Solution structures and dynamics of the sterile α Motif (SAM) domain of the deleted in liver cancer 2 (DLC2): a monomeric structure with membrane-binding properties

Abstract

The Deleted in Liver Cancer 2 (DLC2) gene encodes a Rho GTPase-activating protein (GAP) with growth suppressor function. In addition to the RhoGAP domain, this molecule also contains a sterile α motif (SAM) and a lipid-binding StAR-related lipid-transfer (START) domain. To gain an insight into the function of DLC2, we have expressed and purified a recombinant 13C/15N doubly-labeled DLC2 SAM domain. The three-dimensional solution structure and dynamics of the SAM domain of DLC2, DLC2-SAM, were investigated by NMR spectroscopy together with molecular dynamic simulated annealing. We showed that DLC2-SAM is distinct from all other known SAM domains by the presence of a four-helix bundle. The spatial arrangement of four α-helices is also unique. Backbone dynamics studies showed a restricted motion of four helices and a high mobility of the N-and C-termini as well as loops. Although it has been shown that some members of the SAM domain family can form dimers and oligomers, both NMR and biochemical analyses indicated that DLC2-SAM exists as a monomer in solution. We also examined the interaction of DLC2-SAM domain with sodium dodecyl sulfate (SDS) micelles by NMR and CD spectroscopic techniques. 2D [1H,15N] HSQC spectra of DLC2-SAM in the presence of SDS displayed shifts of most residues although still well dispersed, in agreement with slightly decreased α-helical content based on CD spectra of DLC2-SAM in the absence and presence of SDS, suggesting that DLC2-SAM may interact with membrane lipids in vivo with secondary structure changes of the protein. Finally, we demonstrated that DLC2-SAM is dispensable for the self association of full-length DLC2 in vivo.