Structure-function studies of the 8-kDa dynein light chain : a multifunctional regulatory protein
by Jing-Song Fan
xviii, 178 leaves : ill. (some col.) ; 30 cm
The focus of this dissertation is the structure and function of the 8-kDa dynein light chain (DLC8). I chose DLC8 to study as it acts as a multifunctional regulator protein in eukaryotes....[ Read more ]
The focus of this dissertation is the structure and function of the 8-kDa dynein light chain (DLC8). I chose DLC8 to study as it acts as a multifunctional regulator protein in eukaryotes.
The interaction between DLC8 and neuronal nitric oxide synthases (nNOS) was investigated by a combination of NMR and biochemical techniques. The DLC8-binding region of nNOS adopts a random coil conformation in solution and it was precisely mapped to a 17-residue peptide fragment. A synthesized peptide was used to confirm that this fragment is sufficient for DLC8 binding.
The solution structure of DLC8 was determined by NMR techniques. DLC8 forms a dimer in solution under physiological condition. Two monomers related by a two-fold axis form a rectangular dimer. Two pairs of α-helices cover opposite faces, and each pair of helices packs against a β-sheet with five antiparallel β-strands. Each β-sheet contains four strands from one monomer and a fifth strand from the other monomer. Therefore, the DLC8 dimer is assembled using a three-dimensional domain swapping mechanism. The highly charged helical faces contribute to the solubility of the protein. Inter-subunit hydrogen bonds between antiparallel β-strands, together with the extensive hydrophobic interactions contributed by amino acids in the dimer interface render DLC8 to be extremely stable in solution.
Two identical target-binding grooves were identified on both sides of the DLC8 surface. NMR structures of two different DLC8/target complexes showed that the target peptides (nNOS and Bim) fit snugly into the binding-grooves of the protein by forming β-sheet with the β2-strand of DLC8. The complex structures revealed that DLC8 employs significantly different strategies in engaging targets with diverse amino acid sequences. Additionally, the backbone dynamics of DLC8 in both apo- and target-bound form were characterized by NMR relaxation measurements. The results indicated that in apo-form, the amino acid residues in the target-binding grooves display significant conformational exchanges at a timescale of millisecond-to-microsecond. Such slow timescale conformation fluctuations disappear upon formation of complexes with the target peptides. It is likely that the unique surface-charge properties as well as the malleable nature of the concave target-binding grooves enable DLC8 to bind to diverse targets without obvious binding specificities.
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