生物大分子结构与功能第5章蛋白质的柔性结构

1、第五章：第五章： 蛋白质的柔性结构蛋白质的柔性结构天然折叠的蛋白分子往往不是以一种构象状态存在的。在晶体结构中我们看到的往往仅是一种状态的构象，它是蛋白质分子的一个平均构象。实际上，蛋白质分子始终是处于一种呼吸的状态。蛋白质结构中所有的原子都在运动，这些原子的运动通常是随机的，但有时可以是集合性的运动。这种集合性的运动引起分子中的原子团在相同的方向上产生运动，造成蛋白质分子中的侧链可以从一种构象转化为另一种构象。某些环区域也并不总是固定在一种单一的构象状态，螺旋也可以互相产生滑动，完整的结构域之间也可以改变它们的堆积接触以打开或关闭结构域之间的距离。通常这些运动都是比较小的，有时小到仅有1/1

2、0 的运动，但有时这种集合性运动可以很大，大到足以具有重要的生物学意义。这样大的集合性运动在x-射线晶体学研究中所表现出来的是电子密度的水平低，甚至在某些情况下看不到电子密度的存在。产生这样的运动的区域通常在晶体学中被表述为柔性(flexibility)运动或无序运动或无序(disorder)。核磁共振实验对于这样的区域的测定可以作为一种互补，因为核磁共振实验可测出这些区域的各种不同的构象，通过理论计算也可以计算出这些分立的或集合性运动这叫作分子动力学模拟。分子动力学模拟。分子动力学模拟已经表明，每一个分立的残基的集合性运动仅在皮秒(10-12 秒)的时间尺度，而环区域的运动在纳秒(10-9

3、秒)的尺度。这种运动对于许多蛋白质的功能是非常重要的。象电子转移和配基结合或释放反应均以这样的时间尺度发生，并通常伴随着蛋白质原子的运动。例如，当肌红蛋白呼吸时，通道在溶剂和被包埋在分子内部的结合部位之间打开，以允许氧原子在纳秒的时间尺度范围与肌红蛋白结合或者释放出来。除了蛋白质中原子小的呼吸运动之外，在分子的功能态之间也会发生大的构象变化。不同的ph 和配基的存在和缺失以及环境中的微小的变化，往往能够稳定蛋白质的不同构象态。这些构象变化可以是活性部位的氨基酸侧链的构象变化到环区域的运动等。同时结构域之间的相对取向和寡聚蛋白中四级结构也会发生变化，这样的运动通常是与功能相关的。例如酶的催化，肌

4、肉运动和能量转换等。真核细胞周期的五个相(g0, g1, s,g2 和m 相)例例1：细胞周期调节蛋白激酶的构象变化：细胞周期调节蛋白激酶的构象变化在s 相，dna 合成，dna 被复制并且染色体翻倍。在m 相，有丝分裂父代细胞的二倍化染色体通过有丝分裂的纺锤体分开，这样每个子代细胞接收到相同组分的染色体。 一个细胞分裂的完整周期是m g1 s 和g2。通过g1 s 和g2 相，细胞的蛋白质合成机器大分子和细胞器被建立起来，同时细胞的体积增大。在有丝分裂时，染色体和细胞质被分为两个相等的部分。此外，还有一个静止相g0 相，发生在细胞的未分裂状态。由cyclin 的降解对cdks 的调节细胞周期

5、的进程取决于一系列的叫作cyclin依赖的蛋白激酶(cyclin-dependent protein kinases, cdks)的连续激活作用。图中显示两种类型的cyclin-cdk 复合物，一种是触发s 相，另一种触发m 相。在这两种情况下cdk 的激活需要与cyclin 的结合，它们的非活性依赖于cyclin的降解在脊椎动物的细胞中至少有四种不同cdks ，控制着细胞周期的活动。不同的催化亚基都属于密切相关的基因家族，不同的cdk 的一个或几个cyclin 分子都是该家族的成员。cdks 作为一个延迟开关，控制着从g1 相到s 相从g2 相到m 相以及所有构成细胞周期的其它步骤人的体细胞

6、中调制dna复制的cdk2-cyclin a的结构提供了详细的结构信息以及cyclina 激酶的功能。cyclin a 的功能片段的晶体结构于1995 年由louise johnson 实验室解出，非活性的cdk2 的结构1993 年已由sung-hokim 实验室解出，活性的cyclin a 片段与cdk2 复合物的结构也于1995年由nicola pavletich 实验室解出。通过对这些结构的分析和结构比较，揭示出cyclin a 是如何结合到cdk2 上，并如何在cdk2 的活性部位引起大的构象变化，使cdk2 蛋白质从一种非活性的状态转变为活性状态的。而在此过程中 cyclin a

7、的结构则没有发生构象变化cyclin a 依赖型激酶cdk2 的结构cyclin a依赖型激酶cdk2 有两个结构域，n-端结构域由一段螺旋折叠片组成，在螺旋中pstaire的氨基酸顺序（红色）在所有的cdks 蛋白激酶中都是高度保守的；c-端结构域主要由螺旋组成，并含有一段柔性的环区域称作t-loop （黄色）环区域，含有一个苏氨酸残基，在完全活性的酶中该苏氨酸残基被磷酸化。cyclin a 的结构cyclin a 活性片段残基173-432 的结构由两个非常相似的结构域构成。每个结构域都由五段螺旋组成。该活性片段的作用几乎与完整的cyclin a 分子的作用相同。在所cyclin a 中第

8、一个结构域具有十分保守的氨基酸顺序被称作cyclin-box ，而第二个结构域的氨基酸顺序则不相同。因此尽管cyclin a 片段的两个结构域结构几乎相同但仅有一个cyclin-box 序列。活性的cdk2 蓝色和cyclin a 复合物的结构在cyclin a-cdk2 复合物中，主要是cyclin a 与cdk2 中的pstaire螺旋和t-loop 相互作用，cyclin-box 螺旋2-6 与cdk2 的pstaire 深红色螺旋和t-loop 黄色作用。在该复合物中，cyclin a 的结构与单个cyclin a 是相同的，而cdk2 的结构则发生了很大的构象变化，包括petaire

9、 螺旋t-loop 和atp 的结合部位（浅红色）。整个n 端结构域相对于c端的结构域的取向发生了变化，此外pstaire 螺旋向cdk2 的活性部位靠近并旋转了90， 以便主要的催化残基glu 51 指向裂缝，而不是象在单个的cdk2 结构中那样远离此裂缝。cdk2 与cyclin a 结合的构象变化一旦与cyclin a结合，pstaire 螺旋橙色转动90， 并改变位置以使得glu 51变为指向活性部位。该pstaire螺旋的一些主链原子由于这种一致性运动位移了8.0 的距离。t-loop发生了大的位置重排某些环区域上的氨基酸残基的位移可达20 。左图：在非活性态，pstaire 螺旋红

10、色的取向使glu 51 指向远离atp 的结合部位，而t-loop 封住了与底物的结合部位，以阻止蛋白结合到cdk2 上。右图： 在活性的cyclin a-cdk2 复合物结构中，pstaire 螺旋发生了重新定向以使得glu51 残基指向活性部位并与另一个与催化有关的残基lys 33 形成盐键，t-loop改变了构象并与另一个残基asp 145 一起与活性部位中的镁离子配位，此时底物的结合部位被打开，蛋白可以结合底物。cyclin-cdk2 复合物可以磷酸化ser/thr 残基并进而激活所结合的蛋白。在自由cdk2 t-loop结构中的螺旋在复合物中变为一条 链。cyclin 结合引起结合引

11、起cdk2 的结构变化的结构变化(a)活性部位位于n 端结构域（蓝色）和c 端结构域（紫色）之间的裂缝中，在非活性状态此活性部位被t-loop 所封闭。(b)在活性的cyclin 结合状态的cdk2结构中,tloop的结构发生了变化,活性部位被打开,thr 160 适合于磷酸化.由于cyclin a 的结合所引起的cdk2 的构象变化,不仅暴露了活性部位的裂缝以使atp 和蛋白底物能够与之结合,而且活性部位的残基发生了重排,以形成酶的催化作用。此外thr 160 被暴露出来，并准备被磷酸化以提高催化活性。简而言之蛋白质结构的柔性调节了cdk 家族的酶活性，因而控制了细胞周期。structura

12、l basis of inhibition of cdk-cyclin complexes by ink4 inhibitorsphilip 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, usagenes dev. 2000 14: 3115-3125th

13、e cyclin-dependent 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 kina

14、se, and by the 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 re

15、duced in size. 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

16、 of p18cdk6k-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

17、 lie on opposite 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

18、side of the kinase. (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 comp

19、lex has a large 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 inactivecdk6 structure, the n and c lobes are rotated 13away from each other, resulting in the misalignment of atp-binding residues. the

20、 n-lobe pstaire 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;

21、 russo et 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 ofthe catalytic cleft (residues 99102), which would hydrogen bond to atp, is displaced by several ngstroms.the cdk2cyclina structure (je

22、ffrey et 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 bycorrectly aligning certain active site residues and reorganizing the polypeptide substrate binding site. in the p18cdk6k-c

23、yclin complex, not only does the k-cyclin fail to carry out most of these conformational changes but p18 causes the misalignment of additional residues involvedin atp binding and catalysis.structure of the cdk6k-cyclin interface(a) the pstaire helix of cdk6 is a central feature of the cdk6k-cyclin i

24、nterface. theviewpoint shown corresponds approximately to that in b. three sets of interactions are shown: hydrogen bonds between the cdk6main-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 betwe

26、en k-cyclin and the cdk6 c lobe. p18 is colored yellow, thecdk6 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 bindingand cata

28、lysis are displaced in the p18cdk6k-cyclin complex relative to the active cdk2cyclina conformation. cdk2and cdk6 were superimposed on their clobes. cdk6 is shown in cyan, p18 in yellow, cdk2 in gray. movement of activesite residues is indicated by red arrows.p18 displaces the n lobe relative to the

29、clobe, causing the hydrophobic residues (ile19, val 27, ala 41, leu 152) that sandwichthe adenine ring of atp to move by up to4.5 . the p18 inhibitor also distorts theedge of the active site via phe 82, affectinghydrogen bonding interactions with theedge of the atp ring. the related shift ofthe psta

30、ire helix on the other side of theactive site displaces an active site residue(glu 61). the t loop of cdk6 diverges fromthat of cdk2 between phe 164 and val 181the ink4-induced conformational changes in cdk6 would interfere with the binding of atp and polypeptide substrate and would also misalign an

31、y 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 longer pstaire helix

32、 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 of smaller amino acid

33、s (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 the orientation of th

34、e n and c lobes of the kinase and in the positions of the pstaire helix and t loop. comparedto 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 approximately perpendicular to the

35、 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 multiple contacts with the pst

36、aire 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-cyclin complex and the active

37、 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 gravityis shifted by 15 relative to cyclina. this is cau

38、sed 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-cyclinleads to a lack of significant contacts between k-cyclin and the c lobe and t loop of cdk6 (fig. 4b). in the cdk2cyclina compl

39、ex, 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 minor contacts with the c lo

40、be.conformation of cdk6schematic representation of the different conformations of the cdk. cdks undergo extensive conformational changes onbinding of activating or inhibiting subunits. the major determinants of activity are the positions and conformation of the pstairehelix and t loop, as well as th

41、e relative disposition of the kinase n and c lobes. the pstaire helix adopts a position further awayfrom the catalytic cleft in inactive cdks (labeled as out) than in active cdks (in). the pstaire helix conformation correlates withthe location of a conserved active site residue (cdk2, glu 51; cdk6,

42、glu 61) either inside or outside the catalytic cleft.例二：肽与钙调蛋白例二：肽与钙调蛋白(calmodulin)的结合的结合钙调蛋白是一个含有148 个氨基酸残基的钙结合蛋白，它与钙依赖性的信号通道的过程有关。钙调蛋白可结合到多种蛋白中，像激酶钙泵蛋白，以及一些运动性蛋白等，以调节这些蛋白的活性。这些蛋白的钙调蛋白结合区域大约由20 个相邻的残基组成，虽然它们的氨基酸顺序变化很大，但它们都有形成螺旋的强烈倾向，单个的和与多肽结合的钙调蛋白的结构表明，多肽的结合引起了钙调蛋白分子中大的构象变化。calmodulin (cam) (an abb

43、reviation 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, metabolism, apoptosis, smooth mu

44、scle 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 organelle membranes. many of

45、 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肌浆网. cam undergoes a conformational change upon binding to calciu

46、m, 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 modulate its actions. calmoduli

47、n can also bind to edema factor toxin from the anthrax炭疽 bacteria.与肽结合的钙调蛋白的构象变化(a) 在自由状态下钙调蛋白是一个由两个结构域（红色和绿色）组成的哑铃状分子。每个结构域都有两个与钙结合的ef 手（ef-hand） (b)在结合肽的状态， 螺旋连接子-helix linker 已被切开，分子的两端紧靠在一起，并形成一个致密的球状复合物。每个结构域的内核结构基本上没有变化，结合肽形成一段螺旋，每个结构域内含有两个ef 手，每个ef 手结合一个钙离子。这两个结构域显然在空间上是互相靠近的，并在 螺旋连接子的两端分开。当钙

48、调蛋白与它的配基结合时实际上仅有5 个基团改变了构象。这是螺旋连接子中的5 个保守残基，这5 个残基发生了解旋并形成一个环区域，虽然在此环区域之后仍是一个螺旋，但其方向发生了很大的变化。第二个螺旋以完全不同的取向与第一个螺旋靠近多肽，构象如此小的局部变化引起了如此大的结构域之间的变化，这是由配基引起蛋白变化的最大的一种蛋白。there are 4 helix-loop-helix (ef-hand) motifsupon binding of some target sequences to calmodulin, the two domains come together to form a

49、 hydrophobic channelcalmodulin 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 calcium binding, hydrophobic methyl gr

50、oups from methionine residues become exposed on the protein via conformational change. this presents hydrophobic surfaces, which can in turn bind to basic amphiphilic两性的 helices (baa helices) on the target protein. these helices contain complementary hydrophobic regions. the flexibility of calmoduli

51、ns 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 calmodulin as part of their quaternary stru

52、cture, 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 eukaryotesonce in the cytosol, the ca+ typically binds to a small protein, calmodulin. once four ca+ bind to

53、 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 concentrat

54、ions of ca2+ .a subsequent increase in the ca2+ concentration induces aconformational change in the bound calmodulin, regulating the activity of the target protein. how does calmodulin bind to proteins?a transformation of the corresponding iq12 region of scallop muscle myosin-ii. martin & bayley

55、, 2002. disease states characterized by unregulated growth, such as cancer, are correlated with elevated levels of ca+-bound camsome anti-calmodulin drugscams hydrophobic surface can bind different aromatic moleculescalmodulin 1 (phosphorylase kinase) is a protein that in humans is encoded by the ca

56、lm1 gene.calmodulin 1 is the archetype of the family of calcium-modulated (calmodulin) proteins of which nearly 20 members have been found. they are identified by their occurrence in the cytosol or on membranes facing the cytosol and by a high affinity for calcium. calmodulin contains 148 amino acid

57、s and has 4 calcium-binding motifs. its functions include roles in growth and the cell cycle as well as in signal transduction and the synthesis and release of neurotransmitters. calmodulin 1 has been shown to interact with akap9,3 trpv1, 4 androgen receptor, 5 iqgap167 and ppef1calmodulin 2 (phosph

58、orylase kinase) is a protein that in humans is encoded by the calm2 gene, calm2 has been shown to interact with akap9calmodulin3 (phosphorylase kinase) is a protein that in humans is encoded by the calm3 gene calmodulin-like protein 1calmodulin-like protein 2calmodulin-like protein 3calmodulin-like

59、protein 4calmodulin-like protein 5calmodulin-like protein 6 例三：例三：serpin 抑制丝氨酸蛋白酶的作用抑制丝氨酸蛋白酶的作用1 抗胰蛋白酶属于在血浆中发现的丝氨酸蛋白酶抑制剂家族的成员，统称叫作serpin 。该家族的其它成员是抗凝血酶(antithrombin) 和血浆酶原激活子抑制剂(plasminogen activator inhibitor, pai) ，两者都是血液凝集连锁反应的调节子。所有的serpin 分子都是同源的，且都有非常相似的三维结构。这些serpin 分子在各种不同状态下的一般折叠是相同的，但是柔性环区

60、域的位置则变化很大。卵白蛋白的serpin 折叠由三个反平行的 折叠a b 和c 构成。红色区域是serpin 相应的活性部位，该部分像一个结构的把手一样穿出卵白蛋白，可把非切断形式的卵白蛋白结构考虑为规范的serpin 的结构。 折叠片a ( -sheet a) 有五段 链，柔性的环区域起始于折叠片a 的链5 的末端，然后形成一段位于分子顶端的螺旋，并靠近 折叠片c 的边缘，最终在折叠片b 的起始链结束。三种状态下活性部位环区域红色的图解三种状态下活性部位环区域红色的图解活性形式下，环区域从分子的主要部分穿出，与丝氨酸蛋白酶的活性部位发生作用(b) 作为抑制蛋白酶的结果，serpin 分子在环区域的