High-resolution structures of annexin A5 in a two-dimensional array

Journal Pre-proofs High-resolution structures of annexin A5 in a two-dimensional array Seokho Hong, Soohui Na, Ok-Hee Kim, Soyeon Jeong, Byung-Chul Oh, NamChul Ha PII: DOI: Reference:

S1047-8477(19)30212-6 https://doi.org/10.1016/j.jsb.2019.10.003 YJSBI 7401

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Journal of Structural Biology

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Please cite this article as: Hong, S., Na, S., Kim, O-H., Jeong, S., Oh, B-C., Ha, N-C., High-resolution structures of annexin A5 in a two-dimensional array, Journal of Structural Biology (2019), doi: https://doi.org/10.1016/j.jsb. 2019.10.003

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High-resolution structures of annexin A5 in a two-dimensional array Seokho Hong1, Soohui Na1,3, Ok-Hee Kim2, Soyeon Jeong1, Byung-Chul Oh2, and NamChul Ha1, *

1

Department of Agricultural Biotechnology, Research Institute of Agriculture and Life

Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University, Seoul 08826, Republic of Korea 2

Department of Physiology, Lee Gil Ya Cancer and Diabetes Institute, Gachon University

College of Medicine, Incheon 21999, Republic of Korea 3

Current address: Skin Care Research Team, LG Household & Healthcare Ltd., Seoul

07795, Republic of Korea

To whom correspondence should be addressed: Nam-Chul Ha (e-mail: [email protected], Tel: +82-2-880-4853, Fax: +82-2-873-5095)

1

Abstract Annexins are soluble cytosolic proteins that bind to cell membranes. Annexin A5 selfassembles into a two-dimensional (2D) array and prevents cell rupture by attaching to damaged membranes. However, this process is not fully understood at the molecular level. In this study, we determined the crystal structures of annexin A5 with and without calcium (Ca2+) and confirmed the Ca2+-dependent outward motion of a tryptophan residue. Strikingly, the two structures exhibited the same crystal packing and 2D arrangement into a p3 lattice, which agrees well with the results of low-resolution structural imaging. Highresolution structures indicated that a three-fold interaction near the tryptophan residue is important for mediating the formation of the p3 lattice. A hypothesis on the promotion of p3 lattice formation by phosphatidyl serine (PS) is also suggested. This study provides molecular insight into how annexins modulate the physical properties of cell membranes as a function of Ca2+ concentration and the phospholipid composition of the membrane.

Keywords: annexin, crystal structure, two-dimensional array, membrane rupture Abbreviations: phosphatidyl serine (PS), annexin A (AnxA)

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Introduction Annexins are found in most eukaryotes with 12 members of the annexin A (AnxA) family (AnxA1–11, AnxA13) found in vertebrates (Gerke and Moss, 2002). Annexins comprise a large superfamily of proteins that are recruited to cellular membranes in a Ca2+-dependent manner by interactions with anionic phospholipid head groups. Annexins are present mostly as monomers in solution with various oligomeric states being adopted for specific functions, including the repair of ruptured membranes via formation of a twodimensional (2D) ordered array on the membrane surface (Bouter et al., 2015). Annexins are also involved in the promotion of exocytosis, intracellular signaling, vesicle transport during cell division, and the formation of multivesicular bodies (Boye et al., 2018; Degrelle et al., 2017; Lizarbe et al., 2013). Annexins consist of an N-terminal region and C-terminal core domain (Gerke and Moss, 2002). The N-terminal region is variable in length and contains a diverse sequence of amino acids. In contrast, the C-terminal core domain is conserved among all annexin proteins and consists of four similar repeat units, called annexin repeats. The only exception is AnxA6, which contains eight annexin repeats. Each annexin repeat consists of ~70 amino acids and is made up of five α-helices, each containing a Ca2+binding site that is structurally distinct from other Ca2+-binding motifs, including EFhands (Concha et al., 1993; Gerke and Moss, 2002; Sopkova et al., 1993). The four repeat units in the core domain adopt a circular arrangement, forming a slightly curved disc shape. The convex face of the curved disc of the core domain is oriented and bound to the membrane at the Ca2+-binding sites, with the N-terminal region located on the concave face of the disc. 3

AnxA5 is the most well-studied annexin protein, and it exhibits both extra- and intracellular functions (Kundranda et al., 2004). The first crystal structure of AnxA5 was determined by Huber et al. (Huber et al., 1990) and showed a pseudo-two-fold symmetry of the four repeat units (Repeats I–IV) in the core domain. Many crystal structures of AnxA5 indicate a tight homotrimeric assembly in crystal packing (Burger et al., 1994; Colloc'h et al., 2007; Concha et al., 1993; Lewit-Bentley et al., 1992; Mo et al., 2003; Sopkova et al., 1993). Specifically, a Ca2+-binding loop in Repeat III can adopt one of two conformations: inward (Huber et al., 1990; Lewit-Bentley et al., 1992) or outward (Concha et al., 1993; Mo et al., 2003; Sopkova et al., 1993). The trimer of AnxA5 exists as a hexagonal disc owing to the three-fold symmetry of the monomers and the pseudotwo-fold symmetry within each monomer. AnxA5 can further assemble into a 2D array depending on the ambient Ca2+ concentration, as revealed by low-resolution electron microscopy (Burger et al., 1994; Mosser et al., 1991; Oling et al., 2000; Olofsson et al., 1994; Voges et al., 1994). A 2D array of AnxA5 on a disrupted membrane prevents tear expansion and promotes the resealing of the membrane (Bouter et al., 2011). To date, two different 2D lattices, the p6 and p3 lattices, have been observed on membranes (Oling et al., 2000; Reviakine et al., 2000; Reviakine et al., 1998; Voges et al., 1994). Previous studies have established that the p6 lattice forms first upon phosphatidyl serine (PS) and Ca2+ binding, and that, the p6 lattice transforms into the p3 lattice in high PS and high Ca2+ concentrations (Reviakine et al., 2001; Richter et al., 2005). The p3 lattice is more tightly packed than the p6 lattice and contains different molecular contacts. Although annexin structures, including that of AnxA5, have been determined previously, a 2D array of annexins has been not observed by X-ray crystallography. The lack of high-resolution imaging has limited our understanding of the role(s) of AnxA5 at 4

the molecular level. Herein, we describe the crystal structures of human AnxA5 in its 2D array conformation. These structural analyses suggest conformational changes in AnxA5 into sheet-like arrays in response to Ca2+ concentration and provide insight into the role(s) of PS and Ca2+ binding during the formation of 2D arrays.

Material and Methods Construction of the recombinant plasmid The human ANXA5 gene with a C-terminal 10xHis tag gene was inserted into the Nco1 and Xho1 enzyme sites of pET28a (Novagen).

Protein expression and purification E. coli BL21 (DE3) cells harboring the recombinant plasmid were grown in 3.0 L Luria– Bertani medium (Merck, USA) supplemented with 50 μg/mL kanamycin at 37°C until the OD600 reached ~0.7 and were then induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 18°C for 15 h. The cells were harvested by centrifugation at 5,000 × g for 7 min and resuspended in 60 mL lysis buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and 2 mM β-mercaptoethanol. The cell lysate was disrupted by sonication and centrifuged at 10,000 × g to remove cell debris. The supernatant was loaded onto an open column packed with Ni-NTA affinity resin (GE Healthcare, USA). The resin was washed with 300 mL lysis buffer supplemented with 20 mM imidazole. After washing the resin, the protein was eluted with 25 mL lysis buffer supplemented with 250 mM imidazole. Eluted recombinant proteins were further purified by anion exchange 5

chromatography using a HiTrap Q column (GE Healthcare) with a linear gradient of increasing NaCl concentrations. The fractions were collected and loaded onto a Superdex 200 HiLoad 26/600 size exclusion chromatography column (GE Healthcare) that had been

pre-equilibrated

with

buffer

containing

10

mM

4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) pH 7.0, 150 mM NaCl, 50 μM CaCl2, and 2 mM β-mercaptoethanol. The final purified samples were concentrated using the Vivaspin® centrifugal concentrator with a 30-kDa cutoff (Sartorius, Germany) and stored at −80°C until use.

Crystallization and data collection A single crystal of the Ca2+-unbound AnxA5 structure was obtained by hanging-drop vapor diffusion using a precipitant solution consisting of 0.2 M sodium phosphate dibasic dihydrate, 20% (v/v) polyethylene glycol 3350 (PEG 3350), and 2 mM Tris (2carboxyethyl) phosphine (TCEP). Equal volumes (1 μL) of the protein and reservoir solution were mixed and equilibrated against a 500-μL reservoir solution at 14°C for 1 week. The crystal was cryo-protected in reservoir solution and flash-cooled in a liquid nitrogen stream at −173°C. A single crystal of the Ca2+-bound AnxA5 structure was obtained by the same method using another precipitant solution consisting of 0.2 M potassium citrate tribasic pH 8.3, 0.1 M HEPES pH 7.5, 17% (v/v) PEG 3350, 50 μM CaCl2, and 2 mM TCEP. The crystal was cryo-protected in the precipitant solution supplemented with 25% (v/v) glycerol. X-ray diffraction data were collected on the 5C Beamline at the Pohang Accelerator Laboratory (PAL) (Pohang, Republic of Korea).

Structural determination and refinement 6

X-ray diffraction data were processed using HKL2000 software (Otwinowski and Minor, 1997). The structures of AnxA5 were determined by the molecular replacement method using MOLREP (Vagin and Teplyakov, 2010) in the CCP4 package (Winn et al., 2011) based on the structure of human AnxA5 (PDB code: 2XO3) as a search model. The final structures of AnxA5 were refined using the PHENIX software suite (Adams et al., 2010).

Accession numbers: Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 6K22 and 6K25.

Results Crystal structures of AnxA5 under low-salt conditions To date, most AnxA5 proteins have been crystallized in the presence of a high concentration of sulfate ion. Sulfate ions contribute to protein crystallization by mediating the interactions among protomers or trimers (Campos et al., 1998; Colloc'h et al., 2007; Mo et al., 2003; Oling et al., 2000). Unfortunately, the binding of sulfate ions results in a step between planes containing the trimers, preventing the formation of planar 2D conformations. To avoid this effect of the sulfate ions, we attempted to crystallize AnxA5 in the absence of sulfate ions. Crystals were obtained under two different conditions with the full-length human AnxA5 protein with a 10xHis tag at the C-terminus (Fig. 1). The crystals had the same space group (P6322) with similar unit cell parameters and crystal packing. Both crystals contained one molecule in an asymmetric unit. One of the resulting structures was determined at a resolution of 2.7 Å and contained three Ca2+ ions. This structure is hereafter referred to as the Ca2+-bound structure. The other structure, acquired 7

at a resolution of 2.4 Å resolution, did not contain Ca2+ and is hereafter referred to as the Ca2+-unbound structure (Table 1). Overall, both structures are similar to previously determined structures acquired under high-salt conditions (Huber et al., 1990). The attached 10xHis tag was not ordered in the structure. The annexin core domain adopts a diamond-shaped configuration consisting of four annexin repeats (Repeat I: residues 17–88, Repeat II: 89–159, Repeat III: 168–246, Repeat IV: 247–317) and the N-terminal region (residues 1–16). Repeats I– IV are arranged in a circular plane with an internal pseudo-two-fold symmetry (Figs. 1A and 1B). Structural superposition of Repeat III of the two AnxA5 structures revealed significant conformational variations (Fig. 1C). Trp187 in Repeat III of the Ca2+-bound structure was exposed to the surface, whereas the same residue in the Ca2+-unbound structure was buried in the hydrophobic core of the protein. It has been proposed that Trp187 at the surface of AnxA5 plays a dual role in interactions with the phospholipid bilayer and in the formation of a 2D array at the membrane surface (Campos et al., 1998; Pigault et al., 1999). Ca2+ ion in Repeat III of the Ca2+-bound structure is held within a cage defined by the side-chain carboxyl group of Glu228 and the backbone carbonyl groups of Gly183 and Gly188 (Fig. 1D; top). This configuration seems to induce the outward-facing Trp187 conformation described previously (Sopkova et al., 1993). In contrast, the side chain of Trp187 in the Ca2+-unbound structure is buried in the hydrophobic core defined by Leu179, Phe194, Phe198, and Leu237 (Fig. 1D; bottom).

2D arrays with a p3 lattice of the AnxA5 Both of the AnxA5 crystal structures obtained in this study indicated planar 2D 8

conformations. The crystal packing arrangements were consistent with a p3 lattice when superimposed onto low-resolution electron density images generated by negative stain electron microscopy (Fig. 2A and Supplementary Fig. 1) (Reviakine et al., 2000). All structural features associated with a 2D array having a p3 lattice are accounted for in the high-resolution structures. The Trp187 residues are on one face of the 2D array, whereas the N-terminal regions are on the opposite face (Fig. 2B). Since the 2D array in crystal packing represents the physiological oligomeric state, we analyzed the molecular contacts required for the formation of 2D arrays. Three different three-fold axes, i.e., the first, second, and third three-fold axes, were identified in the 2D arrays (Fig. 2C) and are located near Repeat II, Repeat III, and Repeat IV, respectively (Fig. 2C; see below for details). Each three-fold axis generates different interactions representing different combinations of three subunits.

The first three-fold axis near Repeat II Trimers generated by the first three-fold axis have been identified in many of the previously determined crystal structures of AnxA5 (Colloc'h et al., 2007; Mo et al., 2003). Repeat II of each protomer is close to the first three-fold axis of the trimer (Fig. 3A). Salt bridges between the acidic amino acids of Repeats II and III of one of the protomers and basic amino acids from Repeat I of the other protomer constitute the major interactions resulting in the trimeric unit (Fig. 3A) (Bouter et al., 2011). Despite significant structural differences in Repeat III of the Ca2+-bound Trp187-outward and Ca2+-unbound Trp187inward structures (Fig. 1C), the differential regions in Repeat III are not involved in the first three-fold interaction. Therefore, conformational variation may not affect trimer formation via the first three-fold interaction. 9

The second and third three-fold axes The second three-fold axis is found near the tip of Repeat III (Fig. 2C and Fig. 3B) and features interactions never seen before in annexin crystal structures. In the Ca2+-bound structure, Gln177 and Gln181 form a circular network of hydrogen bonds with water molecules at the center of the three-fold axis (Fig. 3B). The exposed Trp187 side chains are grouped together near the three-fold axis along with the bound Ca2+ ion (Fig. 3B and Fig. 4B). A comparison with the Ca2+-unbound structure is given below. The third three-fold axis is located near Repeat IV in the 2D array of both the Ca2+-bound and unbound structures, with only two polar interactions between the subunits (Fig. 3C). Lys26 forms an ionic interaction with Glu223 of the adjacent subunit, and Tyr297 forms a hydrogen bond with the backbone carbonyl group of Ala293.

Ca2+- and PS-dependent structural changes near the second three-fold axis The region surrounding the second three-fold axis in the 2D array of AnxA5 exhibited significant structural differences between the Ca2+-bound Trp187-outward and Ca2+unbound Trp187-inward conformations. Unlike the Ca2+-bound structure (Fig. 4B), the Ca2+-unbound structure did not feature any direct contacts in the vicinity of the second three-fold axis (Fig. 4A) and exhibited relatively smaller, or shrunken, features. These observations suggest that two different structures of AnxA5 combine to form a cooligomer on the p3 lattice 2D array. PS, together with Ca2+, strongly induces the outward orientation of Trp187. To examine this relationship more closely, we modeled the p3 lattice 2D array with the previously determined PS-bound structure (PDB code: 1A8A) (Swairjo et al., 1995). 10

According to the PS complex structure of AnxA5 (Swairjo et al., 1995), PS is bound near Trp187, and both are localized at the second three-fold axis. In the modeled 2D array of PS-bound structure, the distance between the Trp187 side chains was reduced compared to the PS-unbound structures (Figs. 4B and 4C). These observations suggest that PS binding induces the outward motion of Trp187 and strengthens the oligomerization at the second three-fold axis. Therefore, in the presence of Ca2+, a PS-rich membrane would promote both AnxA5 oligomerization and formation of the compact p3 lattice 2D array.

Discussion Two crystal structures of AnxA5 were determined under low-salt conditions. We first confirmed previous observations indicating that the binding of Ca2+ in Repeat III induces the outward motion of Trp187 toward the cell surface under low-salt conditions. Importantly, crystal-packing interactions showed that the 2D array of AnxA5 forms regardless of the orientation of Trp187, consistent with the p3 lattice observed previously in low-resolution images. At least two different 2D array patterns were characterized in AnxA5. Ca2+ is required to form the 2D array, and the presence of PS in the membrane is a prerequisite for the formation of the p3 and p6 lattice. Different 2D patterns would result in differences in membrane rigidity on an annexin-coated surface. It was reported previously that the p6 lattice is formed first upon Ca2+ and PS binding, and then transformed into p3 lattice when the cell membrane contains high concentrations of PS (Brisson et al., 1999; Reviakine et al., 2001; Richter et al., 2005). The p3 lattice features three different three-fold interactions of AnxA5 monomers. 11

The first three-fold interaction was characterized previously via the high-resolution crystal structures and was proposed to be a crucial interaction for p6 lattice formation. Using electron crystallography analysis and high-speed atomic force microscopy, Oling et al. and Miyagi et al. revealed that the p6 lattice 2D array is formed by the first threefold interaction and pairwise contact of Repeat III with the two-fold interaction (Miyagi et al., 2016; Oling et al., 2001). Interactions with the second three-fold axis were found only in the p3 lattice and were deemed key interactions for the preferential formation of the p3 lattice. These previous observations are consistent with modeling studies, which indicate that PS binding would increase the strength of interactions along the second three-fold axis.

This study elucidated the molecular features that result in 2D array formation in AnxA5 and provides a molecular explanation for the Ca2+-dependent formation of a p3 lattice in a PS-rich membrane. This work also broadens our understanding of how annexins can modulate the physical properties of cell membranes depending on the Ca2+ concentration and the phospholipid composition of the membrane.

Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT) (2017R1A2B2003992 and 2017M3A9F6029755 to NCH). This research was also supported by the National Research Foundation of Korea (NRF-2017H1A2A1042661: Global Ph.D. Fellowship 12

Program to SH). We made use of the 5C Beamline at Pohang Accelerator Laboratory (Pohang, Republic of Korea).

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Figure legends Figure 1. AnxA5 monomer structures under two different crystallization conditions A. The asymmetric unit of the Ca2+-bound structure exhibits the Trp187-outward conformation with Ca2+ binding. Three Ca2+-binding sites were observed. Each annexin repeat unit is labeled (I, II, III, and IV), and Trp187 (stick model; magenta) and Ca2+ ions (sphere; orange) are shown. The region within the box is enlarged in (D). B. The asymmetric unit of the Ca2+-unbound structure exhibits the Trp187-inward conformation. Each annexin repeat unit is labeled (I, II, III, and IV), and Trp187 (stick model; magenta) is shown. The region within the box is enlarged in (D). C. Structural superposition of Repeat III of monomers with different structures (Ca2+-bound form in green, Ca2+-unbound form in pale blue). D. The boxes represent Ca2+ binding and the Trp187 conformation in the Ca2+-bound structure (top), and the hydrophobic core around Trp187 in the Ca2+-unbound structure (bottom), at the same positions as in (A) and (B). The residues (stick model; green in Ca2+-bound structure and pale blue in Ca2+-unbound structure) interacting with Trp187 (stick model; magenta) and Ca2+ ions (sphere; orange) are shown.

Figure 2. AnxA5 2D array formation A. The 2D array of the Ca2+-bound structure (foreground) is superimposed on the p3 lattice of the electron microscopy image (background) (Reviakine et al., 2000). The electron microscopy image was reprinted from Reviakine et al. (Reviakine 19

et al., 2000) with permission from the Journal of Structural Biology, Elsevier. B. Side view of the 2D array of the Ca2+-bound structure. Trp187 residues (magenta) are exposed to the phospholipid surface, which is oriented toward the cell membrane, whereas the N-terminal regions (blue) are on the opposite surface. C. The 2D array of the Ca2+-bound crystal structure with three three-fold axes. The first, second, and third three-fold axes are indicated by black, red, and blue triangles, respectively. Homotrimers of AnxA5 with the first three-fold axes are shown with black regular hexagons, and three representative protomers in the second three-fold axis are shown with red diamonds. Representative interfaces for each three-fold axis are boxed in the same color code as the three-fold axis. The region within the box is enlarged in Fig. 3.

Figure 3. Interfaces for the first, second, and third three-fold axes in the 2D array A. A side view of the interfacial interactions of adjacent protomers in the first threefold axis is shown in the black box of Fig. 2C with salt bridges (dotted line; black) and Ca2+ ions (sphere; orange). B. The interactions in the second three-fold axis around Trp187 in Repeat III of the Ca2+-bound crystal. A close-up view of the Trp187 cluster in the red box of Fig. 2C is shown with hydrogen bonds (dotted line; black) mediated by water molecules (sphere; red) and Ca2+ ions (sphere; orange). C. Polar interactions in the interfaces of the third three-fold axis are shown around Repeat IV. A close-up view of the blue box in Fig. 2C represents polar interactions (dotted line; black) between adjacent protomers with residues (stick) and Ca2+ ions (sphere; orange). 20

Figure 4. Structural comparison of the second three-fold interactions from a different state. Three protomers are shown with the second three-fold interactions in the 2D array of the Ca2+-unbound crystal (A; blue), Ca2+-bound crystal (B; green), and modeled PS-bound crystal (C; brown). All protomers are shown in a surface representation with Trp187 residues (magenta). The orange spheres are Ca2+, and the ball-and-stick models in (C) are phosphatidyl serine molecules bound near Trp187 residues.

Supplementary Figure 1. The 2D array of the Ca2+-unbound crystal The 2D array of the Ca2+-unbound crystal is shown with three three-fold axes. The first, second, and third three-fold axes are indicated by black, red, and blue triangles, respectively. The homotrimer of AnxA5 with the first three-fold axis is shown with black regular hexagons, and the protomers in the second three-fold axis are shown with red diamonds.

Table 1. X-ray diffraction and refinement statistics Data collection Beam line Wavelength (Å) Space group Cell dimensions a, b, c (Å) Resolution (Å) Rmerge I/σI Completeness (%) Redundancy

AnxA5 (Ca2+-bound)

AnxA5 (Ca2+-unbound)

PAL 5C 1.2325 P6322

PAL 5C 0.9795 P6322

92.8, 92.8, 134.0 50.0-2.75 (2.80-2.75) 0.145 (0.390) 13.4 (3.5) 98.8 (87.4) 16.8 (7.4)

94.5, 94.5, 136.7 50.0-2.40 (2.44-2.40) 0.083 (0.385) 21.2 (3.59) 94.8 (93.6) 15.7 (6.9)

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Refinement Resolution (Å) No. reflections Rwork/Rfree No. of Total atoms Wilson B-factor (Å) R.M.S deviations Bond lengths (Å) Bond angles (°) Ramachandran plot Favored (%) Allowed (%) Outliers (%) PDB ID

46.4-2.74 (3.14-2.74) 8874 0.25/0.31 2537 39.14

38.9-2.40 (2.59-2.40) 12988 0.23/0.29 2525 29.47

0.00 0.41

0.01 0.40

96.5 3.5 0 6K22

96.2 3.5 0.3 6K25

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