摘要
蛋白质是生物体各种生理过程的主要执行者。在生理条件下,不同蛋白质特定序列的氨基酸长链能够折叠成特定的三维结构,行使其生理功能。因此漫长的进化过程必然使蛋白质的结构具有功能意义。随着蛋白质结构测定技术的日益成熟,越来越多的蛋白质三维结构被解析出来,这些结构信息揭示了很多重要生理过程的分子机制,也为药物的研制提供了关键的理论基础。然而,随着当前结构生物学和生物物理化学的逐步深入发展,人们发现仅有蛋白质三维结构的信息往往不足以理解其生物学功能。这是因为结构解析给出的是单一的一个静态结构(如晶体结构),而蛋白质在行使其生理功能时往往会发生构象变化,产生一系列瞬态构象。换言之,溶液中的蛋白质作为一种软物质可以在其平衡构象附近振荡,甚至可以远离平衡构象,而晶体结构得到的只是蛋白质势能面上的一个点。要全面理解蛋白质的功能和揭示生理过程的分子机制我们不但需要了解蛋白质的静态结构,更需要了解蛋白质的结构柔性和各个瞬态构象之间的转换,即动力学性质。因此需要在传统的结构生物学研究中加入时间的维度,将结构-功能关系扩展到结构-动力学-功能关系的研究。蛋白质动力学行为的时间尺度很宽,其涵盖范围从快速而局部性的原子波动到慢速而整体性的诸如蛋白质折叠的构象变化。许多蛋白质分子实现其生物功能时都需要发生大幅度的构象变化,比如通过线团(loop)结构的摆动和二级结构单元的运动实现对活性位点构象的调整或者对底物分子进行识别,这类运动的时间尺度大致在纳秒到微秒(10-9到10石秒)级别,运动幅度一般在1到5A。更复杂的运动则往往需要通过结构域的整体运动来完成,这种结构域间的相对转动和平动的时间尺度大致在微秒到毫秒(10-6到10-3秒)级别,运动幅度一般会达到5到10 A左右。各时间尺度上的运动从原子振动到大幅度的蛋白整体运动会发生相互耦合,使得对蛋白质动力学和功能关系的研究变得越发复杂。
和实验手段相比,计算机模拟方法在研究蛋白质动力学方面具有独特的优势。只要有一个高精度的蛋白质实验结构作为起点,计算方法就能够“全面”地描述蛋白质的动力学(以分子动力学模拟为代表),我们可以跟踪单个蛋白质分子在每个时刻每个原子的精确位置和相应的能量。尽管蛋白质不同的构象状态和它们之间的转换速率可以通过实验方法检测,但是对构象之间转换路径上各个状态的高精度描述却是实验无法达到的,因为这些状态都是寿命很短,出现几率很低的高能构象。计算方法则可以克服这些局限。另一方面,计算机模拟中包含了体系粒子之间的所有相互作用力和相应的能量,因此尽管实验方法可以告诉我们蛋白质怎样运动,计算方法却可以告诉我们蛋白质为什么这样运动。目前计算方法的主要缺陷是传统的分子动力学方法能够模拟的动力学时间尺度是皮秒到纳秒范围,对蛋白质的一些慢速(微秒到毫秒)构象变化过程的模拟则非常困难。为了克服这一困难,计算生物学家发展了多种方法和策略来简化力场或施加外力以加速构象变化过程。
我们选取了三个大小构造不一但都能够发生大尺度构象变化的蛋白质体系来研究结构单元和构象变化之间的关系。这些体系包括含有四个结构域的ABC转运体蛋白输入体BtuCD和输出体MsbA、PSD-95蛋白中含有两个结构域的串列体PDZ12以及脂质化修饰的膜融合SNARE蛋白Ykt6。在实际应用过程中,除了常规分子动力学模拟之外我们也使用了靶向分子动力学模拟和正则模式分析的方法,分别通过外加力场加速构象变化以及分析局域势能面形态的方式来获得蛋白质体系大幅度构象变化的信息。
ABC转运体蛋白超家族在生物体中能够利用ATP分子结合和水解的能量实现底物分子跨细胞膜的转运。转运的过程需要发生大幅度的构象变化,在面向内(inward-facing)构象(孔道向内打开向外关闭)和面向外(outward-facing)构象(孔道向内关闭向外打开)之间切换。ABC转运体蛋白由两个跨膜结构域与两个核苷结合结构域组成,其运作的核心机制在于其跨膜结构域与核苷结合结构域的运动耦合。前者内部底物转运孔道的开闭和后者在ATP分子的结合和水解时表现出的二聚和解离贯穿着整个底物转运的过程。就此,我们以BtuCD输入体和MsbA输出体为研究对象采用了正则模式分析和靶向分子动力学模拟的方法进行了研究。
维生素B12输入体BtuCD蛋白具有20个跨膜螺旋,孔道的大部分由一对TM5螺旋组成,而跨膜结构域则通过L线团结构与核苷结合结构域相连。我们发现不论是从面向外到面向内还是从面向内到面向外的构象变化过程,核苷结合结构域的运动总会通过L线团与转运孔道细胞内侧端的运动同步进行。在从面向外构象到面向内构象的变化中,核苷结合结构域的打开会导致L-线团的打开,继而引起孔道主体TM5螺旋细胞内侧端的打开。其逆过程也基本相似。同时,我们也发现正逆向的变化过程并没有经历同一条构象转化路径,而是存在着一定的差异。在正方向过程中,TM5螺旋内侧端会平稳地经由对称运动的方式打开,而逆方向过程则往往会有一段更明显的非对称运动阶段。我们的研究揭示了BtuCD蛋白各个结构域构象变化之间的耦合关系,给出了BtuCD输入体转运机制的详细图像,并特别指出了从面向内到面向外的变化过程会经历一个非对称的中间状态。
MsbA蛋白是细菌中脂质A和脂多糖分子的输出体。它与多类药物抗性蛋白P糖蛋白序列高度同源且结构相似,对它的转运机理的研究将为癌症治疗药物的设计提供直接的参考依据。通过研究其面向外到面向内的构象变化过程我们再度证实了核苷结合结构域与跨膜结构域的耦合机制,但其具体的构象变化过程与BtuCD体系完全不同。核苷结合结构域的打开变化首先会经过序列高度保守的X线团抵达孔道细胞内侧端的四螺旋束,然后随着四螺旋束的打开再经由跨膜螺旋TM6将细胞内侧的变化情况向细胞外侧传递。在转运过程中各结构单元紧密配合,由核苷结合结构域开始经由X-线团、四螺旋束以及跨膜螺旋TM6直到细胞外侧端,这些结构单元共同参与了整个变化过程。这一系列过程也首次指出了在输出体蛋白中序列高度保守的X线团在传导结构域构象变化方面的重要作用。对MsbA构象变化过程的研究证实了长期以来人们关于ABC转运体蛋白转运机理的一个假设,即蛋白质的大范围构象变化是由核苷结合结构域的构象变化引发的,而构象变化信号在结构域之间的传递呈现出清晰的时间空间顺序。
与ABC转运体蛋白相比,PSD-95蛋白的N端PDZ12串列体的结构较为简单。它含有两个球状的PDZ结构域PDZ1和PDZ2,之间通过一段序列保守的五肽连接片段串列而成。两个PDZ结构域都能够特异性地与目标蛋白的C末端结合,作为信号传导、膜受体集聚以及细胞极性维持等生理过程中的重要一环。实验发现,在未结合肽段时,PDZ12串列体的两个PDZ结构域之间的取向相对固定,而在结合肽段之后则不再具有这种固定取向。这预示着PDZ12串列体在结合肽段后发生了大幅度的构象变化。我们利用分子动力学模拟直接比较了无肽段结合状态和肽段结合状态,结果发现12纳秒的模拟很好地表现出肽段结合对PDZ1和PDZ2结构域之间相对运动的影响。无底物结合态PDZ12的PDZ结构域相对取向夹角的取值范围较小,约有50°,相比之下,肽段结合后夹角的摆动幅度可以达到150°。也就是说,肽段的结合加大了PDZ结构域相对取向的运动自由度。从能量变化的角度来讲,自由肽段与蛋白质分子的结合会降低肽段的自由度,从而降低整个过程的熵变不利于反应的进行。PDZ12串列体则显示其两个PDZ结构域和五肽连接片段之间存在着一套联动机制,这使它能够在肽段结合后通过提高结构域间相对运动的自由度来弥补肽段结合带来的熵损失,更利于蛋白与肽段的结合。通过结构域之间的相对运动来调控蛋白和配体的相互作用强度是一种全新的蛋白质动力学与功能关系的模式,我们预计这一调控方式具有在多结构域模块的支架蛋白中普遍存在。
介导膜融合的SNARE蛋白Ykt6的结构更为简单,由一个球状的longin结构域和一段60个氨基酸残基左右的SNARE核心区两部分组成,但它在生理过程中依然会发生复杂的构象变化。其结构中,SNARE核心区在囊泡运输过程囊泡与目标膜层的对接和融合中起着核心作用,而longin结构域则有着调控SNARE核心区构象的功能。实验发现,在体系中加入DPC脂质分子会使得Ykt6蛋白整体处于关闭构象,类似生理状态下法尼酰化Ykt6蛋白的自抑制状态。这时,SNARE核心区包裹在longin结构域外侧,在longin结构域和SNARE核心区的界面上形成疏水槽用以容纳脂质分子疏水侧链。而当体系中没有脂质分子时,Ykt6蛋白则呈现出比较广泛的运动范围,在多个构象之间运动切换。通过对含DPC分子的体系(DPC-Ykt6),无底物结合态体系(apo-Ykt6)和法尼酰化体系(far-Ykt6)各50纳秒的模拟,我们发现DPC分子能够用其亲水端和疏水端同时与Ykt6蛋白作用,从而稳定蛋白质分子的结构并使之只在局部构象空间中运动。相比之下,apo-Ykt6体系由于没有疏水链的存在疏水槽发生坍缩形变,整个蛋白的构象分布也更广,表现出与实验结果相似的多构象切换特点。far-Ykt6体系则保持了DPC-Ykt6体系主要的结构特点,在构象空间上显示出与DPC-Ykt6体系相似的集中分布,说明体系在50纳秒的分子动力学模拟后已经达到收敛。蛋白质分子处在稳定的构象状态并与apo-Ykt6体系存在构象重叠。我们的结果解释了DPC分子对Ykt6蛋白构象的稳定机制,同时也证明了DPC-Ykt6结构能够很好地代表法尼酰化Ykt6蛋白的结构。以往人们公认脂质化修饰的功能是帮助蛋白在膜上定位,而脂质化修饰主动调节蛋白构象变化则是一种全新的功能模式。我们发现脂质化修饰对Ykt6构象变化的调控符合蛋白质构象调控中的“构象选择”模型,即蛋白质自身“预存”存在多个构象,构象的调控是通过与其他蛋白或配体相互作用,移动构象分布平衡来实现的。
我们的工作表明蛋白质分子的构象柔性和动力学性质具有重要的功能意义,长期进化的结果使蛋白质具有高度精密的整体协调性来调节蛋白质分子的大幅度构象变化,并且这种调节模式具有多样性的特点。BtuCD和MsbA转运体蛋白能够协调自身各结构单元,通过核苷结合结构域与跨膜结构域的耦合机制进行底物分子的运输。PDZ12串列体能够协调肽段结合位点和五肽连接片段的动态运动性质,通过提高PDZ结构域之间的相对运动自由度弥补结合肽段造成的熵损失。Ykt6蛋白则能够对脂质长链进行响应,调控自身在构象空间上的分布。此外,我们的工作也表明分子动力学模拟是研究蛋白质分子大幅度构象变化非常有力的工具,而靶向分子动力学模拟和正则模式分析也是非常有效的研究手段。随着计算机运算能力的发展和算法的进一步优化,计算模拟的作用必将更为强大,同时也会与实验手段一起在揭示蛋白质分子构象变化规律上发挥更大的作用。
Proteins play important roles in all kinds of biological process. They have specific 3-dimensional structures which are folded from peptides with certain amino acid sequences under physiological conditions. The structures get functional meaning in evolution. Nowadays with the development of protein structure determination methods, more and more high-resolution protein structures are obtained. They have revealed the molecular details of many important biological processes and are the theoretical bases of drug discoveries. However, new problems come out with the progress of structural biology and biophysical chemistry. People find that a 3-dimensional structure (such as a crystal structure) usually represents a static state, but proteins are flexible and always undergo conformational changes in physiological processes, producing a series of transient states. In other words, a protein in solution can be viewed as a soft material oscillating near its equilibrated state, or even going far from the equilibrated state, but a crystal structure is only a single point on the potential surface. To have a deeper understanding of protein function, we should not only determine the static structures, but also try to reveal the dynamics, including the structural flexibility and the transitions between different conformational states. So it becomes very necessary to introduce time dimension to conventional structural biological studies and extend the structure-function relationship to the structure-dynamics-function relationship. The dynamics of proteins cover a very broad time scale, ranging from fast and localized atomic fluctuations to slow and global motions such as protein folding. Large-scale motions are always related to protein functions. For example, active site conformation adaptation may be consisted of loop motions and secondary structure motions, spanning a characteristic time scale from nanoseconds to microseconds (10-9-10-6 s) and a range of amplitudes from 1 to 5 A. A more complicated conformational transition involves domain motions. It takes place in a time scale from microseconds to milliseconds (10-6-10-3 s) and falls into a range of amplitudes from 5 to 10 A or so. However, different types of motions from fast fluctuations to slow global motions are interdependent and coupled to one another. This complicates the studies on protein dynamics.
Computational simulation has its own advantages over experiments in protein dynamics studies. It can start with a high-revolution structure and give a "full-scale" description of the dynamics of the protein (molecular dynamics, as an example). It traces the position and velocity of every atom at every moment and interprets every state on the conformational transition pathway with high accuracy, while experiments often ignore these states as they are of high energy, ephemeral and sparsely-populated. Computational simulation also involves all the particle interactions and monitors energy variations. When experiment tells us how protein moves, computational simulation can tell us why it moves. At present, molecular dynamics are limited in time scale from picoseconds to nanoseconds, still far from some slow motions falling in the range of microseconds to milliseconds. But there are different methods and strategies to overcome this difficulty, such as replacement of all-atom force field with a coarse-grained one or introduction of external force to accelerate the conformational transition.
We select three systems, all of which undergoes large-scale motions under physiological conditions, to study the structure-dynamics-function relationship. These systems have very different structures, including ABC transporters such as importer BtuCD and exporter MsbA (with four domains), PDZ12 tandem of PSD-95 (witktwo domains) and SNARE protein Ykt6 (with one domain). We use conventional molecular dynamics simulation, together with targeted molecular dynamics simulation and normal mode analysis to study the large-scale conformational changes of these systems.
ABC transporters constitute one of the largest superfamily in organism and they use the energy from nucleotide binding and hydrolysis to translocate substrates across cell membranes. They undergo large-scale conformational changes, switching between inward-facing and outward-facing conformation during the translocation process. ABC transporters are made up of at least four domains, two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). ATP binding and hydrolysis drive the opening and closing motion of nucleotide-binding sites and the motions in NBDs are proposed to be coupled to changes of the translocation pathway between TMDs. Here we focus on import system BtuCD and export system MsbA to study the translocation mechanism.
Vitamin B12 importer system BtuCD has twenty transmembrane (TM) helices and TMDs are connected to NBDs by L-loops in TMDs in crystal structures. We found that in the transitions between the inward-facing and the outward-facing conformation, NBD motions are always coupled with TMD motions at the cytoplasmic side through L-loops. In the outward-facing forward to inward-facing transition, the opening of nucleotide-binding sites stretches L-loops and expands the cytoplasmic gate. This coupling mode is similar in the backward transition. We also found that the forward and backward transitions are not the same. The cytoplasmic gate moves more symmetrically in the forward process, while its motion becomes more asymmetrical in the backward process. Our work reveals a detailed coupling mode of the domains in BtuCD and indicates an asymmetrical intermediate state in the transition from the inward-facing to outward-facing conformation.
MsbA is a bacterial lipid efflux and it is homology to P-glycoprotein, the multidrug resistance ABC exporter in human. Studies on the translocation mechanism of MsbA would be very instructive to cancer drug design. We again found the coupling between TMDs and NBDs by simulating the outward-facing to inward-facing transition, but the details in MsbA are totally different from BtuCD. The conformational change follows a clear spatio-temporal order. The opening of NBD dimer interface is the first event and the highly conserved X-loop transmits the changes to the cytoplasmic tetra-helix bundle. Then the breaking of tetra-helix bundle network induces large-scale rearrangement of the cytoplasmic side and TM6 helix brings these changes to the periplasmic side. Different parts of the structure, such as NBD, X-loop, tetra-helix bundle and TM6 helix closely cooperate and together buildup the signal transition pathway. The sequential transition process points out the functional importance of the highly conserved X-loop and approves the hypothesis that the large-scale conformational change of the transporter is triggered by the motion of nucleotide-binding sites.
PDZ12 tandem contains two N-terminal PDZ domains of PSD-95 and the domains are closely connected by a conserved peptide linker of five amino acids. Both PDZ domains can bind specifically to a short peptide at the extreme C terminus of target proteins. This is crucial for PSD-95 to organize signal transduction complexes, cluster membrane receptors, and maintain cell polarities. In PDZ12 tandem, the two PDZ domains have limited freedom of rotation relative to each other, but this restrained interdomain orientation disappears when the tandem is in complex with its binding peptide. This means ligand binding increases the inter-PDZ mobility remarkably. We performed two independent molecular dynamics simulations of 12 ns for the ligand-free and-bound forms of PDZ12 and found their dynamic properties are remarkably different. In the peptide-bound system, the interdomain orientation is not restrained and the protein samples a much larger conformational space than the free form. This means the conformational flexibility of PDZ12 tandem increases dramatically upon peptide binding by losing the relative interdomain orientation. The case of PDZ12 represents a new mode of "induced-fit" effect. The dynamics variation upon ligand binding is attributed to the changes of interdomain mobility in addition to the local induced fit within an individual domain. By utilizing this domain cooperativity, PDZ12 gains extra compensatory conformational entropy favoring its target binding. We anticipate that this may be one of the general strategies adopted by multidomain scaffold proteins to facilitate its target recognition.
SNARE Ykt6 is an essential protein involved in multiple membrane fusion reactions. It is very flexible and can change between open state and closed state. It is consisted of an N-terminal longin domain, a conserved central 60-70 amino acid "SNARE core" and a C-terminal "CCAIM" motif. The SNARE core mediates the specific targeting and fusion of different classes of transport vesicles to their distinct membrane destinations, and the longin domain is capable of regulating the activity of SNARE core. The unlipidated Ykt6 shows multiple interconverting conformational states in solution and the states can be shifted into one homogenous conformation by addition of a long acyl chain fatty acid, DPC. This homogenous conformation is attributed to a closed state similar to farnesylated Ykt6 under physiological conditions. In this structure, the SNARE core folds around the longin domain and forms a hydrophobic groove at their interface to accommodate the entire aliphatic tail of DPC. We characterized the unlipidated state, the DPC-binding state and the farnesylated state of Ykt6 by molecular dynamics simulations. The unlipidated state shows a collapsed hydrophobic groove and is wandering among different conformational states as experiments revealed. DPC interacts with Ykt6 by both its hydrophobic and hydrophilic end and localizes the protein in one of the unlipidated conformational state. Farnesylation shows a similar effect with DPC by stabilizing the closed state and also traps the protein in a local area in the conformational space. Thus, the population of conformational states is changed upon lipidation and one of the states is selected to take the dominance. We propose this process fits the "conformational selection" mechanism. Lipidation is often used to anchor proteins to membranes, but Ykt6 represents a novel case as lipidation can regulate the conformation of proteins.
Our work shows the dynamics-function relationship is of great importance to proteins. ABC transporter BtuCD and MsbA closely couple the motions of their TMDs and NBDs to translocate the substrates across cell membrane. PDZ12 tandem is endowed with cooperativity between PDZ domains. The interdomain mobility gets increased to create conformational entropy upon ligand binding. SNARE Ykt6 can response to lipid binding or lipidation with its hydrophobic groove and shift the population of conformational states. Our work reveals that molecular dynamics simulation is a very powerful tool for the studies on large-scale motion of proteins and targeted molecular dynamics simulation and normal mode analysis are also very useful methods. We anticipate that computational simulation will be more powerful with the development of cpu speed and the optimization algorithms and plays a more important role in the exploration of structure-dynamics-function relationship of proteins.
引文
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