The NMR structure of the murine DLC2 SAM domain reveals a variant fold that is similar to a four-helix bundle

Abstract

Background: The tumor suppressor DLC2 (Deleted in Liver Cancer -2) participates in cell signaling at the mitochondrial membrane. DLC2 is characterized by a SAM (sterile alpha motif) domain, a Rho GTPase activating protein (GAP) domain, and a START lipid transfer domain.

Results: Towards understanding the function of DLC2, we have solved the NMR solution structure of the SAM domain. The DLC2-SAM domain structure reveals an atypical four-helix composition that is distinct from the five-helix SAM domain structures that have been determined to date. From structural alignments, helix 3 of the canonical SAM domain appears to be replaced by shorter, extended secondary structure that follows a similar path. Another difference is demonstrated by helices 1 and 2 that form a helical hairpin that is situated approximately parallel to the canonical helix 5.

Conclusion: The DLC2-SAM domain adopts a structure that is topologically more similar to an anti-parallel four-helix bundle than a canonical SAM domain. This alternate topology may allow the DLC2-SAM domain to interact with a novel set of ligands.

Figure 1

Delineation of the minimal DLC2-SAM domain. (a) By sequence homology, the DLC2-SAM domain is defined by exons 2–4. Of four protein fragments expressed, three were suitable for further biophysical analyses. (b) Far UV CD spectra. (c) Thermal stability was determined by monitoring ellipticity at 220 nm, a wavelength characteristic of α-helical secondary structure.

Figure 2

A comparison of DLC2-SAM domain and other SAM domains. (a) A representative structure of the DLC2-SAM domain in ribbon form and as ensemble of the 10 lowest energy solutions. A short helix H3 (cyan) typically observed in SAM domains such as (b) S. cerevisiae Ste50 [PDB: 1Z1V] is absent in the DLC2-SAM domain. (c) Sequence alignment of murine DLC2 with SAM domains known to participate in protein-protein interactions. Listed below DLC2 are SAM domains from Drosophila Polyhomeotic (Ph), Drosophila Sex-Comb-on-Midleg (Scm), S. cerevisiae Ste11 and Ste50. Hydrophobic amino acids that contribute to the hydrophobic core are highlighted.

Figure 3

A comparison of the DLC2-SAM domain (in teal) and FELIX (in magenta), a model of a engineered four-helix bundle [PDB: 1FLX]. (a, b) Two views of a best-fit superimposition of the DLC2 and FELIX structures. While helix H4 of DLC2 is much shorter than its analogous helix (H3) in FELIX, its observed length is consistent with other SAM domains. (c) A sequence alignment highlighting identical (black) and homologous (grey) residues.

Figure 4

A NMR relaxation study of the DLC2-SAM domain. (a) Per residue ¹⁵N T₁ longitudinal relaxation times. (b) Per residue ¹⁵N T₂ transverse relaxation times. (c) Per residue heteronuclear NOE ratios.

Figure 5

A molecular surface representation of the DLC2 SAM domain was colored according to charge (Asp/Glu, red; Lys/Arg, blue; aromatics, yellow; all others, white). With helices H2 and H4 immediately towards the viewer, a shallow cleft lined with aromatic residues is apparent.