Crystal structure of the LUFS domain of human single-stranded DNA binding Protein 2 (SSBP2)

Abstract

The human single-stranded DNA binding Protein 2 (SSBP2) is a tumor suppressor implicated in multiple cancer forms. The SSBP2 and related SSBP3/SSBP4 proteins are predicted to be intrinsically disordered excepted for their highly conserved N-terminal LUFS (LUG/LUH, Flo8, and SSBP/SSDP) domain. LUFS domains are found in a number of proteins including some transcriptional co-repressors. Although LUFS domains contain an N-terminal Lis homology (LisH) motif that typically forms a stable dimer, no 3D structure of any LUFS domain is available. Here, we report a crystal structure of the LUFS domain of human SSBP2 at 1.52 Å resolution. We show that the SSBP2 LUFS domain forms a homo-tetramer and reveal how an alpha-helix C-terminal to the LisH motif mediates SSBP2 tetramerization (dimerization of dimers). Conservation of the tetramerization interface among LUFS domains suggests that other LUFS domains may also form tetramers in similar manners.

Keywords: LUFS (LUG/LUH, Flo8 and SSBP/SSDP) domain; Lis homology (LisH) motif; SSBP proteins; X-ray crystallography; single-stranded DNA binding Protein 2; tetramerization; tumor suppressor.

Figure 1

Biochemical characterization of N‐terminal LUFS domain of SSBP2. (A) Evolutionary conservation of the LUFS domain among SSBP2/SSBP3/SSBP4, LEUNG/LUH, Flo8, MSS11, and SOMA proteins.20, 21, 22, 23 The double slash in SOMA sequence represents a 32‐residue hydrophilic loop. Hs, Homo sapiens; At, Arabidopsis thaliana; Ca, Candida albicans; Nf, Neosartorya fumigate. (B) Limited proteolysis analysis of SSBP2 fragments. One subtilisin‐resistant fragment can be obtained through limited proteolysis of SSBP2(1–94) (left) or SSBP2(10–94) (right), which is about 7 kDa, red box marked. The marked ratios are the molar ratios of subtilisin over SSBP2(1–94) or SSBP2(10–94) used in limited proteolysis. The SDS‐PAGE gel was stained with Coomassie brilliant blue. (C) SEC‐MALS analysis of SSBP2(1–94), SSBP2(10–77) and the F68D/L71A/Y72A triple mutant of SSBP2(1–94) (SSBP2(1‐94)Mut for short). Molecular weights measured by SEC‐MALS are shown. (D) Alignment of SEC chromatograms of SSBP2(1–94) (in red), SSBP2(10–77) (in green), and SSBP2(1–94)Mut (in blue). Calculated molecular weights for SSBP2(1–94) tetramer, SSBP2(10–77) tetramer, and SSBP2(1–94) dimer are labeled.

Figure 2

Crystal structure of the LUFS domain of human SSBP2. (A) Cartoon representation of SSBP2(10–77) dimer observed in each asymmetric unit, in two orthogonal orientations. Each SSBP2(10–77) subunit is colored in gradient, with blue to red representing the peptide chain from N‐ to C‐terminus. (B) Cartoon representation the overall structure of SSBP2(10–77) tetramer in stereo. The two dimers forming each tetramer are related by a “horizontal” two‐fold axis in this orientation. (C) Sequence alignment and structural superposition of different LisH motifs: these of SSBP2 (in green), LIS1 (in magenta, PDB code:1UUJ), FOP (in cyan, PDB code: 2D68) and TBL1X (in yellow, PDB code:2XTC). (D) Key residues involved in the tetramerization of the SSBP2 LUFS domain, shown in stereo.