生物大分子結(jié)構(gòu)與功能第5章蛋白質(zhì)的柔性結(jié)構(gòu).ppt

1、第五章： 蛋白質(zhì)的柔性結(jié)構(gòu),天然折疊的蛋白分子往往不是以一種構(gòu)象狀態(tài)存在的。在晶體結(jié)構(gòu)中我們看到的往往僅是一種狀態(tài)的構(gòu)象，它是蛋白質(zhì)分子的一個(gè)平均構(gòu)象。實(shí)際上，蛋白質(zhì)分子始終是處于一種呼吸的狀態(tài)。蛋白質(zhì)結(jié)構(gòu)中所有的原子都在運(yùn)動(dòng)，這些原子的運(yùn)動(dòng)通常是隨機(jī)的，但有時(shí)可以是集合性的運(yùn)動(dòng)。這種集合性的運(yùn)動(dòng)引起分子中的原子團(tuán)在相同的方向上產(chǎn)生運(yùn)動(dòng)，造成蛋白質(zhì)分子中的側(cè)鏈可以從一種構(gòu)象轉(zhuǎn)化為另一種構(gòu)象。某些環(huán)區(qū)域也并不總是固定在一種單一的構(gòu)象狀態(tài)，螺旋也可以互相產(chǎn)生滑動(dòng)，完整的結(jié)構(gòu)域之間也可以改變它們的堆積接觸以打開或關(guān)閉結(jié)構(gòu)域之間的距離。通常這些運(yùn)動(dòng)都是比較小的，有時(shí)小到僅有1/10 的運(yùn)動(dòng)，但有時(shí)這種

2、集合性運(yùn)動(dòng)可以很大，大到足以具有重要的生物學(xué)意義。,這樣大的集合性運(yùn)動(dòng)在X-射線晶體學(xué)研究中所表現(xiàn)出來的是電子密度的水平低，甚至在某些情況下看不到電子密度的存在。產(chǎn)生這樣的運(yùn)動(dòng)的區(qū)域通常在晶體學(xué)中被表述為柔性(flexibility)運(yùn)動(dòng)或無序(disorder)。,核磁共振實(shí)驗(yàn)對于這樣的區(qū)域的測定可以作為一種互補(bǔ)，因?yàn)楹舜殴舱駥?shí)驗(yàn)可測出這些區(qū)域的各種不同的構(gòu)象，通過理論計(jì)算也可以計(jì)算出這些分立的或集合性運(yùn)動(dòng)這叫作分子動(dòng)力學(xué)模擬。,分子動(dòng)力學(xué)模擬已經(jīng)表明，每一個(gè)分立的殘基的集合性運(yùn)動(dòng)僅在皮秒(10-12 秒)的時(shí)間尺度，而環(huán)區(qū)域的運(yùn)動(dòng)在納秒(10-9 秒)的尺度。這種運(yùn)動(dòng)對于許多蛋白質(zhì)的功能是

3、非常重要的。象電子轉(zhuǎn)移和配基結(jié)合或釋放反應(yīng)均以這樣的時(shí)間尺度發(fā)生，并通常伴隨著蛋白質(zhì)原子的運(yùn)動(dòng)。例如，當(dāng)肌紅蛋白呼吸時(shí)，通道在溶劑和被包埋在分子內(nèi)部的結(jié)合部位之間打開，以允許氧原子在納秒的時(shí)間尺度范圍與肌紅蛋白結(jié)合或者釋放出來。,除了蛋白質(zhì)中原子小的呼吸運(yùn)動(dòng)之外，在分子的功能態(tài)之間也會(huì)發(fā)生大的構(gòu)象變化。不同的pH 和配基的存在和缺失以及環(huán)境中的微小的變化，往往能夠穩(wěn)定蛋白質(zhì)的不同構(gòu)象態(tài)。這些構(gòu)象變化可以是活性部位的氨基酸側(cè)鏈的構(gòu)象變化到環(huán)區(qū)域的運(yùn)動(dòng)等。同時(shí)結(jié)構(gòu)域之間的相對取向和寡聚蛋白中四級結(jié)構(gòu)也會(huì)發(fā)生變化，這樣的運(yùn)動(dòng)通常是與功能相關(guān)的。例如酶的催化，肌肉運(yùn)動(dòng)和能量轉(zhuǎn)換等。,真核細(xì)胞周期的五個(gè)

4、相 (G0, G1, S,G2 和M 相),例1：細(xì)胞周期調(diào)節(jié)蛋白激酶的構(gòu)象變化,在S 相，DNA 合成，DNA 被復(fù)制并且染色體翻倍。在M 相，有絲分裂父代細(xì)胞的二倍化染色體通過有絲分裂的紡錘體分開，這樣每個(gè)子代細(xì)胞接收到相同組分的染色體。,一個(gè)細(xì)胞分裂的完整周期是M G1 S 和G2。通過G1 S 和G2 相，細(xì)胞的蛋白質(zhì)合成機(jī)器大分子和細(xì)胞器被建立起來，同時(shí)細(xì)胞的體積增大。在有絲分裂時(shí)，染色體和細(xì)胞質(zhì)被分為兩個(gè)相等的部分。此外，還有一個(gè)靜止相G0 相，發(fā)生在細(xì)胞的未分裂狀態(tài)。,由cyclin 的降解對CDKs 的調(diào)節(jié),細(xì)胞周期的進(jìn)程取決于一系列的叫作cyclin依賴的蛋白激酶(cycli

5、n-dependent protein kinases, CDKs)的連續(xù)激活作用。,圖中顯示兩種類型的cyclin-CDK 復(fù)合物，一種是觸發(fā)S 相，另一種觸發(fā)M 相。在這兩種情況下CDK 的激活需要與cyclin 的結(jié)合，它們的非活性依賴于cyclin的降解,在脊椎動(dòng)物的細(xì)胞中至少有四種不同CDKs ，控制著細(xì)胞周期的活動(dòng)。不同的催化亞基都屬于密切相關(guān)的基因家族，不同的CDK 的一個(gè)或幾個(gè)cyclin 分子都是該家族的成員。 CDKs 作為一個(gè)延遲開關(guān)，控制著從G1 相到S 相從G2 相到M 相以及所有構(gòu)成細(xì)胞周期的其它步驟,人的體細(xì)胞中調(diào)制DNA復(fù)制的CDK2-cyclin A的結(jié)構(gòu)提供

6、了詳細(xì)的結(jié)構(gòu)信息以及cyclinA 激酶的功能。Cyclin A 的功能片段的晶體結(jié)構(gòu)于1995 年由Louise Johnson 實(shí)驗(yàn)室解出，非活性的CDK2 的結(jié)構(gòu)1993 年已由Sung-hoKim 實(shí)驗(yàn)室解出，活性的cyclin A 片段與CDK2 復(fù)合物的結(jié)構(gòu)也于1995年由Nicola Pavletich 實(shí)驗(yàn)室解出。通過對這些結(jié)構(gòu)的分析和結(jié)構(gòu)比較，揭示出cyclin A 是如何結(jié)合到CDK2 上，并如何在CDK2 的活性部位引起大的構(gòu)象變化，使CDK2 蛋白質(zhì)從一種非活性的狀態(tài)轉(zhuǎn)變?yōu)榛钚誀顟B(tài)的。而在此過程中 cyclin A 的結(jié)構(gòu)則沒有發(fā)生構(gòu)象變化,cyclin A 依賴型激酶

7、CDK2 的結(jié)構(gòu),cyclin A依賴型激酶CDK2 有兩個(gè)結(jié)構(gòu)域，N-端結(jié)構(gòu)域由一段螺旋折疊片組成，在螺旋中PSTAIRE的氨基酸順序（紅色）在所有的CDKs 蛋白激酶中都是高度保守的；C-端結(jié)構(gòu)域主要由螺旋組成，并含有一段柔性的環(huán)區(qū)域稱作T-loop （黃色）環(huán)區(qū)域，含有一個(gè)蘇氨酸殘基，在完全活性的酶中該蘇氨酸殘基被磷酸化。,Cyclin A 的結(jié)構(gòu),Cyclin A 活性片段殘基173-432 的結(jié)構(gòu)由兩個(gè)非常相似的結(jié)構(gòu)域構(gòu)成。每個(gè)結(jié)構(gòu)域都由五段螺旋組成。該活性片段的作用幾乎與完整的cyclin A 分子的作用相同。在所cyclin A 中第一個(gè)結(jié)構(gòu)域具有十分保守的氨基酸順序被稱作Cyc

8、lin-box ，而第二個(gè)結(jié)構(gòu)域的氨基酸順序則不相同。因此盡管cyclin A 片段的兩個(gè)結(jié)構(gòu)域結(jié)構(gòu)幾乎相同但僅有一個(gè)Cyclin-box 序列。,活性的CDK2 藍(lán)色和cyclin A 復(fù)合物的結(jié)構(gòu),在cyclin A-CDK2 復(fù)合物中，主要是Cyclin A 與CDK2 中的PSTAIRE螺旋和T-loop 相互作用，cyclin-box 螺旋2-6 與CDK2 的PSTAIRE 深紅色螺旋和T-loop 黃色作用。在該復(fù)合物中，cyclin A 的結(jié)構(gòu)與單個(gè)cyclin A 是相同的，而CDK2 的結(jié)構(gòu)則發(fā)生了很大的構(gòu)象變化，包括PETAIRE 螺旋T-loop 和ATP 的結(jié)合部位（

9、淺紅色）。,整個(gè)N 端結(jié)構(gòu)域相對于C端的結(jié)構(gòu)域的取向發(fā)生了變化，此外PSTAIRE 螺旋向CDK2 的活性部位靠近并旋轉(zhuǎn)了90， 以便主要的催化殘基Glu 51 指向裂縫，而不是象在 單個(gè)的CDK2 結(jié)構(gòu)中那樣遠(yuǎn)離此裂縫。,CDK2 與cyclin A 結(jié)合的構(gòu)象變化,一旦與cyclin A結(jié)合，PSTAIRE 螺旋橙色轉(zhuǎn)動(dòng)90， 并改變位置以使得Glu 51變?yōu)橹赶蚧钚圆课弧Ｔ揚(yáng)STAIRE螺旋的一些主鏈原子由于這種一致性運(yùn)動(dòng)位移了8.0 的距離。T-loop發(fā)生了大的位置重排某些環(huán)區(qū)域上的氨基酸殘基的位移可達(dá)20 。,左圖：在非活性態(tài)，PSTAIRE 螺旋紅色的取向使Glu 51 指向遠(yuǎn)離

10、ATP 的結(jié)合部位，而T-loop 封住了與底物的結(jié)合部位，以阻止蛋白結(jié)合到CDK2 上。 右圖： 在活性的cyclin A-CDK2 復(fù)合物結(jié)構(gòu)中，PSTAIRE 螺旋發(fā)生了重新定向以使得Glu 51 殘基指向活性部位并與另一個(gè)與催化有關(guān)的殘基Lys 33 形成鹽鍵，T-loop改變了構(gòu)象并與另一個(gè)殘基Asp 145 一起與活性部位中的鎂離子配位，此時(shí)底物的結(jié)合部位被打開，蛋白可以結(jié)合底物。cyclin-CDK2 復(fù)合物可以磷酸化Ser/Thr 殘基并進(jìn)而激活所結(jié)合的蛋白。 在自由CDK2 T-loop結(jié)構(gòu)中的螺旋在復(fù)合物中變?yōu)橐粭l 鏈。,cyclin 結(jié)合引起CDK2 的結(jié)構(gòu)變化,(a)活

11、性部位位于N 端結(jié)構(gòu)域（藍(lán)色）和C 端結(jié)構(gòu)域（紫色）之間的裂縫中，在非活性狀態(tài)此活性部位被T-loop 所封閉。,(b)在活性的cyclin 結(jié)合狀態(tài)的CDK2結(jié)構(gòu)中,Tloop的結(jié)構(gòu)發(fā)生了變化,活性部位被打開, Thr 160 適合于磷酸化.由于cyclin A 的結(jié)合所引起的CDK2 的構(gòu)象變化,不僅暴露了活性部位的裂縫以使ATP 和蛋白底物能夠與之結(jié)合,而且活性部位的殘基發(fā)生了重排,以形成酶的催化作用。此外Thr 160 被暴露出來，并準(zhǔn)備被磷酸化以提高催化活性。簡而言之蛋白質(zhì)結(jié)構(gòu)的柔性調(diào)節(jié)了CDK 家族的酶活性，因而控制了細(xì)胞周期。,Structural basis of inhibi

12、tion of CDK-cyclin complexes by INK4 inhibitors,Philip D. Jeffrey, Lily Tong, and Nikola P. Pavletich Cellular Biochemistry and Biophysics Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA Genes Dev. 2000 14: 3115-3125,The cyclin-depen

13、dent kinases 4 and 6 (Cdk4/6) that drive progression through the G1 phase of the cell cycle play a central role in the control of cell proliferation, and CDK deregulation is a frequent event in cancer. Cdk4/6 are regulated by the D-type cyclins, which bind to CDKs and activate the kinase, and by the

14、 INK4 family of inhibitors.,The structure reveals that p18-INK4c inhibits the CDKcyclin complex by distorting the ATP binding site and misaligning catalytic residues. p18INK4c also distorts the cyclin-binding site, with the cyclin remaining bound at an interface that is substantially reduced in size

15、. These observations support the model that INK4 binding weakens the cyclins affinity for the CDK. This structure also provides insights into the specificity of the D-type cyclins for Cdk4/6.,Overall structure of the p18Cdk6K-cyclin complex and comparison with Cdk2cyclinA Schematic view of p18Cdk6K-

16、cyclin. p18 is shown in yellow, Cdk6 in cyan, K-cyclin in purple. The T loop and PSTAIRE elements of Cdk6 are highlighted in red, and the helices of the first cyclin repeat are labeled. N and C termini are labeled where visible. The p18Cdk6 and K-cyclinCdk6 interfaces do not overlap and lie on oppos

17、ite sides of the kinase, burying a total of 4350 2 of surface area. (B) Top view of the p18Cdk6K-cyclin complex, approximately orthogonal to view in A. The ankyrin repeats of p18 are numbered. The PSTAIRE helix is central to the Cdk6K-cyclin interface, but the T loop packs on the other side of the k

18、inase. (C) View of Cdk2cyclinA complex superimposed on the C lobe of Cdk6 in the same orientation as in A. Both the PSTAIRE helix and T loop, in red, pack against cyclinA. (D) View of superimposed Cdk2cyclinA complex from same viewpoint as B.,The Cdk6 structure in the p18Cdk6K-cyclin complex has a l

19、arge number of conformational changes compared with the active conformation of Cdk2 (Jeffrey et al.1995; Fig. 2C,D) or of other protein kinases. In this inactive Cdk6 structure, the N and C lobes are rotated 13away from each other, resulting in the misalignment of ATP-binding residues. The N-lobe PS

20、TAIRE helix, which contains an invariant active site residue (Glu 61),is displaced by 4.5 away from the active site and is rotated by 16. A C-lobe loop (T loop, residues 162182), which contains the threonine that is phosphorylated (Thr 177) on the full activation of the kinase (Morgan 1995; Russo et

21、 al. 1996) and that forms part of the polypeptide substrate-binding site (Brown et al. 1999), is displaced by 30 . Finally, an additional loop at the back of the catalytic cleft (residues 99102), which would hydrogen bond to ATP, is displaced by several ngstroms.,The Cdk2cyclinA structure (Jeffrey e

22、t al. 1995) showed that cyclinA binding to Cdk2 caused conformational and positional changes in the PSTAIRE helix and T loop and that these changes activated the kinase by correctly aligning certain active site residues and reorganizing the polypeptide substrate binding site. In the p18Cdk6K-cyclin

23、complex, not only does the K-cyclin fail to carry out most of these conformational changes but p18 causes the misalignment of additional residues involved in ATP binding and catalysis.,Structure of the Cdk6K-cyclin interface,(A) The PSTAIRE helix of Cdk6 is a central feature of the Cdk6K-cyclin inte

24、rface. The viewpoint shown corresponds approximately to that in B. Three sets of interactions are shown: hydrogen bonds between the Cdk6 main-chain preceding the PSTAIRE helix and the conserved LysGlu pair of K-cyclin (K106, E135); the conserved Ile 59 of Cdk6 inserts into a hydrophobic pocket in K-

25、cyclin; residues at the end of the PSTAIRE helix, one turn longer in Cdk4 and Cdk6 than in Cdk2, interact with residues on the N-terminal helix of K-cyclin and may play a role in cyclinCDK specificity. (B) Surface representation of p18Cdk6K-cyclin complex illustrating the minimal interactions betwee

26、n K-cyclin and the Cdk6 C lobe. p18 is colored yellow, the Cdk6 N lobe is cyan, the Cdk6 C lobe is blue, and the K-cyclin is purple. The only contacts between K-cyclin and the C lobe of Cdk6 arise from interactions with the N-terminal helix of K-cyclin. (C) Surface representation of Cdk2cyclinA in t

27、he equivalent orientation as that in A, showing significantly greater interactions between the C lobe of the Cdk2 and the cyclinA, giving rise to a much more extensive cyclinCDK interface.,The ATP-binding site of p18Cdkl6K-cyclin and Cdk2cyclinA. Active site residues implicated in ATP binding and ca

28、talysis are displaced in the p18 Cdk6K-cyclin complex relative to the active Cdk2cyclinA conformation. Cdk2 and Cdk6 were superimposed on their C lobes. Cdk6 is shown in cyan, p18 in yellow, Cdk2 in gray. Movement of active site residues is indicated by red arrows. p18 displaces the N lobe relative

29、to the C lobe, causing the hydrophobic residues (Ile 19, Val 27, Ala 41, Leu 152) that sandwich the adenine ring of ATP to move by up to 4.5 . The p18 inhibitor also distorts the edge of the active site via Phe 82, affecting hydrogen bonding interactions with the edge of the ATP ring. The related sh

30、ift of the PSTAIRE helix on the other side of the active site displaces an active site residue (Glu 61). The T loop of Cdk6 diverges from that of Cdk2 between Phe 164 and Val 181,The INK4-induced conformational changes in Cdk6 would interfere with the binding of ATP and polypeptide substrate and wou

31、ld also misalign any weakly bound substrates with respect to phosphotransfer.,The differences with respect to Cdk2cyclinA arise from contacts at the C terminus of the PSTAIRE helix caused by a three residue insertion in Cdk6 (residues 7072) resulting in one additional helical turn of 3.10 type. The

32、longer PSTAIRE helix of Cdk6 would collide with the N-terminal helix of cyclinA (Thr 70 and Phe 71 of Cdk6 would clash with Met 189 and Tyr 185 of cyclinA).The longer Cdk6 PSTAIRE helix is accommodated in K-cyclin by a small shift of the N-terminal helix relative to cyclinA and by the substitution o

33、f smaller amino acids (Asn 24 of K-cyclin instead of Tyr 185 of cyclinA). This results in contacts between Thr 70 and Phe 71 in the Cdk6 insertion and Asn 24, Ile 28, and Phe 32 of K-cyclin.,The structure of Cdk6 in the p18Cdk6K-cyclin complex differs from the structure of cyclinA-activated Cdk2 in

34、the orientation of the N and C lobes of the kinase and in the positions of the PSTAIRE helix and T loop. Compared to the Cdk2cyclinA complex, the kinase N and C lobes of the p18Cdk6K-cyclin complex are rotated by13 about an axis that passes through the back of the catalytic cleft and is approximatel

35、y perpendicular to the plane of the ATP that would bind there.,The rotation of the N lobe and the PSTAIRE helix away from the C lobe is also associated with the T loop not adopting the conformation needed for substrate binding and kinase activity. In the Cdk2cyclinA complex, the T loop makes multipl

36、e contacts with the PSTAIRE helix, the cyclin, and other parts of the C lobe. As these contacts would not be possible in p18Cdk6K-cyclin because of the misalignment of the lobes and PSTAIRE helix.,Despite the overall similarities in the N lobe-cyclin interactions between the inhibited p18Cdk6K-cycli

37、n complex and the active Cdk2cyclinA complex, there is a large difference in the position and orientation of the cyclin relative to the kinase C lobe. When the two complexes are compared by superimposing their CDK C lobes, K-cyclin is rotated by 40, and its center of gravity is shifted by 15 relativ

38、e to cyclinA. This is caused in part by the rotation between the kinase N and C lobes in p18Cdk6K-cyclin and in part by the rotation of the PSTAIRE helix relative to the N lobe. The shift in K-cyclin leads to a lack of significant contacts between K-cyclin and the C lobe and T loop of Cdk6 (Fig. 4B)

39、. In the Cdk2cyclinA complex, there are extensive contacts between the first cyclin repeat and the T loop and between the N-terminal helix and other parts of the Cdk2 C lobe (Fig. 4C; Jeffrey et al. 1995). In the inhibited Cdk6K-cyclin complex, there are no contacts with the T loop and only a few mi

40、nor contacts with the C lobe.,Conformation of Cdk6,Schematic representation of the different conformations of the CDK. CDKs undergo extensive conformational changes on binding of activating or inhibiting subunits. The major determinants of activity are the positions and conformation of the PSTAIRE h

41、elix and T loop, as well as the relative disposition of the kinase N and C lobes. The PSTAIRE helix adopts a position further away from the catalytic cleft in inactive CDKs (labeled as out) than in active CDKs (in). The PSTAIRE helix conformation correlates with the location of a conserved active si

42、te residue (Cdk2, Glu 51; Cdk6, Glu 61) either inside or outside the catalytic cleft.,例二：肽與鈣調(diào)蛋白(Calmodulin)的結(jié)合,鈣調(diào)蛋白是一個(gè)含有148 個(gè)氨基酸殘基的鈣結(jié)合蛋白，它與鈣依賴性 的信號通道的過程有關(guān)。鈣調(diào)蛋白可結(jié)合到多種蛋白中，像激酶鈣泵蛋 白，以及一些運(yùn)動(dòng)性蛋白等，以調(diào)節(jié)這些蛋白的活性。這些蛋白的鈣調(diào)蛋白結(jié)合區(qū)域大約由20 個(gè)相鄰的殘基組成，雖然它們的氨基酸順序變化很 大，但它們都有形成螺旋的強(qiáng)烈傾向，單個(gè)的和與多肽結(jié)合的鈣調(diào)蛋白 的結(jié)構(gòu)表明，多肽的結(jié)合引起了鈣調(diào)蛋白分子中大的構(gòu)象變

43、化。,Calmodulin (CaM) (an abbreviation for CALcium-MODULated proteIN) is a calcium-binding protein expressed in all eukaryotic cells. It can bind to and regulate a number of different protein targets, thereby affecting many different cellular function.,CaM mediates processes such as inflammation, meta

44、bolism, apoptosis, smooth muscle contraction, intracellular movement, short-term and long-term memory, nerve growth and the immune response. CaM is expressed in many cell types and can have different subcellular locations, including the cytoplasm, within organelles, or associated with the plasma or

45、organelle membranes. Many of the proteins that CaM binds are unable to bind calcium themselves, and as such use CaM as a calcium sensor and signal transducer. CaM can also make use of the calcium stores in the endoplasmic reticulum, and the sarcoplasmic reticulum肌漿網(wǎng). CaM undergoes a conformational c

46、hange upon binding to calcium, which enables it to bind to specific proteins for a specific response. CaM can bind up to four calcium ions, and can undergo post-translational modifications, such as phosphorylation, acetylation, methylation and proteolytic cleavage, each of which has potential to mod

47、ulate its actions. Calmodulin can also bind to edema factor toxin from the anthrax炭疽 bacteria.,與肽結(jié)合的鈣調(diào)蛋白的構(gòu)象變化,(a) 在自由狀態(tài)下鈣調(diào)蛋白是一個(gè)由兩個(gè)結(jié)構(gòu)域（紅色和綠色）組成的啞鈴狀分子。每個(gè)結(jié)構(gòu)域都有兩個(gè)與 鈣結(jié)合的EF 手（EF-hand） (b)在結(jié)合肽的狀態(tài)， 螺旋連接 子-helix linker 已被切開，分子的兩端緊靠在一起，并形成一個(gè)致密的 球狀復(fù)合物。每個(gè)結(jié)構(gòu)域的內(nèi)核結(jié)構(gòu)基本上沒有變化，結(jié)合肽形成一段 螺旋，每個(gè)結(jié)構(gòu)域內(nèi)含有兩個(gè)EF 手，每個(gè)EF 手結(jié)合一個(gè)鈣離子。這

48、兩個(gè)結(jié)構(gòu)域顯然在空間上是互相靠近的，并在 螺旋連接子的兩端分開。,當(dāng)鈣調(diào)蛋白與它的配基結(jié)合時(shí)實(shí)際上僅有5 個(gè)基團(tuán)改變了構(gòu)象。這是螺旋連接子中的5 個(gè)保守殘基，這5 個(gè)殘基發(fā)生了解旋并形成一個(gè)環(huán)區(qū)域，雖然在此環(huán)區(qū)域之后仍是一個(gè)螺旋，但其方向發(fā)生了很大的變化。第二個(gè)螺旋以完全不同的取向與第一個(gè)螺旋靠近多肽，構(gòu)象如此小的局部變化引起了如此大的結(jié)構(gòu)域之間的變化，這是由配基引起蛋白變化的最大的一種蛋白。,There are 4 helix-loop-helix (EF-hand) motifs,Upon binding of some target sequences to calmodulin, th

49、e two domains come together to form a hydrophobic channel,Calmodulin is only active when all four sites are filled.,The binding of the four Ca+ ions is cooperative,Mechanism: Calcium is bound via the use of the EF hand motif, which supplies an electronegative environment for ion coordination. After

50、calcium binding, hydrophobic methyl groups from methionine residues become exposed on the protein via conformational change. This presents hydrophobic surfaces, which can in turn bind to Basic Amphiphilic兩性的 Helices (BAA helices) on the target protein. These helices contain complementary hydrophobic

51、 regions. The flexibility of Calmodulins hinged region allows the molecule to wrap around its target. This property allows it to tightly bind to a wide range of different target proteins.,Calmodulin wraps around a target domain of some proteins only after binding Ca+. Other proteins have bound calmo

52、dulin as part of their quaternary structure, even in the absence of Ca+. In either case, a conformational change induced by binding of Ca+ to calmodulin alters the activity of the target protein.,CAM is highly conserved across all eukaryotes,Once in the cytosol, the Ca+ typically binds to a small pr

53、otein, calmodulin. Once four Ca+ bind to calmodulin, it activates specific proteins inside the cell, such are certain protein kinases.,Ca2+-independent binding of calmodulin to its target proteins by contrast, uses a consensus sequence (IQxxxRGxxxR) called an IQ motif. Some proteins bind calmodulin through their IQ motifs at low concentrations of Ca2+ . A subsequent increase in the Ca2+ concentration induces a conformational change in the bound calmodulin, regulating the activity of the target protein.,How does Calmodulin bind to proteins?,A transformation of the corresponding I