Tài liệu Báo cáo khoa học: S100–annexin complexes – structural insights pptx

The unique N-terminal sequences of some annexins A1, A2 are closely associated with the core domain in the absence of calcium, and are sub-sequently released on binding of the ions.. How

S100–annexin complexes – structural insights

Anne C Rintala-Dempsey, Atoosa Rezvanpour and Gary S Shaw

Department of Biochemistry, University of Western Ontario, London, Canada

Introduction

The fusion of cellular phospholipid membranes is

required for processes such as membrane

reorganiza-tion, exocytosis and vesicular trafficking In this

manner, annexin A1 has been shown to be involved in

the vesiculation and sorting of epidermal growth factor

receptors [1] Annexins perform their function by

reversibly binding to membranes in a

calcium-depen-dent manner through calcium-binding loops on the

convex sides of their highly conserved core domains

[2–4] The unique N-terminal sequences of some

annexins (A1, A2) are closely associated with the core

domain in the absence of calcium, and are

sub-sequently released on binding of the ions S100 proteins, which are dimeric EF-hand calcium-binding proteins, also coordinate calcium ions, but undergo a significant conformational change to expose hydrophobic residues

on their surface [5,6] Identical hydrophobic surfaces

on either side of the S100 molecule are able to bind two separate target molecules, such as the N-terminal sequences of annexin proteins This heterotetrameric interaction allows two membrane-bound annexin pro-teins to be brought into close proximity via an S100 protein To more clearly understand the interactions between annexin and S100 proteins, efforts have been made to determine the interactions and structures of these two protein families Numerous structures of

Keywords

calcium-binding protein; didalcin; EF-hand;

membrane interaction; NMR spectroscopy;

protein interaction; S100A11; S100B;

structure; X-ray crystallography

Correspondence

G S Shaw, Department of Biochemistry,

University of Western Ontario, London, ON

N6A 5C1, Canada

Fax: 1 519 661 3175

Tel: 1 519 661 4021

E-mail: gshaw1@uwo.ca

(Received 16 June 2008, revised 29 July

2008, accepted 5 August 2008)

doi:10.1111/j.1742-4658.2008.06654.x

Annexins and S100 proteins represent two large, but distinct, calcium-binding protein families Annexins are made up of a highly a-helical core domain that binds calcium ions, allowing them to interact with phospho-lipid membranes Furthermore, some annexins, such as annexins A1 and A2, contain an N-terminal region that is expelled from the core domain

on calcium binding These events allow for the interaction of the annexin N-terminus with target proteins, such as S100 In addition, when an S100 protein binds calcium ions, it undergoes a structural reorientation

of its helices, exposing a hydrophobic patch capable of interacting with its targets, including the N-terminal sequences of annexins Structural studies of the complexes between members of these two families have revealed valuable details regarding the mechanisms of the interactions, including the binding surfaces and conformation of the annexin N-termi-nus However, other S100–annexin interactions, such as those between S100A11 and annexin A6, or between dicalcin and annexins A1, A2 and A5, appear to be more complicated, involving the annexin core region, perhaps in concert with the N-terminus The diversity of these interac-tions indicates that multiple forms of recognition exist between S100 pro-teins and annexins S100–annexin interactions have been suggested to play a role in membrane fusion events by the bridging together of two annexin proteins, bound to phospholipid membranes, by an S100 protein The structures and differential interactions of S100–annexin complexes may indicate that this process has several possible modes of protein–pro-tein recognition

individual annexin and S100 proteins have been

determined and, in addition, two structures of the

complexes between the two protein families have been

completed In particular, the structures of the

com-plexes between S100A10 and annexin A2 and

S100A11 and annexin A1 have been solved [7,8], and

reveal a common mode of interaction between these

two proteins However, other types of interaction

between annexins and S100 proteins have been

observed that utilize other portions of the annexin

protein Included amongst this group are interactions

of S100A1, S100A11 and S100B with annexin A6

[9,10], and dicalcin, an S100-like protein, that

inter-acts with annexins A1, A2 and A5 in a

calcium-dependent manner [11] These complexes indicate that

multiple modes of protein–protein recognition may be

present In this review, the structures of annexins,

S100 proteins and the complexes between the two

protein families are used to provide insights into their

complex biology highlighted in the accompanying

review [12]

Structures of the annexin proteins

In humans, there are 12 different annexin proteins,

annexins A1–A11 and A13, that have orthologues in

most vertebrates [13] As of May 2008, there were 63

three-dimensional structures of annexin proteins, most

from X-ray crystallographic methods, deposited in the

Protein Data Bank (http://www.rcsb.org) These

struc-tures include full-length, truncated and mutant forms

of the annexins (particularly annexin A5), as well as

annexin–protein complexes In particular, vertebrate

structures of human annexins A1, A2, A3, A5, A8 and

bovine annexins A4 and A6 have been determined,

some in both the calcium-free and calcium-bound forms

Consistent with the first annexin structure dete-rmined, annexin A5 [14–17], all annexins, except annexin A6, form a core domain consisting of four conserved structural repeat sequences (I–IV), each about 70–75 residues in length Annexin A6 is a unique member of the annexin family possessing two four-repeat core domains connected by a linker region [18], a result of a gene duplication event As shown in Fig 1 for annexin A1 [19], each repeat unit is formed from five a-helices (A–E), arranged such that heli-ces A, B, D and E are roughly antiparallel to each other, with helix C nearly perpendicular to these heli-ces The repeats pack into two distinct arrangements within the core domain The repeat pairs I⁄ IV and

II⁄ III pack together, mostly as a result of hydrophobic interactions between helices B and E in each repeat, arranged in a near-antiparallel fashion [19,20] For example, in annexin A2 [21], residues in helices B and

E from repeat I (V54, V57 and V98, L102) and repeat

IV (I289, V293 and A330, Y333, L334) form a tight nonpolar network between these two repeat units In general, the hydrophobic nature of these residues in the annexin sequences is highly conserved

Calcium binding to the annexins promotes their binding to phospholipid-containing membranes Most structures of annexins show that the coordination of calcium ions by annexins occurs via three residues in the A⁄ B loop that ligate the calcium ion using their backbone carbonyl atoms and the bidentate side-chain

of either an Asp or Glu 38 residues downstream in the

D⁄ E loop (Fig 1) [22] Water molecules satisfy the remaining two coordination sites for each calcium ion

In this manner, each annexin protein coordinates one

Fig 1 Extrusion of the N-terminal helix in annexin A1 on calcium binding Ribbon representations of apo-annexin A1 (1HM6) (A) [19] and

Ca 2+ -annexin A1 (1MCX) (B) [24] The core domain repeats are coloured red for repeat I, blue for repeat II, yellow for repeat III and green for repeat IV The helices of repeat III are labelled A–E The N-terminus of apo-annexin A1 was resolved in the calcium-free crystal structure and

is shown in magenta The extreme N-terminal helix of annexin A1 is associated with repeat III in the absence of calcium, and essentially takes the place of helix D In the presence of calcium, the N-terminal helix is not visible in the structure and is presumed to be expelled from the core domain The calcium ions are shown as orange spheres.

calcium ion per repeat, giving rise to the trademark

annexin sequence pattern GXGT-(38)-D⁄ E [23] In

addition, secondary coordination of calcium ions with

lower affinity has also been noted within the D⁄ E

loops of repeats I and III, as in annexins A1 [24] and

A2 [21], or in the A⁄ B loop near the primary calcium

site in this region In both cases, a larger number of

water molecules are used to satisfy the calcium

coordi-nation Furthermore, these sites appear to show

greater variability within the annexin structures,

proba-bly as a result of differences in crystallization

condi-tions Remarkably, the calcium ions all lie on the same

curved side of the annexin structures [25], forming a

convex surface which has been proposed to interact

with phospholipids (Fig 1)

Unlike the structures of EF-hand signalling

pro-teins, such as troponin-C [26] or members of the

S100 protein family [27,28], the annexins do not

appear to undergo a significant structural change

within their core domains on calcium binding For

example, a comparison of the calcium-free and

cal-cium-bound forms of annexins A1 and A2 shows

only 1.56 and 0.72 A˚ differences between the

back-bone arrangements of these structures The most

significant difference between these structures is a

dis-ruption of the packing of the helices in repeat III of

calcium-free annexin A1 as a result of the presence

of an N-terminal helix The N-terminal extension

ranges between 11 (annexin A6) and more than 50

(annexins A7 and A11) residues, and is only found in

some annexins (A1, A2, A6, A7, A9, A11) However,

annexin A1 is the only member of the group in

which the intact N-terminal sequence is visible in the

X-ray structure [19] In the calcium-free state, this

structure shows that the N-terminus of annexin A1

forms a kinked a-helix in which residues A2–A11

from this helix are buried against helix E (D259–

A271) of repeat III and back on to helix C The

unique feature of this N-terminal helix is that it

essentially replaces helix D from the helical packing

arrangement in repeat III found in the calcium-bound

form of annexin A2 or in other annexin structures

In the presence of calcium, the N-terminal helix is

absent from the annexin A1 structure, suggesting that

it has been extruded from the core structure [24]

This calcium-sensitive extrusion is reminiscent of that

exhibited by the EF-hand protein recoverin, which

releases an N-terminal myristoyl group on calcium

binding [29,30] Studies of peptides derived from the

N-terminus of annexin A1 reveal that an extruded

N-terminus probably has little regular secondary

structure, but undergoes a coil–helix transition on

protein or membrane binding [31,32]

Structures of S100 proteins

The S100 protein family is a group of 25 members, found solely in vertebrates These proteins undergo a calcium-induced structural change during signalling events As a result, the calcium-bound forms of S100 proteins are able to interact with target molecules, giv-ing rise to a variety of biological responses, includgiv-ing protein phosphorylation, cell growth and motility, and gene transcription [5] The structures of several S100 proteins have been determined using NMR spectro-scopy and X-ray crystallography, and show the details

of the calcium-binding sites, dimerization motif and structural changes on calcium binding Unlike the dumbbell shapes of well-studied EF-hand calcium-binding proteins, such as calmodulin [33] and tropo-nin-C [26], the S100 proteins have a more compact, globular structure As shown for S100A11 (Fig 2), each S100 monomer comprises two helix–loop–helix motifs, or EF-hands, connected by a flexible linker The N-terminal calcium-binding site (site I) is termed

a ‘pseudo’ EF-hand, because of the presence of two extra residues in the loop and the coordination of calcium mainly through backbone carbonyls, whereas the tighter binding C-terminal site (site II) is a canon-ical EF-hand, binding calcium through acidic side-chains The majority of S100 proteins form symmetric noncovalent homodimers, a feature that is unique to these proteins within the EF-hand family of calcium-binding proteins Heterodimers, such as that formed between S100A8 and S100A9 [34], are also possible The dimer interface is composed of the antiparallel arrangement of helices I and IV of each monomer The two calcium-binding loops are held in close prox-imity via a short antiparallel b-sheet, and are on the opposite side of the molecule relative to the N- and C-termini

In the calcium-free (apo) structures of several S100 proteins, including apo-S100A11, helices III and IV are nearly parallel to one another, resulting in a num-ber of residues at their interface being inaccessible to the solvent and giving the protein a more ‘closed’ structure [35] On calcium binding, the N-terminus of helix III moves by almost 40 relative to helix IV and becomes nearly perpendicular to helix IV, exposing hydrophobic residues on both helices (V57, M61, L85, A88, F93 in S100A11), as well as on helix I (I12, I16) and the linker region (L45, A47, F48), which were pre-viously buried in the apo state (Fig 2) In S100A11, it has also been noted that helix IV becomes elongated

on calcium binding The large conformational change

of helix III and the exposure of the hydrophobic resi-dues, first shown for S100B [36–38], have become a

trademark of the S100 calcium-binding event and are

responsible for the interactions of these proteins with a

diverse array of target proteins [5] One member of the

S100 family, S100A10, differs from the others as it is

unable to bind calcium ions because of a three-residue

deletion in site I (N28, N29, T30 of S100A11 are

absent in S100A10) and mutations of acidic

calcium-coordinating residues in site II (D68 and E77 of S100A11 are substituted with C and S in S100A10) (see sequences in Fig 3A) Remarkably, the structure

of calcium-free S100A10 [7] is nearly identical (rmsd 0.85 A˚) to that of Ca2+-S100A11 [8] despite the presence (or absence) of calcium ions in the calcium-binding loops (Fig 2)

I II

III

IV I′

II′

III′

IV′

annexin A2

annexin A2

Ca 2+

I

II

III IV I′

II′

III′

IV′

I

II III

IV I′

II′

III′

IV ′

annexin A1 annexin A1

helix II I movement

annexin A1

Ca 2+ -S100A11-annexin A1 complex

apo-S100A10-annexin A2 complex

I II

III

IV I′

II′

III′

IV′

Fig 2 Calcium-induced conformational change of S100 proteins Ribbon representations of apo-S100A11 (1NSH) (A) [35] and Ca 2+ -S100A11 (1QLS) (B) [8] are shown in similar orientations to demonstrate the conformational changes that occur on calcium binding The helices are numbered I–IV for one monomer and I¢–IV¢ for the other Helix I is shown in red (residues E5–Y20 in apo-S100A11 and E7–A23 in Ca 2+

-S100A11), helix II in yellow (K32–E42 and K34–M41), helix III in green (V55–K62 and G56–D66) and helix IV in blue (Q74–V85 and F75–K99) The b-sheets in the calcium-binding loops are shown in cyan and, in Ca 2+ -S100A11, the short a-helix of the linker is shown in grey Calcium ions are shown as orange spheres When calcium binds to S100A11, the largest conformational changes occur in the C-terminal EF-hand, whereas the N-terminal EF-hand remains relatively unchanged Helix III moves  40 with respect to helix IV (green arrow shows the direc-tion of movement), exposing a hydrophobic cleft between helix IV and the linker of one monomer and helix I¢ of the other monomer (C) Binding of the N-terminal region of annexin A1 (magenta) is mediated by hydrophobic residues of the binding cleft on either side of the S100A11 dimer, making contacts with helices III and IV from one monomer and helix I¢ of the partner monomer simultaneously (D) The structure of S100A10, an S100 protein that does not bind calcium, bound to the N-terminal region of annexin A2, is shown to illustrate the similarity to the Ca2+-S100A11–annexin A1 structure When the two S100–annexin structures are superimposed, the rmsd for the polypep-tide backbones is 0.87 A ˚

In S100A10 and Ca2+-S100A11, helices I and IV

comprise the dimer interface as in the other S100

structures; however, helix IV is markedly longer than

in apo-S100A11, extending nearly to the C-terminus

Helix III has a similar orientation in both S100A10

and Ca2+-S100A11, thus exposing very similar

hydro-phobic regions and residue composition (Fig 2) On

the basis of these structural observations, it is clear

that S100A10 and Ca2+-S100A11 should interact with

target proteins in a similar manner

Dicalcin is a unique S100 protein

Dicalcin is an S100-like protein (originally named

p26olf) isolated from the olfactory epithelium of frog

(Rana catesbeiana) [39], which has been implicated in

the calcium-dependent regulation of olfactory neurones

through interaction with a b-adrenergic receptor

kinase-like protein [40] The protein consists of 217 residues arranged in two homologous halves: an N-ter-minus (1–105) and a C-terN-ter-minus (119–217) connected though a Pro-rich linker region (residues 106–118) Based on the sequence alignment of dicalcin with dimeric S100B or S100A11, dicalcin is predicted to be composed of a pair of approximately 100 residue halves arranged in tandem, each comprising N-termi-nal pseudo (EF-A and EF-C) and C-termiN-termi-nal canoni-cal (EF-B, EF-D) EF-hand canoni-calcium-binding sites (Fig 3) Multiple sequence alignment of the two halves

of dicalcin with the EF-hand motifs of 18 different S100 proteins shows a four-residue insertion in each C-terminal EF-hand and a 13-residue insertion in the linker region connecting the N- and C-domains (Fig 3) Despite the four-residue insertions in sites EF-B and EF-D, dicalcin is still able to bind four cal-cium ions [40,41] As a result of the sequence similarity

M2

V3

S4 E5

F6

L7

K8

Q9

A10

W1 1 F12

I13

D14

annexin A1

S1

T2

V3

H4 E5

I6

L7

S8

K9

L1 0

S1 1 L1 2

E1 3

G1 4

annexin A2

C89, E9', I12', E13' , I 16' E5', M8', E9', M12'

L45, A47, F48, L85 , C89, E9', I12'

F38, F41, L78, C82, E5', M8 '

F41, A81, C82, Y8 5 A88, S92

L85, A88, C89, S92 C82, Y85, F86, M90, M12′

S100A1 1 contacts

S100A10 contact s

Fig 3 Similarity of sequences and protein–protein contacts in S100–annexin structures (A) A sequence alignment of S100A11 (pig), S100A10 (human) and dicalcin is shown to allow a comparison of the residues involved in interactions with annexin peptides The helices are shaded in similar colours to those used in Fig 2 and the calcium-binding loop residues are underlined The N-terminal and C-terminal halves of dicalcin were aligned with S100A11 and S100A10 as described by Tanaka et al [42] The shading of the helices for dicalcin corre-sponds to the observed a-helices in a dicalcin model based on the three-dimensional structure of bovine apo-S100B Schematic representa-tions of the contacts between S100A11 and the annexin A1 peptide (B) and between S100A10 and annexin A2 (C) are shown to illustrate the similarities between the two complexes The annexin peptides are shown as helical wheels to illustrate the relative positions of the amino acids in the helices, with the key hydrophobic residues shaded in light purple forming an XOOXXOOX motif The residues of the respective S100 binding partners that make contact (< 6 A ˚ ) with each of the hydrophobic residues are labelled For example, the side-chain

of L7 of annexin A1 is within 6 A ˚ of the side-chains of L45, A47, F48 within the linker of S100A11, L85 and C89 of helix IV and E9¢ and I12¢

of helix I¢ of the other monomer L7 of annexin A2 makes nearly identical contacts with F38, F41, L78, C82, E5¢ and M8¢ of S100A10, as can be seen when the residues are compared in the sequence alignment of the two proteins.

of dicalcin with other S100 proteins, Tanaka et al [42]

proposed a model for apo-dicalcin, in which each half

of the dicalcin protein consists of two tightly packed

EF-hands similar to the fold of an S100 monomer

(Fig 4) The interface for the two halves of dicalcin is

arranged in a four-helix bundle, in which helix I in the

N-terminal domain and helix V in the C-terminal

domain are nearly antiparallel to each other and

roughly perpendicular to helices IV (N-domain) and

VIII (C-domain) The X-type arrangement of these

four helices contains an extensive hydrophobic

inter-face similar to the homodimeric dimer interinter-face of

S100B [43–45] or S100A11 [8,35] However, the

non-identity of the N- and C-terminal portions of dicalcin

might point to fine tuning of the dicalcin structure

which is more reminiscent of a heterodimeric complex,

such as that observed for S100A8⁄ S100A9 [34]

Structures of S100 proteins complexed

with annexins

The first structure of an S100 protein complexed with

an annexin protein was solved by Rety et al in 1999

[7] and comprised S100A10 bound to the first 13

resi-dues of the N-terminus of annexin A2 One year later,

the structure of Ca2+-S100A11 bound to the 14

N-ter-minal residues of annexin A1 was determined [8]

These two structures (Fig 2) provide valuable

infor-mation on how these two protein families physically

interact with one another, and how these interactions

give rise to the biological functions that have been

observed in the cell S100 proteins have long been

known to interact with members of the annexin family,

and these interactions play a role in membrane fusion events [46–48] In particular, the structures reveal a common mode of interaction between these two protein families, as well as key elements for target specificity

Early studies have shown that the binding of annex-ins A1 and A2 to Ca2+-S100A11 and S100A10, respectively, is strongly dependent on the unique N-terminal regions of the annexin proteins [49–52] This was confirmed by the crystal structures of Ca2+ -S100A11 [8] and S100A10 [7] in the presence of N-terminal annexin peptides Despite the number of differences between the sequences of the S100 proteins (Fig 3) and the calcium-bound states of the proteins, and the fact that the two annexin peptides are from different protein sources, both structures contain two annexin peptides per S100 dimer, located in near-iden-tical binding sites on either side of the S100 molecule (Fig 2) Each peptide makes contact with both S100 monomers, resulting in the bridging together of the two monomers by the annexin protein In each case, the peptides form a-helical structures when bound to their S100 binding partners, as predicted previously on the basis of their sequences (acetyl-AMVSEFLKQAW-FID and acetyl-STVHEILSKLSLEG for annexins A1 and A2, respectively) [50,51,53] and the structure of this region in the calcium-free form of annexin A1 [19] Furthermore, N-acetylation of the peptides has been found to be a requirement for S100–annexin interactions [8,50,53], as removal of the acetyl group in annexin A2 results in a 2700-fold decrease in binding affinity to S100A10 [53] Although no direct contacts are made between the acetyl groups and the S100 pro-teins, it has been suggested that the acetyl group aids

in the stabilization of the helix dipole of the annexin N-terminus, and therefore the required helical confor-mation of the peptide The rmsd for the backbones for the entire Ca2+-S100A11–annexin A1 and S100A10– annexin A2 complexes is 0.87 A˚ This is a clear indica-tion of a common mode of interacindica-tion between these members of the S100 and annexin families

Hydrophobic interactions between the annexin N-termini and the S100 proteins play a major role in their interactions The amphipathic nature of the ann-exin peptides presents a series of hydrophobic residues

on one face that interact with S100A10 or Ca2+ -S100A11 In annexin A1, the hydrophobic surface is made up of residues V3, F6, L7 and A10 and, in ann-exin A2, it is made up of residues V3, I6, L7 and L10 (Fig 3) This representation clearly indicates a strong conservation of hydrophobic residues at these positions (XOOXXOOX; X = hydrophobic residue, O = polar residue), which make the largest number of contacts

I

IV

II

III V

VI

VIII VII

Fig 4 Model of apo-dicalcin based on the three-dimensional

struc-ture of bovine apo-S100B [42] The ribbon diagram of apo-dicalcin is

shown to illustrate the first ‘half’ of the dicalcin protein, composed

of helices I–IV (residues 1–105) and the second portion formed

from helices V–VIII (residues 119–217) The ribbon diagram shows

helices I and V (red), II and VI (yellow), III and VII (green), and IV

and VIII (blue) for the two homologous halves of the protein An

extended linker region (residues 106–118, shown in grey) joins the

C-terminus of helix IV with the N-terminus of helix V.

with the S100 proteins In the S100A10–annexin A2

structure, V3 of the annexin A2 peptide interacts with

a large number of residues in helix I¢ (E5¢, M8¢, E9¢,

M12¢) Similarly, E9¢, I12¢, E13¢ and I16¢ of S100A11

are in close proximity to V3 of annexin A1 (Fig 3B)

The importance of this residue is further illustrated by

its substitution with a polar amino acid, which leads to

a complete loss of binding between annexin A2 and

S100A10 [53] However, Ca2+-S100A11–annexin A1

complex formation seems to be less sensitive to

substi-tution, as replacement of V3 with Ala results in little

change in binding affinity [54] The residue at

posi-tion 6 (I or F) makes numerous contacts with helix IV

(C82, Y85, F86, M90 in S100A10 and L85, A88, C89,

S92 in S100A11) The side-chain of L7 makes the

larg-est number of contacts with the S100 proteins by

inter-acting with residues in the linker (F38, F41 in

S100A10 and L45, A47, F48 in S100A11), helix IV

(L78, C82 in S100A10 and L85, C89 in S100A11) and

helix I¢ (E5¢, M8¢ in S100A10 and E9¢, I12¢ in

S100A11) A decrease in the size of the hydrophobic

side-chain at positions 6 and 7 by substitution with

either Ala or Val in both annexins A1 and A2

dramat-ically reduces binding, indicating the close packing of

the S100–annexin interaction [53,54] The residue at

position 10 (A or L) is near helix IV (A81, C82, Y85

in S100A10 and A88, S92 in S100A11) Several

hydro-gen bonds between the peptides and the S100 proteins

stabilize the interaction

The structures of the S100A10–annexin A2 and

Ca2+-S100A11–annexin A1 heterotetramers show how

a single S100 protein may interact with two annexin

proteins [7,8,55] In both cases, the interaction utilizes

the N-terminus of the annexin protein, a region of the

protein that is expelled from the annexin core structure

on calcium binding to the annexin protein As

pro-posed by Gerke and Moss [56], this would provide an

elegant mechanism, whereby calcium binding by an

annexin protein not only promotes its association with

a phospholipid membrane, but also facilitates

interac-tion with either S100A10 or Ca2+-S100A11, allowing

two membrane surfaces to be brought within close

proximity for a fusion or vesiculation event

Insights into other S100–annexin

interactions

Other interactions between the S100 and annexin

fami-lies have been reported Consistent with the

calcium-induced conformational change observed in S100A11,

most of these complexes require the calcium form of

the S100 protein, although there are a few

calcium-independent interactions, including S100A4 and

annexin A2 [57] Some annexins, such as annexin A6, have many possible S100 binding partners, e.g S100A1 [9], S100A6 [58], S100A11 [10] and S100B [9], whereas other S100 proteins can interact with several different annexins For example, S100A6 has been shown to bind annexins A2 [59], A6 [58] and A11 [60] The S100A6–annexin A11 interaction appears to involve a similar pattern of hydrophobic residues (XOOXXOOX) from the N-terminal extension of ann-exin A11 (L52, M55, A56 and M59) [61] as observed for annexins A1 and A2, and this region has been pre-dicted to adopt an amphipathic helix Similar to the S100A10–annexin A2 complex, S100A4 also interacts with the N-terminus of annexin A2 in a calcium-inde-pendent manner [57] Alternatively, the interaction between S100A11 and annexin A6 has been found to involve residues within each of the two core domains

of annexin A6 [10] A similar conclusion has been reached for the interaction of S100A1 and S100B with annexin A6 [9] In the latter case, and for the S100A12–annexin A5 interaction [62], it has also been observed that the extreme C-terminus of the S100 pro-tein is not involved in the annexin interaction This is

in contrast with observations for the S100A10–annexin A2 and Ca2+-S100A11–annexin A1 complexes (Figs 2 and 3), where the C-termini of the S100 proteins are indispensable, probably as a result of the elongated nature of helix IV which extends nearly to the last resi-due in each protein Together, these results indicate that multiple modes of binding between S100 proteins and annexins are possible

On the basis of the similarity of the amino acid sequences of the S100 proteins with those of dicalcin (Fig 3), it is perhaps not surprising that in vivo and

in vitro data show that dicalcin interacts with ann-exins A1, A2 and A5 in a calcium-dependent manner [11] Furthermore, as annexins A1 and A2 utilize the N-terminal helix region to interact with S100A10 and

Ca2+-S100A11, respectively, it may be suggested that

a similar mode of interaction is used for these annexins with dicalcin In this regard, N-terminally truncated forms of annexins A1 and A2 exhibit a calcium-sensi-tive interaction with dicalcin, albeit approximately four- to five-fold weaker than that for the full-length protein, indicating that the annexin N-terminal helix is not the sole binding site [11] This finding is consistent with the sequence of annexin A5 which lacks a corre-sponding N-terminal region to annexins A1 and A2, and yet is able to interact with calcium-bound dicalcin The results may point to two separate regions utilized

by annexins A1 and A2 for their interactions with dicalcin A similar conclusion has been reached for the interaction of Ca2+-S100A11 with annexin A6, where

the N-terminal sequence of annexin A6 is not

neces-sary for the interaction [10]

Uncovering the potential binding sites in the core

domains of annexins A1, A2 and A5 for dicalcin or

annexin A6 for S100A11 has not yet been attempted

Other than the calcium-sensitive extrusion of the

N-terminal helix from annexins A1 and A2, the most

significant structural change that occurs is the

expo-sure of Trp187 in annexin A5 at high calcium ion

concentrations [14,17,63] The exposure of this residue

facilitates binding to the phospholipid membrane, and

so is unlikely to be used also for interactions with an

S100 protein In addition, this residue is not

con-served in annexins A1 and A2, where Lys residues

exist Rather, the most obvious choice for S100

pro-tein binding with the annexins is the opposite ‘side’

of the core domain from the membrane-binding

region An attractive site may be helix C from

domain IV, as this helix sits near the bottom of the

structure (Fig 1) In the absence of calcium, helix C

is protected by the N-terminal helix in annexins A1

and A2 On calcium binding to the annexin protein,

several residues near helix C become mostly exposed

(E305, N309, D310, A313, K317 in annexin A1)

Sev-eral of the analogous residues are also exposed in

annexin A5 However, analysis of this helix does not

reveal the XOOXXOOX motif used in the N-terminal

helix, suggesting that a different mode of interaction

may occur It is also interesting that residues V287–

V298 in annexin A2 match the TRTK-12 consensus

motif observed for peptide binding to S100B [64]

However, most of these residues appear to be buried

in annexin A2 Further experiments are needed to

confirm whether this or some alternative site on the

annexin proteins is used to interact with dicalcin and

other S100 proteins

Although the structures of Ca2+-S100A11 and

S100A10 clearly show the surfaces used to interact

with annexins A1 and A2, respectively, models of

other S100–annexin complexes, such as Ca2+-S100A11

with annexin A6 or calcium-bound dicalcin with

annexins A1, A2 or A5, are not available However,

some information about potential binding sites can be

gleaned by an examination of the S100 protein

struc-tures in the apo- and calcium-bound states For

exam-ple, S100A11 utilizes several residues in helix I (E9,

I12, I13, I16), the linker (L45, A47, F48) and the

extreme C-terminus of helix IV (L85, A88, C89, S92)

to interact with annexin A1 (Fig 3) Many of these

residues are inaccessible in the calcium-free state and

would be expected to provide an interactive surface

not only for annexin A1, but also for other annexin

proteins It will be important to complete site-directed

mutagenesis experiments on S100 proteins, such as S100A11 and dicalcin, to understand the roles of these residues in the affinities and interactions with different annexin proteins

Future perspectives

Of the 25 members of the S100 protein family, seven (S100A1, S100A4, S100A6, S100A10, S100A11, S100A12 and S100B) have been shown to interact with at least one of the 12 annexin proteins In addi-tion, some S100 proteins, such as S100A6, appear to form complexes with several annexin proteins (A2, A5, A6 and A11) More recently, the unique S100 protein dicalcin has been shown to bind to annex-ins A1, A2 and A5 in a calcium-sensitive manner On the basis of the association of annexins with anionic lipid membranes, it is probable that most of these S100 proteins coordinate with annexins to facilitate the association of two membrane surfaces important for cellular events, such as vesicle formation Struc-tural studies have established that calcium binding to the S100 protein (except S100A10) is required in order to facilitate most S100–annexin interactions However, only two three-dimensional structures (S100A10–annexin A2, Ca2+-S100A11–annexin A1) are available that show how this interaction might occur Both structures show that the annexin mole-cule utilizes an XOOXXOOX motif in its extreme N-terminal helix to bridge helices III and IV of one subunit with helix I¢ of the other in the S100 protein Alternatively, several S100–annexin complexes, includ-ing those of S100A1, S100A11 and S100B with ann-exin A6, and dicalcin with annann-exins A1, A2 and A5, appear to require the annexin core domain for optimal binding Future experiments are needed to narrow down the unique regions on the annexins most important for their calcium-sensitive interactions with different S100 proteins Furthermore, the three-dimensional structures of calcium-bound S100 pro-teins complexed with different annexin propro-teins will

be required in order for details of the recognition modes between these important proteins to be identi-fied Together with advances in S100–annexin biology, this information will provide a detailed description of their roles in calcium signalling

Acknowledgements

This work was supported by a grant from the Cana-dian Institutes of Health Research (GSS) and an award from the Canada Research Chairs program (GSS)

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