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The 8-kDa light chain of dynein (DLC8) is ubiquitously expressed in various cell types. Other than serving as a light chain of the dynein complexes, this highly conserved protein has been shown to bind a larger number of proteins with diverse biological functions. DLC8 forms a homodimer via three-dimensional domain swapping of an internal β-strand (the β2-strand) at neutral pH. The protein undergoes non-reversible dimer-tomonomer dissociation when the pH value of the protein solution decreases. The three-dimensional structure of the DLC8 monomer determined by NMR spectroscopy at pH 3.0 showed that the protein is well folded. The major conformational change accompanied by dimer dissociation is in the β2-strand of the protein, which undergoes transition from a β-strand to a nascent α-helix. The monomer form of DLC8 is not capable of binding to target proteins. Insertion of two flexible amino acid residues in the tight β1/β2-1oop dramatically stabilized the monoruer conformation of the protein. NMR studies showed that the mutation altered the conformation as well as the three-dimensional domain swapping-mediated assembly of the DLC8 dimer. The mutant DLC8 was unable to bind to its targets even at physiological pH. The threedimensional structure of the mutant protein in its monomeric form provides the structural basis of the mutation-induced stabilization of the monomer conformation. Based on the experimental data, we conclude that the formation of the β2-strand swapping-mediated dimer is mandatory for the structure and function of DLC8. We further note that the DLC8 dimer represents a novel mode of three-dimensional domain swapping. [ABSTRACT FROM AUTHOR]