The cooperative folding of annexin A2 relies on a transient nonnative intermediate

Abstract

Annexins (Anxs) are a family of highly homologous proteins that bind and aggregate lipid vesicles in the presence of calcium. All members of the family contain a variable N-terminus determining specific functions, followed by a conserved core region responsible for the general calcium-dependent lipid-binding property. The core structure consists of four homologous domains (D_I-D_IV), each consisting of a right-handed super-helix of five α-helices. We present data from a combination of site-directed mutagenesis, NMR, and circular dichroism showing that the G25-D34 region of the N-terminus as well as the contacts between residues D38A, R63A, and Q67A of AnxA2-D_I are crucial for the autonomous folding and stability of D_I of AnxA2. However, we also show that the folding of the full-length protein is very robust in that mutations and truncations that disrupted the folding of AnxA2-D_I did not abolish the folding of full-length AnxA2, only lowering its thermal stability. This robustness of the folding of full-length AnxA2 is likely to be mediated by the existence of at least one transient nonnative intermediate as suggested by our kinetic data using stopped-flow fluorescence experiments. We also show that hydrophobic amino acids in AnxA2-D_I involved in interfacial contacts with AnxA2-D_IV are important for the cooperative folding and stability of the full-length protein. Mutating all of the V57E-V98R-G101Y residues in AnxA2-D_I did not affect the folding of the domain, only its stability, but prevented the cooperative folding of the full-length protein. Our collective results favor a highly cooperative and robust folding process mediated by alternative intermediate steps. Since AnxA2 is a multifunctional protein involved in several steps of the progression of cell transformation, these data on structure and folding pathways are therefore crucial to designing anticancer drugs targeting AnxA2.

Graphical abstract

Figure 1

(A) Ribbon plot of domain I (D_I) of AnxA2 (from Pro21) showing the four conserved residues that appear to constitute a structural tetrad at the N-terminus of D_I. The four conserved residues are F33, D38, R63, and Q67, counting the first M as residue 1. (B) Ribbon plot of D_I of AnxA2 (from P21) indicating the side chains of the three mutated hydrophobic residues (V57, V98, and G101) at the interface of D_I with α-helices in D_II and D_IV of AnxA2. The figures were prepared using Biovia Studio Visualizer (http://accelrys.com/products/collaborative-science/biovia-discovery-studio/visualization.html) and based on the PDB structures 1XJL and 4X9P. (C) Schematic representation of wild-type and different mutated/truncated forms of AnxA2-D_I and full-length AnxA2. To investigate the role of a folded AnxA2-D_I in the folding of the full-length AnxA2, single mutations (F33A, D38A, R63A, and Q67A) were introduced in the context of [25–104] and [35–104] (not F33A) AnxA2-D_I as well as in [1–339] full-length AnxA2 and [35–339] Δ1-34-truncated AnxA2 (not F33A). Furthermore, a multiple mutation (F33A-D38A-R63A-Q67A) was introduced in [1–339] full-length AnxA2. Subsequently, to investigate the role of interfacial hydrophobic amino acid residues in AnxA2-D_I for the folding of full-length AnxA2, single mutations (V57E, V98R, and G101Y) and a multiple mutation (V57E-V98R-G101Y) were introduced in the context of [25–104] AnxA2-D_I and [1–339] full-length AnxA2 as indicated in the figure. The numbers represent the number of the particular amino acid in the bovine and human AnxA2 sequence, and the four domains are shown as black boxes, denoted D_I–D_IV. The color code for the respective mutated amino acids as indicated in (C) is also used in (A) and (B).

Figure 2

Characterization of the solubility of wild-type and mutated (A) [1–339] (wild-type, F33A, D38A, R63A, Q67A, and F33A-D38A-R63A-Q67A), (B) [35–339] (wild-type, D38A, R63A, Q67A), and (C) [1–339] (wild-type, V57E, V98R, G101Y, V57-V98R-G101Y) AnxA2 forms as determined by 10% SDS-PAGE analysis. The 6His-tagged AnxA2 forms were expressed for 3 h at 37°C after induction with 1 mM IPTG. Subsequently, aliquots were retrieved and sonicated in breakage buffer (total, T). After centrifugation at 16,000 g for 15 min, aggregated proteins were recovered as the pellet (P) fraction and the soluble proteins as the supernatant (S) fraction. The protein bands were visualized by Coomassie Brilliant Blue staining. AnxA2 is indicated by an arrowhead to the right.

Figure 3

Impact of mutating amino acid residues (F33, D38A, R63A, Q67A) or deleting the N-terminus on the folding of AnxA2-D_I. ¹H-¹⁵N HSQC NMR spectra of [25–104] AnxA2; folded wild-type (600 μM), F33A (550 μM) and R63A (150 μM) (upper row), and misfolded D38A (275 μM), Q67A (275 μM) as well as misfolded [35–104] AnxA2; wild-type (50 μM), D38A (700 μM), R63A (700 μM), and Q67A (700 μM), were recorded at pH 7, 25°C, and 700 MHz (¹H frequency). To see this figure in color, go online.

Figure 4

Impact of mutating amino acid residues (D38A, Q67A), causing collapse of the folding of AnxA2-D_I, and of deleting the N-terminus on the folding of full-length AnxA2. ¹H-¹⁵N HSQC NMR spectra of [1–339] AnxA2; wild-type D38A and Q67A as well as [35–339] AnxA2, all at 200 μM were recorded at pH 7, 30°C, and 700 MHz (¹H frequency). To see this figure in color, go online.

Figure 5

Mutating amino acid residues (V57E, V98R, G101Y, V57E-V98R-G101Y) in AnxA2-D_I involved in interfacial contact with other domains does not unfold this domain. ¹H-¹⁵N HSQC NMR spectra of [25–104] AnxA2; wild-type (600 μM), V57E (250 μM), V98R (250 μM), G101Y (250 μM), and V57E-V98R-G101Y (150 μM) were recorded at pH 7, 25°C, and 700 MHz (¹H frequency). To see this figure in color, go online.

Figure 6

Typical unfolding and refolding time courses of AnxA2. (A) AnxA2 diluted in buffer (25 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM Tris(2-carboxyethyl)phosphine (TCEP), pH 8.0) was rapidly mixed with GdnHCl to reach 5.4 M final concentration (unfolding); (B) the protein in denaturing conditions (buffer with added GdnHCl) was mixed with refolding buffer to reach 0.5 M final concentration of GdnHCl.

Figure 7

A semilogarithmic plot of the observed (un)folding rate constants (chevron plot) for wild-type AnxA2. The chevron plot of the observed rate of folding and unfolding is shown as a function of final denaturant concentration (GdnHCl). The total chevron plot is fit to a two-state equation.

Figure 8

The chevron plots of wild-type AnxA2 and the indicated AnxA2 variants. Lines represent the best fit to an equation that describes a three-state folding mechanism, implying the presence of an intermediate along the reaction pathway. Chevron plots were globally fitted by sharing kinetic m values for all datasets (see materials and methods and results sections for details).