Solution structure studies of monomeric human TIP47/perilipin-3 reveal a highly extended conformation

Abstract

Tail-interacting protein of 47 kDa (TIP47) has two putative functions: lipid biogenesis and mannose 6-phosphate receptor recycling. Progress in understanding the molecular details of these two functions has been hampered by the lack of structural data on TIP47, with a crystal structure of the C-terminal domain of the mouse homolog constituting the only structural data in the literature so far. Our studies have first provided a strategy to obtain pure monodisperse preparations of the full-length TIP47/perilipin-3 protein, as well as a series of N-terminal truncation mutants with no exogenous sequences. These constructs have then enabled us to obtain the first structural characterization of the full-length protein in solution. Our work demonstrates that the N-terminal region of TIP47/perilipin-3, in contrast to the largely helical C-terminal region, is predominantly β-structure with turns and bends. Moreover, we show that full-length TIP47/perilipin-3 adopts an extended conformation in solution, with considerable spatial separation of the N- and C-termini that would likely translate into a separation of functional domains.

Figure 1

Domain structure of full-length human TIP47/perilipin-3 and the N-terminal constructs generated for this study. The N-terminal regions are highlighted in light blue with yellow and black inserts indicating the 11-mer repeat and Rab9 binding regions, respectively. The C-terminal region corresponding to the mouse crystal structure is in red, with the 4-bundle helix region as a dark blue insert.

Figure 2

A: MALLS results for human TIP47/perilipin-3 and the truncation mutants. UV trace (lines) and MALLS experimental molar masses for full-length TIP47/perilipin-3 (gray), TIP47/perilipin-3_117-434 (red), TIP47/perilipin-3_152-434 (blue), and TIP47/perilipin-3_187-434 (orange; see also Table I). B: Guinier plots from the SAXS data for the same protein constructs as in part A.

Figure 3

Far-UV spectra determining the secondary structure of full-length TIP47/perilipin-3 (gray line) and TIP47/perilipin-3 117-434 (red line), 152-434 (blue line), and 187-434 (orange line). Data were acquired over the range 182-260 nm and the plots have been scaled to molar protein concentration.

Figure 4

A: Scattering data as I(q) versus q for human TIP47/perilipin-3 and the N-terminal truncation mutants. Full-length TIP47/perilipin-3 (gray, 6.85 mg/mL), TIP47/perilipin-3_117-434 (red, 9.25 mg/mL), TIP47/perilipin-3_152-434 (blue, 8.89 mg/mL), and TIP47/perilipin-3_187-434 (orange, 8.22 mg/mL). The plots for TIP47/perilipin-3_117-434, TIP47/perilipin-3_152-434, and TIP47/perilipin-3_187-434 have been arbitrarily shifted on the vertical axis for clarity. The solid lines are the fits to the data of the best-fit dummy atom models for each construct shown in Fig. 5. B: Corresponding P(r) profiles scaled to the ratio of the squares of the molecular weights of each protein.

Figure 5

Alignment of the mouse TIP47/perilipin-3 PAT-C domain crystal structure (PDB 1SZI; ribbons) spatially positioned within the consensus shape-models (spheres) derived from the SAXS data for human full-length TIP47/perilipin-3 (gray); TIP47/perilipin-3_117-434 (red);TIP47/perilipin-3_152-434 (blue) and; TIP47/perilipin-3_187-434 (orange). The mouse homologue spatially superimposes into the molecular shape of the shortest human truncation mutant (TIP47/perilipin-3_187-434) that spans the C-terminal domains of the protein. The space occupied by regions encompassing the N-terminal half of human TIP47/perilipin-3 appears to extend into solution away from the C-terminal domains.