Structure-function studies of the 8-kDa dynein light chain: A multifunctional regulatory protein

The 8-kDa dynein light chain is the smallest subunit of dynein motor complexes. It can potentially act as a multifunctional regulatory protein. The interaction between DLC8 and neuronal nitric oxide synthases 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 corresponding to the DLC8-binding region was used to confirm that this fragment is sufficient for DLC8 binding.;The solution structure of DLC8 was determined to high-resolution by NMR techniques. DLC8 forms a dimer in solution under physiological conditions. Two monomers related by a two-fold axis form a rectangular dimer. Each five-stranded beta-sheet contains four strands from one monomer and a fifth strand from the other monomer. Two identical target-binding grooves were identified on each side of the surface of DLC8 dimer. NMR structures of two different DLC8/target complexes showed that target peptides (nNOS and Bim) fit snugly into the binding grooves of the protein by forming beta-sheet with the beta2-strand of DLC8. The complex structures reveal that DLC8 uses 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 indicate 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 property as well as malleable nature of the concave target-binding grooves enable DLC8 to bind to a large array of targets without obvious binding specificities.