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䑗倂Ί䍚䞭䍡䍡▵⪱⪱

List of papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Urosev, D., Ma, Q., Tan, A. L., Robinson, R. C., Burtnick, L. D. (2006) The structure of gelsolin bound to ATP. J Mol Biol. 357, 765-72.

II Ma, Q., Wang, H., Nag, S., Chumnarnsilpa. S., Lee, W. L., Hernandez- Valladares, M., Burtnick, L. D., Robinson, R. C. Ca-binding by domain 2 plays a critical role in gelsolin activation and stabilization. Submitted.

III Chumnarnsilpa, S., Ma, Q., Lee W. L., Burtnick L. D., Robinson, R. C. The crystal structure of the C-terminus of adseverin: Implications for ac- tin binding. Submitted.

IV Ma, Q., Lee, W. L., Chumnarnsilpa, S., Burtnick, L. D., Robinson, R. C. Calcium, pH and ATP regulation of the gelsolin superfamily of proteins. Manuscript.

Reprint was made with permission from the publisher.

Contents

Introduction...... 9 The cytoskeleton ...... 9 Actin...... 9 Actin dynamics...... 11 Actin-binding proteins...... 12 The structural basis of actin dynamics at the cell membrane...... 13 Cytoskeleton and pathology ...... 14 Gelsolin family proteins...... 15 Gelsolin...... 16 Adseverin...... 18 Villin...... 19 CapG...... 20 Aim ...... 21 Present investigation ...... 22 Paper I ...... 22 Paper II ...... 23 Paper III...... 23 Paper IV ...... 24 Discussion and future perspectives...... 25 Conclusion ...... 27 Acknowledgements...... 28 References...... 29

Abbreviations

ABP Actin-binding protein ADF Actin depolymerizing factor ADP Adenosine-5'-diphosphate Arp2/3 Actin-related protein 2/3 ATP Adenosine-5'-triphosphate CapG Capping protein, gelsolin-like CapZ Capping protein from the Z-disc F-actin Filamentous actin FAF Familial amyloidosis (Finnish type) G-actin Globular actin LPA Lysophosphatidic acid PI Phosphatidylinositol PIP Phosphatidylinositol 4- monophosphate PIP2 Phosphatidylinositol 4,5-biphosphate PS Phosphatidylserine VASP Vasodilator-stimulated phosphopro- tein WASP Wiskott-Aldrich syndrome protein

Introduction

The cytoskeleton

The cytoskeleton is a three dimensional network of filamentous proteins. These filaments play important structural roles in providing the cell with an internal scaffold, and hence, maintaining cell shape. The cytoskeleton is composed of 3 main components: microfilaments (actin), intermediate fila- ments and microtubules (tubulin). The system is not still, but dynamic, un- dergoing continuous cycles of polymerization and depolymerization and entering into interactions with many other proteins. This dynamic property enables cells to move. The cytoskeleton is also found to be involved in many other processes, including: cytokinesis, signal transduction and apoptosis.

Actin

The structure of monomeric actin (G-actin)

Actin is highly conserved among species within eukaryotes with more dis- tant homologs existing in prokaryotes. Actin is one of the most abundant proteins in eukaryotic cells, comprising up to 20% of the total protein in striated muscles. In non-muscle cells, actin is mainly located in the cyto- plasm with a lower level present in the nucleus [1, 2]. There are three classes of actin existing in multi-cellular organisms: the , , and -isoforms [3, 4].

9

Figure 1. The structure of G-actin

G-actin is composed of 375 amino acids and has a molecular weight of 42 kDa. The first actin atomic resolution structure was resolved at 2.4 Å in complex with DNase I in 1990 [5]. The single polypeptide chain forms two major domains or four subdomains with a nucleotide-binding cleft in the center (Fig. 1). This site usually binds to ATP, ADP or ADP-Pi and a diva- lent cation (Ca2+ or Mg2+) [6, 7]. It has been speculated that the ATP is re- sponsible for the “closed” conformation of actin by the interaction of its - phosphate with a network of side chains across the cleft [8]. However, in the ADP form, the -phosphate does not exist and the interacting residues con- sequently move apart creating the “open” conformation [9]. The groove formed at the interface between subdomain 1 and 3 is rich in hydrophobic residues. This creates the major interaction surface for proteins that bind to actin [10]. G-actin is conventionally orientated with its “pointed” end di- rected at the top of the page and the “barbed” end toward the bottom (Fig. 1). Both the N- and C-terminus of actin are located at the barbed end of G-actin in subdomain 1 [5].

The structure of filamentous actin (F-actin)

Jean Hanson and Jack Lowy first described the helical nature of F-actin more than forty years ago. Presently, there is no F-actin atomic resolution crystal structure available. The natural heterogeneity in filament length does not favor crystallization. Among the low resolution models, the Holmes’ F- actin model has been the most widely accepted [11]. In this model, the F- actin filament is a two-stranded helix where every 13 monomers eclipse each other in what appears to be a crossover. Each monomer rotates 166°, with a pitch of 59 Å, 6 turns in total for each 13 monomer stretch. However, the

10 conformational changes that occur during the G-actin to F-actin transition are not accounted for in this model. Very recently, Oda et al. have derived a higher resolution structure of F- actin from a well oriented sol of rabbit skeletal muscle actin by analyzing the X-ray fibre diffraction pattern. This model predicts that the G- to F-actin conformational transition involves the relative rotation of the two major domains by 20°, which results in the flat conformation of F-actin [12]. The flattening seems to be associated with the polymerization and is supported by many other biochemical experiments. Low resolution (25-30 Å) electron microscope observations suggest that the F-actin filament may exist in different conformations modulated by the type of bound ions, nucleotide, isoform of actin, toxins such as phalloidin and actin-binding proteins, for instance cofilin [13-16]. Thus, F-actin is gen- erally viewed as a dynamic, responsive structure rather than a static entity. F-actin is a polar structure, with two structurally different ends. In the elec- tron microscope, filaments decorated with myosin heads point to the same direction, referred to as "pointed" and "barbed" ends. This polarity is essen- tial to actin’s function in cell motility.

Actin dynamics

Under physiological conditions, actin monomers spontaneously polymerize to form microfilaments (F-actin) [17]. Polymerization is a multi-step process and contains the following main features: 1) Two monomers slowly associ- ate to form an unstable dimer. 2) Occasionally the unstable dimer gains an- other monomer to form a stable trimer. 3) An elongation phase during which a trimer serves as a site for nucleation and the further growth of the actin filament. The first two steps are highly unfavourable. Only the trimer is more likely to rapidly associate than to dissociate [18, 19]. Polymerization continues until it reaches steady state where the rate of actin monomers join- ing the filaments equals that of monomers leaving the filaments. At this point the concentration of free monomers is in equilibrium with the popula- tion of actin filaments and this concentration of monomers is referred to as the critical concentration [20]. At steady state the actin filaments are in a continuous state of assembly/disassembly. The critical concentration is de- pendent on the solvent condition, the presence of actin-binding ligands such as toxins and actin-binding proteins [13, 21-23].

ATP-G-actin ATP-F-actin ADP-Pi-F-actin ADP-F-actin + Pi

ATP bound G-actin spontaneously polymerizes to form F-actin. After as- sociation, actin hydrolyzes its bound ATP to ADP + Pi, eventually releasing

11 Pi [24, 25]. Association and dissociation of actin monomers occurs at either end of the filament. However, the critical concentration for actin at the pointed end is 12 to 15 times higher than that at the barbed end under physiological conditions [26]. In vitro, when the rate of monomer associa- tion and dissociation equal each other, treadmilling occurs where the barbed end keeps elongating and, at the same time, the pointed end is shortened, however, the filament length remains approximately constant [27, 28].

Actin-binding proteins

In vivo, the assembly and disassembly of the actin cytoskeleton is regulated by a plethora of actin-binding proteins (ABPs). They are can be classified into the following groups:

Monomer binding proteins Nucleotide exchanging proteins e.g. profilin. Profilin facilitates nucleotide exchange from ADP-actin to ATP-actin [9, 29-31]. Monomer sequestering proteins e.g. thymosin 4 [32-35] and DNase I [36- 38]. Proteins that can sequester actin monomers. Thymosin 4 forms a pool of ready to use ATP-actin complexes, which can be exchanged with profilin for polymerization. Nucleating proteins e.g. gelsolin [39-41], Arp2/3 [42, 43] with WASP [44- 46], spire [47, 48] and formins [49-51]. These proteins accelerate formation of nuclei by binding to actin monomers upon which filaments grow rapidly.

Depolymerizing proteins These proteins induce the conversion of ADP-F-actin to ADP-G-actin, e.g. ADF/cofilin [52-55] and twinfilin [56-58].

Capping proteins These proteins cap the end of filaments and prevent the exchange of mono- mers. Barbed end capping proteins e.g. capZ [59-61], gelsolin [39-41] and formins [49-51]. Pointed end capping proteins e.g. tropomodulin [62-64] and Arp2/3 [42, 43, 65].

Severing proteins Proteins that bind to the side of the filament and cut them into two frag- ments, e.g. gelsolin [39-41], adseverin [66-68] and villin [69-71].

12 Bundling and cross-linking proteins These proteins usually contain at least two F-actin binding sites or are mul- timeric proteins that contain only a single binding domain per subunit. Actin bundling can be parallel or antiparallel in the alignment of F-actin into linear arrays. Bundling proteins include: -actinin [72-74], espin [75, 76] and VASP [77-79]. Cross-linking proteins such as filamin [80-82], Arp2/3 [42, 43, 65], fimbrin [83-85] and spectrin [86-88] arrange actin filaments into orthogonal arrays.

Stabilizing proteins These proteins bind along the sides of actin filaments and stabilize the fila- ment against spontaneous depolymerization, e.g. tropomyosin [89-91], nebu- lin [92], caldesmon [93] and calponin [94].

Motor proteins These proteins use actin as a scaffold or track upon which to move, e.g. my- osin [95].

The structural basis of actin dynamics at the cell membrane

The assembly and disassembly of actin filament networks at the leading edge of motile cells involves many actin-binding proteins. In Pollard’s model [96], the barbed ends of actin filaments point towards the cell membrane. Activation of the WASP/Scar [44-46] family proteins leads to activation of the Arp2/3 complex and this initiates the nucleation of new filaments on the side of older filaments. Phosphatidylinositol 4,5-biphosphate (PIP2) also dissociates the capping proteins, gelsolin and capping protein, to promote filament growth. Cofilin is able to sever filaments to also increase the num- ber of barbed ends. Onto the barbed ends of these filaments profilin-bound ATP-actin monomers are then added and the growing actin filaments push the cell membrane forward. The system also contains a recycling mechanism. The growth of barbed ends can be stopped by capping protein. ATP hydrolysis and Pi dissociation at the pointed ends create an aged population of filaments that are recog- nized by severing proteins like gelsolin which cut the filaments into smaller fragments. The gelsolin-capped filaments are then recognized by ADF/cofilin. ADF/cofilin further pulls the fragments apart into monomers. Exchange of the actin from ADF/cofilin to profilin occurs. Profilin promotes G-actin nucleotide exchange from ADP to ATP and binds tightly to ATP- actin. By doing so, a pool of ready-to-use ATP-actin for polymerization is

13 prepared. Thymosin 4 regulates the amount of unpolymerizable ATP-actin monomers by competing with profilin for ATP-actin. In this model, the molecular mechanism of cell membrane movement is based on the fact that through the polymerization and depolymerization of actin filaments, the energy of the hydrolysis of ATP-actin is converted into mechanical force.

Figure 2. Actin dynamics at the cell membrane.

Cytoskeleton and pathology

The cytoskeleton plays important roles in cell mechanics, locomotion, dif- ferentiation and neoplastic transformation. The function of the cytoskeleton is tightly regulated by many actin-binding proteins. Mutations of these pro- teins can result in the development of significant pathologies. Because of the many vital roles of actin, mutations in actin itself can cause dramatic effects on survival when no alternative actin gene is available. For instance, tissue- specific isoforms of actin are related to many actin-related genetic disorders [97], yet mutations of the ubiquitous cytoplasmic - and -actin isoforms usually don’t disrupt normal cell function [98, 99]. Another example is ADF/cofilin. Recent studies have revealed that under conditions of cellular stress, wild-type ADF/cofilin forms complexes with actin, and these com-

14 plexes appear to initiate or aid in the progression of diseases as diverse as Alzheimer’s disease [100] and ischemic kidney disease [101].

Gelsolin family proteins

This family of proteins is expressed in eukaryotes. The family includes (but is not limited to) gelsolin, adseverin, villin, capG, severin, advillin, supervil- lin and flightless-I. All contain three to six copies of the 120-amino acid module that is folded into a compact domain structure [40]. Gelsolin con- tains six such domains and from sequence analysis, it appears that gelsolin has evolved by gene triplication followed by gene duplication, as gelsolin domain 1 (G1) is most similar to domain G4, domain G2 is most similar to domain G5 and domain G3 is most similar to domain G6 (Fig. 3). Adseverin is the most similar family member to gelsolin, however, adseverin is lacking the C-terminal helix found in gelsolin [67, 102]. The villin subgroup is dif- ferentiated by having a further C-terminal extension, the villin head piece [103-105]. CapG is a smaller, three-domain protein that cannot sever but can cap the barbed end of an actin filament [106-108].

Gelsolin 1 2 3 4 5 6

Adseverin 1 2 3 4 5 6

Villin 1 2 3 4 5 6

CapG 1 2 3

Figure 3. Domain structure of the gelsolin superfamily members studied in this thesis

15 Gelsolin

Domain structure and regulation

Gelsolin was named based on its ability to transform gel-like actin filaments into a sol-like form in a calcium dependent manner [109]. Gelsolin is a pro- tein containing two compact halves, each half consists of three domains (domains G1-G3 and domains G4-G6). The three dimensional structure of calcium-free gelsolin was determined by X-ray crystallography and it was revealed that, in the absence of calcium, gelsolin folds into a compact globu- lar structure in which the putative actin-binding sequences are buried and cannot bind to actin [40]. From the structure of the G4-G6/actin complex, a model of the calcium activation of gelsolin was proposed in which calcium binding induces a conformational rearrangement involving domain G6 being flipped over and translated by about 40 angstroms relative to domains G4 and G5. This structural reorganization tears apart the continuous -sheet core of domains G4 and G6, exposing the actin-binding site on G4 used for cap- ping of actin filaments [41]. There are two types of calcium-binding sites involved in the gelsolin-actin complex: type I sites share coordination of Ca2+ with actin, while type 2 sites are solely contained within gelsolin. Cal- cium activation of gelsolin is a complicated process and there is ambiguity in the mechanism and the level of calcium required for activation. The effect of calcium ions have been analyzed by several different methods, including limited proteolysis, dynamic light scattering, circular dichroism [110], small- angle x-ray scattering [111] and synchrotron footprinting [112]. It is gener- ally agreed that, at submicromolar concentrations of calcium, calcium ions bind to type 2 sites leading to the release of the G6 helix latch which masks the F-actin binding site on G2 in the absence of calcium [40, 111, 112]. Ac- tin binding occurs only after micromolar levels of calcium are available [110]. For severing to take place, even higher levels of calcium concentra- tion are required (for instance, up to 10 μM half-maximal activity in vitro [112-114]). It is not clear how many calcium-binding sites are filled during the calcium activation of gelsolin and the binding to actin. Gelsolin is also reported to be activated at low pH [115, 116, 117] and this may involve a different mechanism compared with that of calcium activation. Besides cal- cium and pH, the activity of filament depolymerization of gelsolin is also regulated by PIP2. PIP2 uncaps gelsolin from barbed ends of filaments by binding to at least three regions of gelsolin [118, 119], two of these sites are located at the polypeptide hinge between domains G1 and G2, and the third site is found in G5-G6 [116, 120-121]. Ca2+ increases the affinity of gelsolin for PIP2 by exposing its major N-terminal PIP2 binding sites through a con- formational change in the C-terminal half [120, 121].

16 Actin monomer binding and nucleation

Gelsolin can bind to a single actin monomer [110, 122, 123] and to two actin monomers in a calcium dependent manner. This complex can form a nucleus for actin polymerization [124-126]. Besides the two monomer actin-binding sites on G1 and G4, gelsolin also has one site for F-actin binding located in domains G2-G3. At [Ca2+] < 10-7 M, no interaction with actin can be ob- served. Gelsolin undergoes conformational changes enabling it to bind to at least one actin at 10-7 - 10-6 M [Ca2+]. The stable 2:1 actin/gelsolin complex is formed only when [Ca2+] > 10-5 M [127, 128].

Filament binding and severing

Following activation, the gelsolin C-terminal latch is released from contact- ing G2, which contains the F-actin binding site. Firstly, domain G2 binds to the side of the filament. The G1-G2 interface is dissociated to extend the G1- G2 linker which in turn directs G1 for insertion between two actins in the longitudinal axis. G4-G6 reaches across the filament to bind to the other strand. Severing takes place after sufficient actin-actin contacts are broken [40, 122, 129].

Filament capping and uncapping

Gelsolin caps the barbed end of actin filaments after severing and prevents monomers from being added to the filaments [123]. Gelsolin is removed by interaction with polyphosphoinositides.

Gelsolin functions in vivo

Gelsolin has been reported to be generally localized to actin rich structures, for instance, at the cell periphery in platelets [130], along stress fibres in fibroblasts [131], in the I-Z-I region in muscle cells [132] or associated with actin filaments and cell membranes of macrophages and platelets. Plasma gelsolin (83 kDa), an isoform of cytoplasmic gelsolin (80 kDa), exists in plasma where it severs actin filaments released into the circulation. The gel- solin:actin complexes then exchange their actin monomers with vitamin D- binding protein and these are eventually removed from the blood circulation [133]. Cells of gelsolin gene knockout mice display a variety of motility and ac- tin defects. Gelsolin null fibroblasts have excessive actin stress fibers and

17 migrate slower than wild-type fibroblasts. This is consistent with an inability to sever and remodel actin filaments [134]. Furthermore, platelet shape changes are decreased and longer time is needed for blood to clot, which is in line with the requirement of F-actin severing for platelet activation [135]. PIP2 drives signaling, cytoskeleton organization, and membrane traffick- ing [136] by regulating the gelsolin severing activity. Polyphosphoinositides are precursors of intracellular second messengers. Membrane ruffling is ac- tivated by the GTPase Rac. Gelsolin null fibroblasts have increased Rac expression [137], and Rac has been shown to dissociate gelsolin-actin com- plexes in wild-type cells [138]. These data suggest that gelsolin is a down- stream effector of Rac. Gelsolin has been isolated in complexes with increasing number of li- pases and kinases, for instance phospholipase C1 and C [139]. Gelsolin also binds to lysophosphatidic acid (LPA) with high affinity [140]. LPA inhibits the F-actin severing activity of gelsolin and uncaps gelsolin from filament ends, and does so cooperatively with PIP2. Taken together, the phospholipase-gelsolin and the phosphoinositide-gelsolin interactions are now recognized to modulate lipid signaling events. Gelsolin is also a substrate for caspase-3, which is a core effector that is activated during apoptosis [141]. The cleavage product of gelsolin by cas- pase-3 (residues 1-352, the N-terminal half of gelsolin) severs actin fila- ments in a Ca2+ independent manner [142]. This gelsolin fragment disman- tles the membrane cytoskeleton to cause blebbing, a hallmark of apoptosis. Many cancer cells have significantly down regulated gelsolin expression levels [143, 144]. It is not clear how this is related to gelsolin’s effects on remodeling of motility, lipid signaling pathways or apoptosis. Finally, gelsolin-related amyloidosis is an inherited disease characterized by hyper-elastic skin and amyloid deposits in the cornea and cranial nerve. It is a result from Asp187Asn or Asp187Tyr mutations in gelsolin. These mutations reveal a cleavage site between Arg172 and Ala173 that is sensitive to a trypsin-like protease, which leads to the production of the amyloido- genic 173-243 fragment [145, 146].

Adseverin

Adseverin (Scinderin), is a ~80 kDa member of the gelsolin protein family. Adseverin is present in chromaffin cells, platelets and a variety of secretory cells [147-149]. Adseverin is localized to the cortex in these cells where the transit of vesicles between compartments is regulated by actin filament sev- ering activity of adseverin [150-151]. Nucleotide and amino acid sequence analyses indicate that adseverin shares 63% homology with gelsolin [152- 153]. It contains six domains and there are strong similarities between A1

18 and A4, A2 and A5, and A3 and A6. It seems that this protein is also derived from a gene triplication followed by gene duplication of an ancestral gene similar to that of gelsolin. These six domains are speculated to fold into two compact halves and have a similar three dimensional structure as gelsolin. The actin binding sites of adseverin have been identified in A1, A2 and A5. A1 and A2 are responsible for the actin filament severing activity and A5 nucleates actin polymerization [151-152]. Adseverin shows actin-filament severing, barbed end capping, and nucleating activity in a Ca2+ dependent manner [68, 151]. The actin filament severing activity of adseverin resides in its N-terminal half and is regulated by phospholipids. Its C-terminal half binds to G-actin in a calcium dependent manner but does not sever actin filaments [102, 151]. Three actin [150, 151], two PIP2 [150,152] and two Ca2+ binding sites were identified in this protein. Moreover, adseverin redis- tribution and activity is also regulated by pH [68] in resting and stimulated cells.

Villin

Villin is a tissue-specific actin-binding protein of 93 kDa found in the brush border microvilli of epithelial cells [154-155]. Villin shares 50% similarity with gelsolin at the amino acid level [156]. Two actin monomer binding sites are estimated in the villin core based on similarity to the monomer binding sites identified in gelsolin. Villin is a versatile protein which can bind, nu- cleate, sever, cap and crosslink/bundle actin filaments in a calcium-regulated manner. Similar to gelsolin, villin exists in an autoinhibited conformation. The calcium induced conformational changes expose the actin binding sites in villin [157]. In the absence of calcium or at very low calcium level, villin bundles actin filaments [104, 158]. Villin binds to the barbed end of actin filaments and the half-maximum actin capping by villin occurs between 10 to 30 nM Ca2+ [70, 71]. The actin-capping and severing activity of villin is located within the first two domains (V1 and V2) of villin [105, 159], whereas V1 and V4 are involved in the villin nucleation activity [105]. Villin can be tyrosine phosphorylated both in vitro and in vivo [160-161]. Tyrosine phosphorylation decreases the affinity of villin for F-actin binding [160]. In addition, tropomyosin can bind to F-actin and inhibit villin’s association [162]. In vitro, severing of actin filaments was reported at 100 - 200 μM calcium concentration [70]. Tyrosine phosphorylated villin severs actin at nanomolar calcium concentrations and it has been suggested that this is the mechanism by which villin severs actin filaments in vivo [163]. Actin cap- ping and severing by villin is inhibited by direct association of villin with PIP2 [103]. Three PIP2 binding sites are identified in human villin, they are located in domains V1, V2 and V5 [103], which resemble the PIP2 binding

19 sites in gelsolin [121]. Two F-actin binding sites were identified in villin, one is in the core and the other is in the headpiece.

CapG

CapG is an ubiquitous 39 kDa barbed end F-actin binding and capping pro- tein, particularly abundant in macrophages [106]. CapG contains three gel- solin homologous domains and it is the only member of this family which is unable to sever actin filaments [164]. Like other gelsolin family members, capG is also calcium regulated in its actin binding activity. CapG exists in both the cytoplasm and the nucleus [165]. CapG is phosphorylated in vivo and it has been suggested that phosphorylation of capG may be involved in its function in the nucleus [106].

20 Aim

The aim of this work was to determine the crystal structure of the gelsolin family of proteins in complex with actin, to reveal their in vitro biological functions through relevant biochemical and biophysical experiments, and finally to explain the biological functions by using the structural information obtained.

21 Present investigation

Paper I

The calcium activation and PIP2 regulation of gelsolin is reasonably well studied. However, ATP, another gelsolin ligand has received less attention. In this paper, we presented the crystal structure of ATP-bound inactive plasma gelsolin and revealed that ATP binds across the two halves of gel- solin involving an intricate network of polar and hydrophobic interactions. The negatively charged phosphate groups of ATP interact with the positively charged basic residues of gelsolin domain G5, while there are both charged and hydrophobic interactions formed between the adenine ring of ATP and gelsolin domain G3. Comparison of the ATP-bound G4-G6 portion of the structure with that of activated G4-G6 demonstrated that the amino acid residues crucial to bind ATP in the inactive state are not available in the activated state. However, the ATP adenine-binding pocket in the G1-G3 half of the structure showed undisrupted integrity in comparison to the structure of activated G1-G3 bound to actin. ATP binding stabilizes the gelsolin molecule and it takes ten times higher concentrations of Ca2+ to activate gelsolin when ATP is bound. This suggests that ATP might be a modulator of calcium activation of gelsolin. The ATP phosphate groups lie in a posi- tively charged pocket on the surface of gelsolin. This area overlaps with the reported binding site for PIP2, another gelsolin regulator. As part of a col- laboration, PIP2 was modeled into the ATP-binding site. The binding energy estimated for the interaction between the modeled PIP2 head group and gel- solin is similar to that calculated for ATP binding to gelsolin from the struc- ture. The biological relevance of the ATP and PIP2 potentially binding to the same site is unclear. However, this may suggest that ATP may modulate the dissociation of gelsolin from the PIP2-rich membrane.

22 Paper II

In this paper we presented the structure of Ca-free inactive human cytoplas- mic gelsolin and the complex of calcium activated G1-G3 bound to actin. The human gelsolin structure is almost identical to that of Ca-free horse gel- solin. It is composed of six homologous domains and fold into a compact globular conformation in the absence of calcium. The major difference is that there is no disulfide bond between Cys188 and Cys201 in human cyto- plasmic gelsolin. From the comparison of human Ca-free gelsolin and Ca- activated actin-bound N-terminal/C-terminal gelsolin, we hypothesized that two calciums are needed to bind to domains G2 and G6 respectively for gel- solin to open the G2-G6 latch and expose the F-actin binding site on domain G2. My involvement in this work was in proving this point. Thermal stabil- ity assays and pyrene F-actin severing assays provided evidence that only two molar equivalents calcium are required for gelsolin activation and com- plete F-actin severing activity. The human G1-G3/actin structure differs from the horse G1-G3/actin in that the disulfide bond between Cys188 and Cys201 is present, which was absent in horse G1-G3. Furthermore, in the human G1-G3/actin structure, a calcium ion was found to bind to the type-2 site in G2, whereas the calcium-binding site is unoccupied in the horse gel- solin. The structure of the G2 calcium-binding site reveals that this Ca2+ sta- bilizes the G2:G3 interface by coordinating Asp259 from the G2-G3 linker. The thermal denaturing curve of G2-G3 also displayed increasing stability upon calcium binding. From these data we proposed that in familial amyloi- dosis Finnish-type (FAF) the mutations Asp187Asn or Asp187Tyr cause loss of G2 Ca2+-binding that leads to prolonged intermediate activation states in which the protease, furin, can cut gelsolin.

Paper III

In this paper we presented the first structure of the calcium activated C- terminal half of adseverin (A4-A6). The structure contains three bound cal- cium ions, where each domain binds to a single type 2 calcium binding site. The A4-A6 domains are arranged to form an L-shaped entity, which highly resembles that of active gelsolin G4-G6. The calcium-binding sites in A4-A6 are conserved compared to those in G4-G6. This is enforced by that fact that in the thermal denaturation assays, both A4-A6 and G4-G6 displayed almost identical destabilizing and stabilizing profiles upon calcium activation. Ad- severin shares 60% sequence homology with gelsolin. Gelsolin contains a C- terminal extension which masks the F-actin binding site on domain G2 in the absence of calcium. This extension is absent in adseverin. We constructed a

23 calcium-free adseverin model which predicts that the calcium activation will be required to break the interaction between domains A2 and A6 in order to expose the actin-binding site on domain A2. Pyrene-actin depolymerizing assays demonstrated that only one calcium ion is required for adseverin to sever actin filaments, as opposed to the two needed by gelsolin. The differ- ence may be due to the C-terminal extension of gelsolin that needs to be removed from the surface of G2 before F-actin binding can take place. We hypothesize that, in the absence of this extension, the calcium requirement for activation is lowered.

Paper IV

In this paper, we comparatively analyzed the effect of calcium, ATP and pH on protein structure and actin binding activities for four members of the gel- solin protein family: gelsolin, adseverin, villin and capG. In an EGTA- buffed thermal denaturation assay, all members that contain six gelsolin domains destabilize and then restabilize upon calcium activation. This is consistent with the idea that the two halves of these molecules go through intermediate conformations where the two halves separate in response to calcium. Two molar equivalents of calcium for gelsolin and adseverin, and at least three for villin, and one for capG are required to reach the minimum stability in a limited calcium assay. The levels of calcium required for F- actin depolymerization differed from those needed for isolated activation, as judged by thermal denaturation assays. This indicates that besides the direct calcium effect, interactions between F-actin and the gelsolin family are in- volved in activating gelsolin family members. At low pH, all four proteins are destabilized. However, in a pyrene actin depolymerizing assay, only gelsolin and adseverin were able to disassemble actin filaments at a pH be- low 6.5 in the absence of calcium. Interestingly, ATP was found only to bind to gelsolin but not the other three proteins.

24 Discussion and future perspectives

Gelsolin is an actin-binding protein that nucleates actin polymerization and severs and caps actin filaments. All of these functions are calcium depend- ent. The calcium regulation of gelsolin is a multifaceted process, which in- volves complicated conformational changes of the gelsolin structure. It is generally agreed that the calcium activation of gelsolin can be viewed in three major steps starting from a calcium free state, where gelsolin displays a compact globular shape. The second stage occurs at a concentration of Ca2+ between 0.1 to 5 M, which evolves into a third state at Ca2+ concentration between 10 M to 1 mM. However, there is a discrepancy in the concentrations of Ca2+ required for these processes. Gelsolin was shown to release its interaction between do- mains G2 and G6 and open up its two halves when activated at nanomolar calcium concentrations. Two high affinity calcium-binding sites were identi- fied by equilibrium dialysis with a Kd 1 M [166]. Similar experiments identified two calcium-binding sites in G4-6, a high affinity site located in G5-6 (Kd 0.2 M) and a lower affinity site in G4-5 (Kd 2 M) [167]. The number of calcium ions required for activation, and the order of calcium binding, is still unclear. In the second paper, we reported that the G2 cal- cium-binding site is occupied in the human G1-G3/actin complex. It is also well recognized that G4-G6 is responsible for Ca2+ regulation within whole gelsolin. From our detailed structural analysis we speculate that these two critical calcium ions will bind to G2 and G6. In order to provide stronger evidence for this, biochemical assays were performed. For the first time, we used a thermal denaturation assay to eluci- date the calcium effect on gelsolin activation. The theory behind this assay is that gelsolin is stable, either when it is in a compact Ca2+-free state, or when it is a fully activated rigid body. At intermediate points, where there is sepa- ration of the two halves, the molecule will be least stable and this conforma- tional change is captured and represented as the minimum in Tm. In the EGTA-buffered Ca2+ titration assay, gelsolin displayed a destabilization phase followed by a restabilization phase between 16 to 63 nM Ca2+. This shows that it is the occupancy of the high affinity Ca2+ sites that are releasing the G2-G6 latch, which is in line with previous reports using other methods. In the Ca2+ titration assay without EGTA, we observed that only two calcium ions are required for gelsolin activation. An actin depolymerizing assay also

25 showed that the severing is complete at a 2:1 ratio of Ca2+ to gelsolin. Muta- tional analysis of the Ca2+ binding sites within G2 and G6 are now planned. Adseverin is similar to gelsolin in needing two calcium ions to reach the minimum in thermal stability, however, it differs from gelsolin in needing only one calcium ion for complete actin depolymerization in the pyrene- actin assay. Calcium-induced thermal stability transitions of the N-terminal half of adseverin occurred slightly earlier in the EGTA-buffered thermal shift assay than for the C-terminal half indicating a higher affinity for cal- cium. We speculate that the first calcium ion will bind to domain A2 prefer- entially to dissociate the A2-A6 interface. Again mutagenesis experiments need to be conducted to confirm the priority calcium-binding sites. The overall structure of adseverin A4-A6 highly resembles that of gel- solin G4-G6. Both C-terminal halves contain very similar calcium-binding and predicted actin-binding sites. It takes A4-A6 two molar equivalents of calcium to completely depolymerize actin filaments in the pyrene-actin as- say. Previous experiments had reported that there is an F-actin binding and nucleating site at A5 residues 511-518 [168]. However, this stretch of actin- binding residues is buried, forming in part a strand within the A5 -sheet. We speculate that the reported A5 nucleation activity is due to a truncation artifact since only A5-A6 was tested but not the whole C-terminal half. We expect that domain G5, in a G5-G6 construct, may behave similarly and we propose to generate this construct. Adseverin appears to be preferentially activated at lower levels of calcium with respect to gelsolin. However, both gelsolin and adseverin can be acti- vated by low pH, and they are both able to sever actin filaments at pH < 6 in the absence of calcium. In contrast, villin and capG are not released from Ca2+ regulation at low pH. Furthermore, all the gelsolin family members did not completely fill their total number of calcium-binding sites in order to interact with actin. These results suggest that subtle differences between the activities of members of the gelsolin family and affinities of calcium-binding sites will lead to an increase in the range of in vivo activities. Finally, the calcium activation of gelsolin is sensitive to ATP. The data presented here shows that gelsolin, and not adseverin, capG or villin, is able to bind to ATP, which causes stabilization of its structure. The structure presented here may provide the basis for designing a gelsolin inhibitor.

26 Conclusion

The present structures together with biochemical experiments have allowed for a better understanding of the mechanisms of activation of the gelsolin family of proteins by calcium and pH, and of inhibition by PIP2 and ATP. These data have helped us to distinguish subtle structural and functional differences among this family of proteins. Both these similarities and differ- ences give tantalizing insights into the physiological roles of these proteins.

27 Acknowledgements

First of all, I would like to express my heartfelt thanks to my supervisor, Ass. Prof. Robert C. Robinson for giving me the opportunity to make my dream come true - becoming a life science scientist. For his stimulating sugges- tions, encouragement and great patience in trying to make a decent scientist out of me.

I also like to thank my second supervisor Prof. Lars Rask, and my examiner Prof. Kristofer Rubin for co-supervision.

My work could not have been done without collaborations with all my co- authors, especially Prof. Leslie Burtnick.

I would still be disorganized and less efficient without sincere help and sup- port from sister-like Maria.

Big thanks to Mårten for giving me a hand in proofreading my thesis, sug- gesting scholarship applications and being my free Swedish translator.

I am grateful to Albert for his kind invitation to be a friend of his family. They cheer me up constantly and provide me a home away from home.

Thank you my sunshine young friends: Wei Lin, Khay Chun, Shalini and Alvin. Without you guys, my life would be boring. Also thanks to Hema, Raj and Bala for your friendship smiles.

My unreserved appreciation goes to my dear friends Xiao Yi, Ya Qing and Zuo La, for your listening ears when I am down and generously allowing me to share the happiness of your lives. To Tan Ah Geam for your good news from the Bible and my only homemade meals in Singapore every Sunday. To my Irish mountain friend Declan Lunny for your jungle guiding and al- ways saying “you can do it”.

Finally, I’d like to thank myself, Qing, you are a good girl - despite many difficulties you make it through. Please be courageous and believe in your- self, beautiful days are always ahead.

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