number of genes involved in nearly all aspects of cellular function. The interactions between the Sp1 zinc fingers and its cognate GC-rich DNA sequence have been further explored by biochemical and biophysical methods. Three crystal structures of the entire Sp1 consensus sequence d(GGGGCGGGG) with two or three additional base pairs on either the 5' or 3' ends and overhangs have been determined. Despite the different lengths of DNA in the pseudo-dodecamers and pseudo-tridecamer, all three structures form A-DNA duplexes that share a common set of crystal contacts, including a T * (G · C) base triplet and a 5&feet;-overhang that flips out and away from the helical axes to form a Hoogsteen base pair with the 3&feet;-overhang of a symmetry mate. The global conformations of the three structures differ, however, in the widths of their respective major grooves, the lengths of the molecules, and the degree of crystal packing. The structures were determined from crystals grown in an unusual precipitant for A-DNA, polyethylene glycol (PEG) MW 400, and polyamines or ions; cobalt hexamine for the pseudo-13mer, and spermidine for the pseudo-dodecamers. As the Sp1 binding site is a target for anticancer drugs, pseudo-dodecamer crystals were soaked with one such anticancer compound, P4N. Though not visualized unambiguously in the electron density maps, the drug influences the structure, as evident from significant differences in the lattice constants, crystal packing, and overall conformation of the structure.