The neuroendocrine protein 7B2 is intrinsically disordered

Abstract

The small neuroendocrine protein 7B2 has been shown to be required for the productive maturation of proprotein convertase 2 (proPC2) to an active enzyme form; this action is accomplished via its ability to block aggregation of proPC2 into nonactivatable forms. Recent data show that 7B2 can also act as a postfolding chaperone to block the aggregation of a number of other proteins, for example, α-synuclein. To gain insight into the mechanism of action of 7B2 in blocking protein aggregation, we performed structural studies of this protein using gel filtration chromatography, intrinsic tryptophan fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Gel filtration studies indicated that 7B2 exists as an extended monomer, eluting at a molecular mass higher than that expected for a globular protein of similar size. However, chemical cross-linking showed that 7B2 exhibits concentration-dependent oligomerization. CD experiments showed that both full-length 27 kDa 7B2 and the C-terminally truncated 21 kDa form lack appreciable secondary structure, although the longer protein exhibited more structural content than the latter, as demonstrated by intrinsic and ANS fluorescence studies. NMR spectra confirmed the lack of structure in native 7B2, but a disorder-to-order transition was observed upon incubation with one of its client proteins, α-synuclein. We conclude that 7B2 is a natively disordered protein whose function as an antiaggregant chaperone is likely facilitated by its lack of appreciable secondary structure and tendency to form oligomers.

Figure 1

Domain structures for the proteins used in our study.

Figure 2

Size exclusion chromatography and cross-linking. (a and b) Size exclusion chromatogram of 21 kDa 7B2 at 0.5 and 2 mg/mL (blue line), respectively. The inset of panel a shows the size exclusion chromatogram of eukaryotic 21 kDa 7B2 at 0.5 mg/mL. The molecular mass standards, thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobulin (17 kDa), and vitamin B₁₂ (1.35 kDa), are shown with asterisks. (c and d) Chemical cross-linking (0.1% glutaraldehyde) of gel filtration experiments using 0.5 and 2 mg/mL 21 kDa 7B2, respectively.

Figure 3

Chemical cross-linking of 27 and 21 kDa 7B2s: concentration dependence of multimer formation. (a) The 27 kDa 7B2 was cross-linked with 0.1% glutaraldehyde at final concentrations ranging from 7.5 to 30 μM. (b) The 21 kDa 7B2 was cross-linked with 0.1% glutaraldehyde at final concentrations ranging from 7.5 to 30 μM. (c) Carbonic anhydrase, at 30 μM, cross-linked with 0.1% glutaraldehyde. Bands corresponding to monomer (M), dimer (D), and trimer (T) are marked on the gels with arrows.

Figure 4

Tryptophan (left) and ANS (right) fluorescence spectra of 7B2 variants. (a and b) Wild-type rat 27 kDa 7B2. (c and d) Wild-type rat 21 kDa 7B2. (e) C. elegans 27 kDa 7B2.

Figure 5

Far-UV CD spectra of 7B2 and variants. Wild-type rat 27 kDa 7B2 (black dashed line), wild-type rat 21 kDa 7B2 (black solid line), and 27 kDa C. elegans 7B2 (gray solid line) in 10 mM sodium phosphate and 0.4 mM octyl glucoside (pH 6.5).

Figure 6

PONDR analysis of 27 kDa 7B2 and C. elegans 27 kDa 7B2 (signal peptides not included). The black bar shows a disordered region >40 residues in length.

Figure 7

NMR of 27 kDa 7B2. These data depict the ¹H–¹⁵N HSQC spectrum for 27 kDa 7B2.

Figure 8

¹³C–¹H natural abundance carbon HSQC spectrum for 27 kDa 7B2.

Figure 9

7B2 in complex with client protein. ¹H–¹⁵N TROSY spectrum for 27 kDa 7B2 complexed with α-synuclein. The black peaks were obtained from spectra acquired without α-synuclein, and the red peaks were obtained from spectra acquired after incubation with α-synuclein.