Structure of human annexin a6 at the air-water interface and in a membrane-bound state

Abstract

We postulate the existence of a pH-sensitive domain in annexin A6 (AnxA6), on the basis of our observation of pH-dependent conformational and orientation changes of this protein and its N- (AnxA6a) and C-terminal (AnxA6b) halves in the presence of lipids. Brewster angle microscopy shows that AnxA6, AnxA6a, and AnxA6b in the absence of lipids accumulate at the air-water interface and form a stable, homogeneous layer at pH below 6.0. Under these conditions polarization modulation IR absorption spectroscopy reveals significant conformational changes of AnxA6a whereas AnxA6b preserves its alpha-helical structure. The orientation of protein alpha-helices is parallel with respect to the interface. In the presence of lipids, polarization modulation IR reflection absorption spectroscopy experiments suggest that AnxA6a incorporates into the lipid/air interface, whereas AnxA6b is adsorbed under the lipid monolayer. In this case AnxA6a regains its alpha-helical structures. At a higher pressure of the lipid monolayer the average orientation of the alpha-helices of AnxA6a changes from flat to tilted by 45 degrees with respect to normal to the membrane interface. For AnxA6b no such changes are detected, even at a high pressure of the lipid monolayer-suggesting that the putative pH-sensitive domain of AnxA6 is localized in the N-terminal half of the protein.

FIGURE 1

Kinetics of the lateral pressure surface tension (π) increases of the air-water interface in the presence of AnxA6 or its fragments. The subphase contained 20 mM citrate buffer, pH 5.0, 100 mM NaCl, and 0.1 mM EGTA. AnxA6 (trace a) or its N- (trace b) and C-terminal (trace c) fragments were added to a final concentration of 300 nM. One representative trace of at least three experiments is shown. The experiments varied by <5%.

FIGURE 2

PM-IRRAS spectra of AnxA6 (A) and its N- (B) or C-terminal (C) fragments at the air-water interface. Spectra were collected at the different surface pressures of the protein monolayer: 3 mN/m (traces a), 6 mN/m (traces b), 9 mN/m (traces c), 14 mN/m (traces d), and 20 mN/m (traces e). The subphase contained 20 mM citrate buffer, pH 5.0, 100 mM NaCl, and 0.1 mM EGTA. Bulk protein concentration was 300 nM. One set of representative spectra of four independent experiments performed at the same conditions is shown. The experiments varied by <5%.

FIGURE 3

Far-UV CD spectra of AnxA6a (A) and AnxA6b (B). Solid line corresponds to the spectra collected at pH 7.5, dashed line represents pH 6.2 and short-dashed one pH 4.6. To maintain pH values, 10 mM citrate buffer was used, which in addition contained 0.1 mM EGTA. The assay medium of total volume 0.6 ml contained 0.24 mg of AnxA6a or AnxA6b. All measurements were performed at 25°C using a 2-mm optical pathlength cuvette.

FIGURE 4

The effect of AnxA6 or its peptide fragments on the surface pressure of a lipid monolayer. The lipid monolayer was made from DMPS:DMPE mixed at a weight ratio of 1:1. The subphase contained 20 mM citrate buffer, pH 5.0, 100 mM NaCl, and 0.1 mM EGTA. After stabilization on the initial surface pressure of the lipid monolayer at 27 mN/m, the proteins were added in two equal aliquots (at time zero and as indicated by arrow) to reach the final protein concentration of 300 nM. Further additions did not produce any significant effects or even resulted in lipid monolayer collapse. The traces are marked as follows: trace a, AnxA6; trace b, AnxA6a; and trace c, AnxA6b. They represent typical traces chosen from at least three independent experiments performed under the same conditions.

FIGURE 5

Proteolytic digestion of AnxA6 or its peptide fragments in the presence of liposomes at low pH. The reaction was performed in total volume of 40 μl containing 20 mM citrate buffer, pH 5.0, 100 mM NaCl, 0.1 mM EGTA, and 40 μg of protein. Proteolysis was initiated by adding 5 μg of pepsin and terminated by alkalization of the reaction medium by adding 1 μl of 1 M KOH. Then the products of proteolysis were separated by SDS-PAGE. (A) Digestion of AnxA6. Lane 1, standard of AnxA6 (nondigested protein); lanes 2 and 4 show the digestion products formed in the presence of liposomes (+) after 1 and 2 h of digestion, respectively; and lanes 3 and 5, digestion of AnxA6 in the absence of lipids (−) for 1 and 2 h, respectively. (B) The same experiment performed using the N- and C-terminal peptide fragments of AnxA6. Lanes 1–3 represent products of proteolysis of AnxA6a, AnxA6b, and AnxA6, respectively, in the presence of liposomes (+) for 1 h; lanes 4–6 show corresponding nondigested proteins used in the experiment. MW, molecular weight standards. (C) Identification of the products of controlled proteolysis by Western blotting. Lane 1, standards of AnxA6 and its N-terminal fragment AnxA6a; lane 2, products of proteolysis of AnxA6 for 1 h in the presence of liposomes (+), recognized by monoclonal antibodies against the N-terminal part of AnxA6.

FIGURE 6

Labeling of AnxA6 and its N- or C-terminal peptide fragments with ¹²⁵I-TID. The autoradiogram shown is characteristic for three independent experiments. Proteins were incubated with vesicles containing ¹²⁵I-TID at indicated pH in the presence of Ca²⁺ (+) or EGTA (−). After exposition to UV light (for 20 min) samples were separated from unbound reagent by SDS-PAGE. The gels were then exposed to x-ray film. Analysis of the autoradiograms revealed that except for the presented bands and low molecular weight material at the dye front of the gel, no other radioactive material was detected.

FIGURE 7

BAM images of lipid monolayers made of DMPS:DMPE (at a weight ratio of 1:1) recorded after addition of AnxA6 (A), AnxA6a (B), or AnxA6b (C) into the subphase consisting of 20 mM citrate buffer, pH 5.0, 100 mM NaCl, and 0.1 mM EGTA. Reference gray level (GL) was 25 at shutting time (OS) 50; protein concentration was 300 nM. The real image size is 625 × 500 μm. (D) The same experiment performed with AnxA6 at pH 7.4 (20 mM Tris-HCl buffer, 100 mM NaCl) in the presence of 1 mM CaCl₂.

FIGURE 8

PM-IRRAS spectra of AnxA6 (A), and the N- (B) and C-terminal (C) peptide fragments of the protein recorded in the presence of mixed phospholipid film. The concentration of the protein injected into the subphase consisting of 20 mM citrate buffer, pH 5.0, 100 mM NaCl, and 0.1 mM EGTA (A and B) or 20 mM citrate buffer, pH 5.0, 5 mM NaCl, and 0.1 mM EGTA (C), was 300 nM. After stabilization of the lipid/protein monolayers PM-IRRAS spectra were collected. Dotted line represents spectra recorded for pure phospholipid monolayers made of DMPS:DMPE mixed at a weight ratio of 1:1 (corresponding to π = 27 mN/m). Solid line denotes spectra of protein/phospholipid monolayers after subtraction of the lipid spectra to extract the component characteristic for protein. Nonsmoothed spectra typical for at least three independent determinations are shown.

FIGURE 9

The effect of molecular packing of lipid monolayers on AnxA6 (A) or AnxA6a (B) structure and orientation. PM-IRRAS spectra of AnxA6 or AnxA6a were collected at various initial pressures of the phospholipid monolayer: 0 mN/m (no lipid monolayer), traces a; 5 mN/mm, traces b; 15 mN/m, traces c; and 27 mN/m, traces d. In each experiment the same subphase composition, 20 mM citrate buffer, pH 5.0, 100 mM NaCl, and 0.1 mM EGTA, was used. Protein concentration was 300 nM. Phospholipid monolayers were made of DMPS:DMPE at a weight ratio of 1:1. One set of typical spectra from three experiments is shown.