The S100A10-annexin A2 complex provides a novel asymmetric platform for membrane repair

Abstract

Membrane repair is mediated by multiprotein complexes, such as that formed between the dimeric EF-hand protein S100A10, the calcium- and phospholipid-binding protein annexin A2, the enlargeosome protein AHNAK, and members of the transmembrane ferlin family. Although interactions between these proteins have been shown, little is known about their structural arrangement and mechanisms of formation. In this work, we used a non-covalent complex between S100A10 and the N terminus of annexin A2 (residues 1-15) and a designed hybrid protein (A10A2), where S100A10 is linked in tandem to the N-terminal region of annexin A2, to explore the binding region, stoichiometry, and affinity with a synthetic peptide from the C terminus of AHNAK. Using multiple biophysical methods, we identified a novel asymmetric arrangement between a single AHNAK peptide and the A10A2 dimer. The AHNAK peptide was shown to require the annexin A2 N terminus, indicating that the AHNAK binding site comprises regions on both S100A10 and annexin proteins. NMR spectroscopy was used to show that the AHNAK binding surface comprised residues from helix IV in S100A10 and the C-terminal portion from the annexin A2 peptide. This novel surface maps to the exposed side of helices IV and IV' of the S100 dimeric structure, a region not identified in any previous S100 target protein structures. The results provide the first structural details of the ternary S100A10 protein complex required for membrane repair.

FIGURE 1.

Mapping the A10A2 binding site on the C-terminal region of AHNAK by peptide array analysis. A representative peptide array of the Asp⁴⁸⁸⁴–Glu⁵⁸⁹⁰ region of AHNAK was synthesized on a cellulose membrane. Each spot contained an 18-residue peptide shifted by 3 residues from its predecessor until the C terminus was reached. The array was probed with Alexa Fluor 680-A10A2 and imaged at 700 nm. The sequences of AHNAK that showed the best interaction with A10A2 are listed beside and below the array: sequence a (Thr⁴⁹¹⁷–Gly⁴⁹⁴⁰), sequence b (Asp⁴⁹⁵⁹–Pro⁴⁹⁹⁴), sequence c (Pro⁵²²⁰–Ile⁵²⁴³), sequence d (Asn⁵⁶⁰⁷–Ile⁵⁶³³), sequence e (Gly⁵⁶⁵²–Gly⁵⁶⁷⁵), sequence f (Ser⁵⁷⁰³–Thr⁵⁷²⁹), sequence g (Ala⁵⁷⁵⁷–Ser⁵⁷⁸⁰), and sequence h (Leu⁵⁸⁰⁸–Glu⁵⁸³⁷). These sequences were used to define a consensus sequence showing positively charged (+), hydrophobic (ϕ), and variable (X) residues using the Blocks Server (available on the World Wide Web).

FIGURE 2.

An overlay of ¹H-¹⁵N HSQC spectra of annexin A2-bound S100A10 or A10A2 hybrid protein complexes bound to the AHNAK5 peptide. A, ¹H-¹⁵N HSQC spectrum of 0.5 mm ¹⁵N-labeled S100A10 (protomer concentration) bound to 0.5 mm unlabeled annexin A2 peptide (Ac-STVHEILSLKQLEGD) (black) and in complex with 0.25 mm AHNAK5 (Ac-GKVTFPKMKIPKFTFSGREL) (pink). B, ¹H-¹⁵N HSQC spectrum of 0.5 mm uniformly ¹⁵N-labeled A10A2 hybrid protein (protomer) (black) and in complex with 0.25 mm AHNAK5 peptide (cyan). Peaks that exhibited obvious multiplicity in the AHNAK5 complex are labeled according to their one-letter amino acid code and residue number. Spectra were collected on a Varian Inova 600-MHz spectrometer at 35 °C in 90% H₂O, 10% D₂O at pH 7.0.

FIGURE 3.

Stoichiometry of the AHNAK5 peptide with A10A2 measured from ¹H-¹⁵N HSQC experiments. Aliquots of the unlabeled AHNAK5 peptide were added to a solution of 143 μm ¹⁵N,¹³C-labeled A10A2 (dimer concentration), and the resulting changes in peak intensity were monitored. The binding between AHNAK5 and A10A2 is shown as the plot of decrease (A) and increase (B) in peak intensities for Gly⁴⁰ (●), Val⁵¹ (♦), and Gly⁷⁷ (▴) in S100A10 and Leu¹¹² (▾) in annexin A2 as a function of the AHNAK5/A10A2 ratio. C, the normalized changes in peak intensities are shown as a function of AHNAK5 concentration. Data sets for Gly⁴⁰ (●), Val⁵¹ (♦), Gly⁷⁷ (▴), and Leu¹¹² (▾) were globally fit for 1:1 (solid line) and 2:1 (dashed line) AHNAK5/A10A2 stoichiometries, indicating that the dissociation constant (K_d) was <1 μm. Data fitting is described under “Experimental Procedures.”

FIGURE 4.

Mass spectra showing complexes of S100A10, annexin A2 peptide, and AHNAK5 peptides. A, A10A2 hybrid protein mixed with the AHNAK5 peptide, showing masses attributed to the A10A2 protomer (A10A2(m)), A10A2 dimer, and A10A2 complex with a single AHNAK5 peptide. B, S100A10 mixed with annexin A2 and AHNAK5 peptides, showing masses corresponding to the S100A10 protomer (S100A10(m)), S100A10 dimer, S100A10 dimer in complex with two annexin A2 peptides (S100A10:2A2), and S100A10 dimer in complex with two annexin A2 peptides and a single AHNAK5 peptide(S100A10:2A2:AHNAK5). Spectra were obtained under non-denaturing conditions as described under “Experimental Procedures.”

FIGURE 5.

Interaction of AHNAK5 with the A10A2 hybrid protein monitored by fluorescence spectroscopy and isothermal titration calorimetry. A, fluorescence spectra of acrylodan-A10A2 (167 nm) showing the change in acrylodan fluorescence with increasing AHNAK5 peptide concentration. B, binding curve for acrylodan-labeled A10A2 titrated with the AHNAK5 peptide showing the normalized change in fluorescence measured at 500 nm as a function of AHNAK5 concentration. Data were collected from duplicate titrations, and curves were fit globally with a 1:1 ligand binding function (solid line). C, isothermal titration calorimetry experiment for AHNAK5 peptide binding to A10A2. The spectra show the base line-corrected calorimetric titration data for 30 injections of AHNAK5 into a cell containing a solution of A10A2 (top) and the derived binding isotherm for the experiment obtained by integrating the area of each peak after each injection (bottom). The solid line represents the best fit of the data to a single binding site with n = 1.02. The fit yields K_d = 28 ± 2 nm, ΔH = −41.9 ± 0.4 kJ/mol (binding enthalpy), and ΔS = 8.4 J/mol (entropy change).

FIGURE 6.

Binding surface for AHNAK5 on the S100A10-annexin A2 complex. Residues that exhibited the largest differences in chemical shift for pairs of peaks, in the bound AHNAK5 complex with A10A2 hybrid protein, were mapped to the surface of S100A10-annexin A2 derived from its three-dimensional crystal structure (8). Residues that showed the largest changes are shown in magenta and labeled in S100A10 (white) and annexin A2 (blue). Each residue label is followed by either a or b to denote the asymmetric relationship of the dimer caused by AHNAK5 binding. Helices are labeled I–IV and I′–IV′ for each protomer.