Nayar 1 Structure and Function of Thymosin β4 Anita Nayar Senior Comprehensive Paper The Catholic University of America Nayar 2 Abstract The thymosins are a group of proteins from the thymus gland. There are three classes of thymosins based on their pI, α, β, and γ. One of the most prominent of the thymosin family is thymosin β4. This protein contains 43 amino acids and is approximately 5 kDa. Thymosin β4 is found predominately in platelets and has a higher affinity for platelet actin. Thymosin β4 is known to be the main G-actin sequestering peptide and to accelerate wound healing. Analysis of thymosin β4 gave insight into its structure and how it sequesters G-actin. It was found that thymosin β4 has two α-helicies, one at each terminal end of the protein and these two helicies link what is known as the hexapeptide actin binding motif. A hydrophobic cluster is present in the N-terminal αhelix that helps in binding thymosin β4 to actin. Thymosin β4, prevents G-actin from polyermizing into F-actin through these various binding sites to G-actin. It was also discovered that thymosin β4 is unstructured in aqueous solution and upon binding to Gactin, thymosin β4 undergoes a conformational change into a folded structure. Nayar 3 Introduction Up until the early 1960s the thymus gland was thought to be vestigial and functionless. It was during that time that the thymus gland was discovered to be the “master gland” of the immune system.1 The term “thymosin” was given to the group of molecules which, heavily affected the immune system by increasing the number of lymphocytes in the blood from the thymus gland. Research on the thymosins began in 1964 using calf thymus. The first thymosin fraction to be extracted was thymosin fraction-3 (TF3). This fraction was only partially purified but it opened the doors to research of the thymosins. TF3 was further purified through gel filtration, ultrafiltration, and ammonium sulphate fractionation to give rise to a more pure preparation known as thymosin fraction 5 (TF5) which was determined to be biologically active. TF5 has a family of about forty small acidic ploypeptides. The isoelectric points were determined for each of the forty polypeptides. The thymosins were then classified based on their isoelectric points: α for isoelectric points below 5, β for isoelectric points between 5.0 – 7.0, and γ for isoelectric points above 7. The isoelectric points of the classes of the thymosins can be seen in Figure 1. The β-thymosins are a family of highly conserved water-soluble 5kDa polypeptides.2 Thymosin β4 is the most abundant of the β thymosins.1 Nayar 4 Figure 1. The isoelectric focusing for determining the three classes of the thymosin family.1 Thymosin β4 Thymosin β4 is a 43 amino acid, 5kDa polypeptide. Its significant functions include cell proliferation, promotion of angiogenesis (the formation of new blood vessels from a pre-existing vasculature which is needed for both growth and wound repair), acceleration of wound healing. It is also the main G-actin sequestering peptide. The motif Nayar 5 which allows thymosin β4 to bind actin is a seven amino acid sequence found in each thymosin: LKKTETQ.3 I. Structural Determination of Thymosin β4 Amino Acid Sequence of Thymosin β4 The amino acid sequence of thymosin β4 was obtained with subtractive Edman procedures, proteolytic digestion, and a Beckman 890C sequencer.4 Upon reaction with dansyl chloride or phenylisothiocyanate thymosin β4 did not yield an α-amino acid derivative. This indicates that the NH2 terminus is blocked by an acetyl group. Tryptic digestion gave two peptides which lacked a free α-amino group. These two peptides were deduced to be a part of the N-terminus region. The tryptic digestion also produced a peptide which lacks a lysine residue and was therefore placed in the C-terminus region. The side chains were assigned their positions based on their charges determined through high-voltage paper electrophoresis of the peptides derived from Edman degradation and proteolytic digestion. Thymosin β4 contains nine lysine residues and eight glutamic acid residues, however, it does not contain arginine, histidine, valine, tyrosine, and tryptophan.4 The complete sequence of thymosin β4 and the enzymatic cleavage products can be seen in Figure 2. Nayar 6 Figure 2. The complete amino acid sequence of thymosin β4. The F fragments are the result of cyanogen bromide cleavage, the T fragments are the result of tryptic digestion, and the P fragments are the result of partial acid hydrolysis.4 Determination of the Actin Binding Site In order to determine the structure and binding motif of the actin binding site in thymosin β4, mutational analysis was performed. The binding motif observed in virtually all of the β thymosins is 17LKKTET22 and, furthermore, the first three residues are also found in many other actin binding proteins.5 It is important to note that 23Q is considered part of the actin binding motif in thymosin β4 the majority of the other thymosins, however, at least three, do not contain 23Q which is why it is excluded from the conserved motif discussed above.6 Therefore, the binding behavior of the motif was studied when three residues of the six were mutated to L17A, K18E, and K19E. At high actin concentrations, (e.g., 12 μM and 15 μM) wild-type thymosin β4 has 50% cross-links with actin. However, at these high actin concentrations, K19E gave 30% cross-link with actin and K18E only cross-links with actin slightly. Furthermore, when a double mutant of 18K and 19K were exchanged to glutamates (KK18, 19EE), a total loss of inhibition was Nayar 7 observed. Overall, mutant K19E produced an actin- thymosin β4 complex whereas mutant K18E and L17A did not. This shows that these three residues make specific interactions with actin and are essential for preserving the proper binding surface. Another mutant of 18 K was studied, K18A. This mutant did not affect the binding of thymosin β4 as severely as K18E did. This suggests that 18K has electrostatic contact with a negatively charged residue in actin. Moreover, when 18K was replaced with glutamate, a strong repulsive effect was observed, which further confirms that 18K forms electrostatic contact with actin.5 The N-terminus of thymosin β4 (residues 1 through 16) is highly charged. To study the significance of the N-terminus, many of the charged residues were substituted with residues possessing the opposite charge. For example, a lysine was substituted with glutamic acid. This charge reversal did not affect the binding as much as the mutations in the binding site did. However, two mutations did have an unfavorable affect on actin binding.5 These were the mutants of 16K and 14K. K16E had a 12-fold reduction of actin affinity whereas K14E had essentially no affinity for actin. Further analysis of 14K with another mutant K14A showed a similar very low actin binding affinity, and when substituted with a glutamate gave a greater decrease in actin binding. This then suggests that 14K is in an electrostatic interaction with a negatively charged actin residue. Then, the nonpolar residues were studied and analysis showed that substitution of phenylalanine to a smaller, less bulky side chain greatly residues thymosin β4’s affinity for actin. Furthermore, when replaced by either tyrosine or tryptophan, the actin affinity is not Nayar 8 affected as much. Therefore the bulky side chain of phenylalanine is of importance to the binding of thymosin β4 to actin.5 An Intact N-terminal α-Helix is Required for Complete Thymosin β4 Activity5 To determine whether or not the N-terminus in thymosin β4 binds to actin, circular dichroism (CD) measurements were taken.5 These measurements were carried out in both water, and with 60% trifluoroethanol (TFE). It has been observed in NMR studies that thymosin β4 is unstructured in aqueous solutions but the helices are observed in solutions containing fluorinated alcohols6 such as TFE. Therefore the helices in thymosin β4 of residues 4 – 16 and 30 – 40 were more pronounced in 60% TFE solution. The CD measurements of wild type thymosin β4 showed a maximum at a wavelength of 190 nm and double minima at wavelengths 207 nm and 222 nm, which correlate to the two αhelices shown in Figure 3. When CD analysis was performed on the mutants in the same solutions, similar maxima and minima were observed. This shows that the loss of thymosin β4 activity in these mutants is not due to an inability to form a secondary structure. In K11P, a known helix-breaker, proline, replaced 11K as a control and the affinity of thymosin β4 to actin severely diminished. CD analysis showed a decreased intensity for K11P at peak 190nm, which is the marker for a helical conformation. The minima which were associated with a helical conformation also shifted to a lower wavelength for K11P as also shown in Figure 3. This mutation shows that overall loss of activity in thymosin β4 is not entirely due to a change in conformation however, if the integrity of the helix is altered, then thymosin β4 activity can be severely impaired.5 In other words, this shows that thymosin β4 can still form an overall secondary helix, but the Nayar 9 tertiary structure is impaired. If the helix is still present, thymosin β4 can still be functional. However, once the helix is compromised, activity is lost. Therefore, if thymosin β4 cannot form the secondary structures, it cannot function properly. Figure 3. CD measurements of wild-type thymosin β4 in TFE along with mutants K18E, L17A, and double mutant KK18, 19EE (A) and mutated with K11P thymosin β4 in TFE (B). The mutants in (A) only show a slight deviation from the WT and therefore the loss in activity is not due to the fact that they cannot make wild type secondary structures except L19A which shows more intense peaks indicating a stronger N-terminal α-helix or that the helix extends over more residues. (B) shows that when the helix is severely compromised, the activity is thymosin β4 is affected.5 The Proposed Structure of the Actin Binding Site The analysis described above has shown that the sixteen residue N-terminus and the conserved hexapeptide motif are both essential for thymosin β4 to bind to actin. Based Nayar 10 on NMR studies, a final model of the structure was proposed. NMR measurements and analysis suggested a α-helix for residues 4 – 16, an undefined conformation for the hexapeptide binding site at residues 17 and 18, a loop which spans residues 24 through 28, and a less stable second α-helix for residues 30 through 40.5 It is likely that the N-terminus is an α-helix because it can support interaction with actin and can position the hydrophobic cluster to also interact with actin in this conformation. The hydrophobic cluster is formed by three residues: methionine 6, isoleucine 9, and phenyalanine 12. Methionine occurs at position 6 and when methionine is oxidized, thymosin β4 is known as thymosin β4 sulfoxide and the affinity for actin is decreased 20-fold.6 The hydrophilic side of the N-terminal helix consists of 8E, 10E, 11K, 14 K, and 15S of which only 14K makes a salt bridge with actin. This proposed structure is further confirmed with mutant K11P. K11P breaks the helix and renders thymosin β4 essentially inactive. This shows that an intact helix, along with the hydrophobic core, is essential for its activity. Further analysis shows that if the N-terminal helix was made just one residue longer, the placement of 18K and 19K would be shifted in a way that is unfavorable to actin binding.5 This further confirms the necessity of the N-terminal helix for the actin binding to occur and the exact length needed for the N-terminal residue. The final proposed model for the thymosin β4 actin binding site can be seen in Figure 4. It is important to note that the α-helical segment of thymosin β4 is the only completely compacted, structured form of the protein where the hydrophobic segment of thymosin β4 forms an interface with the hydrophobic end of the barbed end (subdomains 1 and 3) of G-actin.7 Nayar 11 Figure 4. The N-terminal helix (residues 4 – 16) is in shown in white, the hydrophobic cluster is shown in yellow surrounded by van der Waals forces, and the important residues for actin binding are in red. The N-terminal helix is the underlined sequence and the hexapeptide motif is the doubly underlined sequence.5 The Actin Binding Site is Conserved in Actin Binding Proteins The entire β- thymosin family (including β- thymosins of various species) binds to actin and therefore has an actin binding motif. It is important to note that thymosin β15 is the only member of the β- thymosin family to have an amino acid substitution in the Gactin binding motif. In thymosin β15, a glutamic acid is substituted with asparagine.6 The motif and other residues such as 14K which are essential for actin binding are conserved throughout the entire β- thymosin family as shown in Figure 5.6 Each lysine that is essential in binding thymosin β4 to G-actin is conserved in the β- thymosin family.7 Nayar 12 Furthermore, other actin binding proteins such as actobindin have a similar, albeit not exactly identical, motif for actin binding.5 Figure 5. The amino acid sequences of the β- thymosin family. The conserved residues are bolded. thymosin β15 is the only member to have a substitution in the actin binding motif.6 NMR Determination of the Structure of Thymosin β4 A NMR technique for the determination of structures of macro molecules is the Nuclear Overhauser Effect (NOE). This technique was used to determine the structure of thymosin 4.8 In order to obtain a basic insight into the structure, 1H – 15N NOE analysis was performed on the protein at 2C and 25C. At both temperatures, thymosin 4 displayed incredibly low values of heteronuclear NOEs (under 0.5 for 2C and under 0 for 25C). Usually a fully structured protein has a heteronuclear NOE value around 0.7. Nayar 13 These data show that in aqueous solution, thymosin 4 is unfolded. When 1H – 15N NOE analysis was performed on the thymosin 4 bound to actin, the value of the heteronuclear NOE is around 0.7 which shows that when bound to actin, thymosin 4 is folded.8 This NOE analysis can be seen in Figure 6. Figure 6. The heteronuclear 1H – 15N NOE analysis of thymosin 4. The represents the thymosin 4 – actin complex, is thymosin 4 at 2C, and is thymosin 4 at 25C.8 Analysis of the NOE peaks gives more insight into the structure of thymosin 4. HN – HN peaks are indicative of helical folding and the most intense HN - H correlation is considered intraresidual. Analysis of the peaks, HN, 15N, and Hα using density functional theory method (CSI) calculations indicate that two α-helicies exist: one at the N-terminus between residues 5D – 17L and one at the C-terminus between residues 31K – 40 A. These two helicies are responsible for linking the extended fragment, the actin binding motif, which is residues 18K – 26N.8 The high correlation values of the NOE of Nayar 14 the thymosin 4 actin complex shows that this region adopts an extended conformation which binds to actin. This is known as the actin binding motif.8 One dimensional 1H-NMR analysis provides the same information about the folding of thymosin β4 to actin.7 The shifts of thymosin β4 alone resemble that of an unstructured protein and the chemical shifts of thymosin β4 bound to actin are in between the shifts of thymosin β4 alone and G-actin alone which indicates that thymosin β4 becomes structured upon actin binding.7 To further confirm that thymosin 4 forms a complex with G-actin, the heteronuclear single quantum coherence (HSQC) spectra was recorded at 25C with thymosin 4 containing [U- 15N] thymosin 4 with an unlabeled actin. A large spreading of amide protons was observed, which is expected of fully structured proteins, and can be seen in Figure 7. Since thymosin β4 is unstructured in aqueous solution, these data show that upon binding to G-actin, thymosin β4 undergoes a conformational change. Furthermore, 26N was observed to undergo the largest shift, which indicates that this amino acid lies on the interface of the thymosin β4 – G-actin complex. These data verify that thymosin 4 forms a tight complex with G-actin.8 Therefore, NMR analysis confirms the structure and binding pattern of thymosin 4 to G-actin, which coincides with the ones proposed in the studies previously described. Nayar 15 1H (ppm) Figure 7. HSQC spectra of [U – 15N] thymosin 4. The red symbols indicate the free thymosin 4 and the black dots indicate thymosin 4 bound to G-actin. The additional black peaks indicate that the intact protein is structured when bound to actin.8 II. Function of Thymosin β4 Thymosin β4 and its Function in Wound Healing It was first thought that thymosin β4 plays a role in wound healing because it is involved in endothelial cell migration and because it is found in abundance in platelets. To test if thymosin β4 is involved in wound healing, six 8 mm punch biopsy wounds were made in rats. These wounds punctured the epidermis and dermis and were in contact with the fat and muscle.9 thymosin β4 was injected topically into 3 mice and intraperitonally in 3 other mice. Saline was injected in the same manner as a control. The injections were made at the time the wound was made and 48 hours later. Figure 8 compares the size of the wounds after four and seven days. Both topical and intraperitonal injections of Nayar 16 thymosin β4 showed a greater decrease in wound size than saline injections. This proves that thymosin β4 is involved in wound repair. It is thought that when the platelets arrive to the injury, they release thymosin β4, among other factors, which attract endothelial cells to being angiogenesis and then begins wound repair.9 Figure 8. a) Topical injection of thymosin β4 shows an 18% decrease in wound size after 4 days and 62% after 7 days compared to the saline injection. b) Intraperitonal injection shows a 42% decrease in wound size after day 4 and 61% after day 7 compared to the saline injection.9 Thymosin β4 as the Main G-Actin Sequestering Peptide Globular actin, also known as G-actin, is responsible for rapid generation of new actin filaments. G-actin contains four subdomains which are all held together by bound ATP or ADP as shown in Figure 9. When G-actin monomers come together they form actin filaments known as F-actin. When G-actin polymerizes into F-actin, each monomer is orientented in the same direction. This shows that the structure is polar with two different ends, one known as the barded end (plus) and the other as the pointed end (minus).10 However, nearly half the intracellular actin present is in its stable monomeric Nayar 17 G-actin form. Monomeric actin is stabilized through its interaction with sequestering factors. Thymosin β4 is known to form a 1:1 complex with G-actin which inhibits saltinduced polymerization into F-actin.2 Thymosin β4 binds to G-actin with a dissociation constant (Kd) between 0.5 and 2.5 μM. This can be seen in Figure 10. Thymosin β4 has a 3 – 5 fold higher affinity for platelet actin over skeletal actin since the platelet actin, in β form, has more amino acid residues on its surface that allows thymosin β4 to interact with.7 A seven-amino-acid sequence has been proposed as the main actin binding motif in thymosin β4. This sequence is: LKKTETQ.3 Furthermore, the first three residues of this motif are essential for the interaction with G-actin as are the three hydrophobic residues 6 M, 9I, and 12F of thymosin β4. NMR analysis showed that the N-terminus of thymosin β4 must be in an α-helix for the interaction between G-actin and thymosin β4 to occur.6 Figure 9. The structure of G-actin showing the 4 subdomains bound to a central ATP.11 Nayar 18 Figure 10. Thymosin β4 (red) interacts with subdomains 3, 1, and 2 of G-actin. When G-actin is released from the complex, it is added to the (+) end of the actin filament.6 Zero-length cross linking was carried out to create isopeptide links between the side chains of thymosin β4 and G-actin. The basis of cross-linking is taken under the premise that certain noncovalent interactions between acidic and basic residues in the structure can be converted into covalent bonds through cross-linking.7 Zero-cross linking with 1-ethyl-3-[3-(dimethyamino)propyl]carbodiimide (EDC) showed that 3K of thymosin β4 cross links with 167E of actin and 18K of thymosin β4 cross links with one of the four N-terminal acidic amino acid residues of actin (1DEDE4). The two contact sites on the barbed end of G-actin are approximately 25 – 30 Å apart. The 12 residue α-helix is approximately 18 Å and binds to both these sites. Therefore the α-helix must be in a stretched helix conformation to span both the sites.7 Also, 38K of thymosin β4 can be cross-linked to 41Q of G-actin. Furthermore, the C-terminus of thymosin β4 can also block Nayar 19 G-actin polymerization into F-actin by binding to the pointed end of actin. These three points of contact are in subdomains 3, 1, and 2 of G-actin, respectively, as can be seen in Figure 9 and 10. Subdomain 2 is known as the pointed end of G-actin and subdomains 1 and 3 are known as the barbed end.7 Thymosin β4 blocks G-actin from polymerizing into F-actin by binding to the barbed and pointed ends of G-actin.7 Additionally, the interaction of G-actin and thymosin β4 depends on either ADP or ATP bound to G-actin. When G-actin is bound to ATP, there is a 50-fold higher affinity to thymosin β4 than the G-actin ADP complex.6 The discussion above states that thymosin β4 is the main G-actin sequestering peptide in the cytoplasm. Thymosin β4 is also the main G-actin sequestering peptide in the nucleus. To first determine the presence of G-actin in the nucleus, DNase I, another actin binding protein, was labeled with Oregon Green in MCF-7 cells. All these cells displayed the Oregon Green-labeled DNase I in the nucleus. Thymosin β4 was fluorescently labeled with Oregon Green cadaverine (OGC) to determine its presence in the nucleus. Some other thymosin β4s were bis-labeled with OGC at 23Q and 36Q. Both of these labels appeared in the analysis of thymosin β4 in the nucleus. When the Kd was determined for the bis-labeled thymosin β4, it did not differ significantly from the unlabeled thymosin β4. In order to further test the distribution of thymosin β4 fluorescently labeled thymosin β4 was also injected into Vero cells. Both MCF-7 and Vero cells showed distribution of the peptide in the nucleus. Then the bis-labeled thymosin β4 was digested using AsnC-endoproteinase. Thymosin β4 contains only one asparagine residue at position 26. Therefore this endoproteinase produced two fragments, the N-terminal fragment containing residues 1 – 26 and the C-terminal fragment Nayar 20 containing residues 27 – 43. Each of the fragments contained a fluorescent label. HPLC analysis showed that the N-terminal fragment containing the actin binding domain was predominant in the nucleus while the C-terminal fragment remained in the cytoplasm as shown in Figure 11. Residues 14 – 16, KSKLKK, is rich in lysine residues and is believed that be a part of thymosin β4’s translocation into the nucleus.2 Figure 11. A and B show the N-terminal in the nucleus and C and D show the Cterminal fragment of thymosin β4 in the cytoplasm. A and C display the fragment of thymosin β4 also injected with rabbit immunoglobin G and B and D display the fragment of thymosin β4 alone.2 Conclusion Many proteins involved in cell cycle regulation are naturally unstructured and fold when they bind to their biological target.8 Thymosin β4 falls into this category. This protein is involved in both wound healing and is the main G-actin sequestering peptide. G-actin is responsible for rapid generation of new actin filaments.2 Thymosin β4 binds to Nayar 21 G-actin in order to prevent G-actin from polymerizing into F-actin. The α-helix of the Nterminus of thymosin β4 binds to subdomains 1 and 3 of G-actin while the α-helix of the C-terminus binds to subdomain 2 of G-actin. In between these two helicies, an actin binding motif of thymosin β4 binds to actin also. Each of these contacts prevents G- actin from polymerization. Thymosin β4 is found predominately in the platelets and even has a higher affinity for platelet actin. Therefore, it is highly possible, especially since thymosin β4 has a role in wound healing, that future drug research can make certain drugs focusing on thymosin β4 to aid in the wound healing process. These types of drugs for example might be able to help people deficient in clotting or shortening the wound healing duration of a serious wound. Nayar 22 References (1) Goldstein, A.; Annals of the New York Academy of Sciences 2007, 1112, 1 – 13. (2) Huff, T.; Rosorius, O.; Otto, A.M.; Müller, C.S.G.; Ballweber, E.; Hannappel, E.; Mannherz, H.G.; Journal of Cell Science 2004, 117, 5333 – 5343. (3) Philip, D.; Huff, T.; Gho, Y.S.; Hannappel, E.; Kleinman, H.K.; The FASEB Journal 2003, 17, 2103 – 2105. (4) Low, T.L.K.; Hu. Shu-Kuang; Goldstein, A.; Proc. Natl. Acad. Sci. USA 1981, 78, 1162 – 1166. (5) Troys, M.V.; Dewitte, D.; Goethals, M.; Carlier, M.F.; Vandekerckhove, J.; Ampe, C.; The EMBO Journal 1996, 15, 201 – 210. (6) Huff, T.; Müller, C.S.G.; Otto, A.M.; Netzker, R.; Hannappel, E.; The International Journal of Biochemistry & Cell Biology 2001, 33, 205 – 220. (7) Safer, D.; Sosnick, T.R.; Elzinga, M.; Biochemistry 1997, 36, 5806 – 5816. (8) Domanski, M.; Hertzhog, M.; Coutant, J.; Gutsche-Perelroizen, I.; Bontems, F.; Carlier, M.F.; Guittet, E.; van Heijenoort, C.; The Journal of Biological Chemistry 2004, 279, 23637 – 23645. (9) Malinda, K.M.; Sidhu, G.S.; Mani, H.; Banaudha, K.; Maheshwari, R.K.; Goldstein, A.; Kleinman, H.K.; The Society for Investigative Dermatology 1999, 113, 364 – 368. (10) Berg, J.; Tymoczko, J.; Stryer, L. Molecular Motors; In Biochemistry; WH Freeman and Company: New York, 2007; pp. 985 – 986. (11) http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/actin.htm