Membrane Protein Modeling

膜蛋白建模

• 批准号: 7048211
• 负责人: HOMER ROBERT GUY
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7048211
• 关键词: bioinformatics; calcium channel; computer assisted sequence analysis; computer simulation; conformation; membrane channels; membrane model; membrane proteins; membrane structure; membrane transport proteins; method development; model design /development; molecular dynamics; nuclear magnetic resonance spectroscopy; physical model; potassium channel; protein folding; protein structure function; site directed mutagenesis; sodium channel; voltage gated channel

The general research aims of my group are to use molecular modeling and bioinformatics to analyze structure, function, and molecular evolution of membrane proteins. Membrane proteins are one of the most important classes of proteins. They comprise about 30% of most genomes and are involved in many biological processes. They are especially important in biomedical research because most targets of current pharmaceutical projects are membrane proteins. Unfortunately, membrane protein structures are difficult to determine experimentally, and most that are determined come from prokaryotes. We fill some of this structural void by developing computational methods of analyzing sequences and developing structural models of membrane proteins. We use computational analyses to do the following:1)Address questions that are not answered by crystal structures. 2)Assist in understanding similarities and differences among homologous proteins.3)Relate structural and sequence information to functional properties.4)Assist in the design and interpretation of experimental studies.Our current projects can be classified into three areas: 1) models of the structure and gating mechanisms of potassium (K+) channels and their relatives; 2) models of the structure and gating mechanisms of the large mechanosensitive channel, MscL; and 3) development of methods to analyze sequences and construct structural models of membrane proteins. Project 1. Models of K+ Channels and Their RelativesPotassium channels and related protein comprise the third largest superfamily of human genes. These proteins are found in almost all cells from bacteria on up. This category of membrane proteins contains several diverse superfamilies of channels including Na+, Ca2+, cyclic nucleotide-gated, TRP and its homologs, glutamate-activated, and Ca2+ release channels plus some K+ symporters and transporters. The smallest of these proteins are 2TM K+ channels that have four identical subunits; each of which has only two transmembrane helices, M1 and M2. A 'P' hairpin segment that spans only the outer half of the transmembrane region is located between M1 and M2. The P segment determines the selectivity of the channel. 6TM K+ channels are more complex, with each alpha subunit having four additional transmembrane segments, S1-S4, that precede the pore-forming S5-P-S6 motif (analogous to the M1-P-M2 motif of 2TM channels) and that forms a voltage-sensing domain in voltage-gated channels. Voltage-gated Ca2+ and Na+ channels have only one alpha subunit; however, it contains four homologous 6TM motifs. One of our goals is to develop structural models of the transmembrane region of at least one member of each major family of K+ channel related proteins. The importance of understanding the structure and functional mechanisms of K+ channels was recognized last year by the awarding of the Nobel Prize in Chemistry to Roderick MacKinnon for his work in solving the crystal structures of two 2TM (KcsA and MthK) and one 6TM (KvAP) K+ channels. An additional 2TM channel structure, KirBac1.1, has also been determined recently. The effort to determine the KirBac1.1 structure was stimulation by our identification of this bacterial homolog of eukaryotic inward rectifying K+ channels. All of these structures are from prokaryotes. We are utilizing these crystallographic data in developing structural models of the gating mechanisms of both the crystallized prokaryotic proteins and of some of their eukaryotic homologs that have been studied extensively and that are important drug targets. The KvAP structure presents a particularly interesting molecular modeling challenge. It is difficult to reconcile the crystal structure of the complete KvAP channel protein and the paddle model of the voltage-dependent gating that MacKinnon's group developed based on this structure with many experimental results and with basic principles of membrane protein energetics. We suspect that the voltage-sensing domain (S1-S4) of this structure is grossly distorted, but that a second crystal structure of an isolated voltage-sensing domain has a native open conformation. We have developed alternative models of the open KvAP channel's structure by attaching the isolated voltage-sensing domain crystal structure to the pore domain (S5-P-S6) from the crystal structure of the complete protein. Using this model as a starting point, we have developed models of resting and intermediate conformations. Our models have the 'traditional' transmembrane topology in which each of the S1-S4 segments transverses the entire transmembrane region in all conformations. Much of the movement of S4 occurs via the helical screw mechanism. We were the first group to propose this topology and gating mechanism shortly after the first voltage-gated Na+ channel sequence was determined in the mid '80's. Molecular dynamics simulations that we have performed of the protein embedded in a lipid bilayer indicate that our models are substantially more stable than is the crystal structure of the complete KvAP protein. Our models of the KvAP channel were constrained to be consistent with experimental results from other Kv channels, primarily the Shaker channel. These constraints are complicated by the facts that these proteins are evolutionarily distant and substantial data have been obtained from Shaker residue positions that are deleted or that may be in a different conformation in the KvAP sequence. To better address these issues, we have developed models of the Shaker channel that are similar to those of our KvAP models, but with some important structural differences. These adjustments make the Shaker models consistent with many experimental observations that are inconsistent with MacKinnon's paddle model of gating. We are using the general structure of our models of the KvAP and Shaker channels in different conformations to model the general backbone folding and gating mechanism of another important class of K+ channels, the Herg channels. Our alignment of the Herg, KvAP, and Shaker sequences is based upon a very large multisequence alignment of all voltage-gated and cyclic nucleotide-gated 6TM channels and upon analyses of correlated mutations among different protein families. We use results of mutagenesis experiments on Herg and closely related EAG channels, results of NMR studies, plus basic modeling principles, to adjust features of these models and to model regions, such as the long S5-P loop in Herg channels for which analogous residues are deleted in KvAP. We are also modeling how the BeKm-1 scorpion toxin binds in the outer vestibule of Herg. The Herg project is being performed in collaboration with Dr. Gea-Ny Tseng, whose lab performs mutagenesis experiments that constrain and test our models. We are also developing models for the structure and gating mechanism of a prokaryotic sodium selective channel, NaChBac. NaChBac was selected because several labs are attempting to solve its structure experimentally. Also, its sequence is intermediate between those of the voltage-gated K+ channels we have modeled and eukaryotic Ca2+ and Na+ channels. Thus models of NaChBac are a logical first step in our efforts to model these important superfamilies of Na+ and Ca2+ channels.We are performing molecular dynamics simulations of all of our membrane protein structures embedded in a lipid bilayer with water on each side. These simulations contain many atoms and are computationally intense. They help us evaluate the feasibility of our structures by comparing the energetically stability of our models to the stability of experimentally determined crystal structures. Project 2: Models of the Mechanosensitive Channel, MscLThis project exemplifies our general approach to modeling the structures and functional mechanisms of membrane proteins. We have modeled the structure of the prokaryote mechanosensitive channel, MscL, as it undergoes a very large

我小组的总体研究目标是利用分子建模和生物信息学来分析膜蛋白的结构、功能和分子进化。膜蛋白是最重要的一类蛋白质。它们约占大多数基因组的30%，并参与许多生物过程。它们在生物医学研究中尤其重要，因为当前制药项目的大多数靶点都是膜蛋白。不幸的是，膜蛋白的结构很难通过实验来确定，而且大多数已经确定的结构都来自原核生物。我们通过开发分析序列的计算方法和开发膜蛋白的结构模型来填补这种结构空白。我们使用计算分析来做以下工作：1)解决晶体结构不能回答的问题。2)帮助理解同源蛋白之间的异同。3)将结构和序列信息与功能属性联系起来。4)协助实验研究的设计和解释。我们目前的项目可分为三个方面：1)钾（K+）通道及其相关通道的结构和门控机制模型；2)大型力敏通道（MscL）的结构和门控机制模型；3)膜蛋白序列分析和结构模型构建方法的发展。项目1。钾离子通道及其相关蛋白构成了人类基因的第三大超家族。这些蛋白质几乎存在于所有细胞中，从细菌开始。这类膜蛋白包含几种不同的通道超家族，包括Na+、Ca2+、环核苷酸门控、TRP及其同源物、谷氨酸激活、Ca2+释放通道以及一些K+同向转运体和转运体。这些蛋白质中最小的是2TM K+通道，有四个相同的亚基；每一个都只有两个跨膜螺旋，M1和M2。位于M1和M2之间的“P”发夹段仅横跨跨膜区域的外半部。P段决定了信道的选择性。6TM K+通道更为复杂，每个α亚基有四个额外的跨膜片段S1-S4，它们在形成孔隙的S5-P-S6基序（类似于2TM通道的M1-P-M2基序）之前，并在电压门控通道中形成电压感应域。电压门控Ca2+和Na+通道只有一个α亚基；然而，它含有4个同源的6TM基序。我们的目标之一是建立每个主要的K+通道相关蛋白家族中至少一个成员的跨膜区域的结构模型。去年，诺贝尔化学奖授予Roderick MacKinnon，以表彰他在解决两个2TM （KcsA和MthK）和一个6TM (KvAP) K+通道的晶体结构方面的工作，认识到理解K+通道的结构和功能机制的重要性。最近还确定了另一个2TM通道结构KirBac1.1。确定KirBac1.1结构的努力受到真核生物向内校正K+通道细菌同源物的鉴定的鼓舞。所有这些结构都来自原核生物。我们正在利用这些晶体学数据来开发晶体化原核蛋白及其真核同源物的门控机制的结构模型，这些同源物已被广泛研究，并且是重要的药物靶点。KvAP结构提出了一个特别有趣的分子建模挑战。完整的KvAP通道蛋白的晶体结构与MacKinnon小组基于该结构建立的电压依赖性门控的桨形模型很难与许多实验结果和膜蛋白能量学的基本原理相一致。我们怀疑这种结构的电压感应域（S1-S4）是严重扭曲的，但隔离电压感应域的第二晶体结构具有天然的开放构象。我们开发了开放KvAP通道结构的替代模型，通过将隔离的电压传感域晶体结构连接到完整蛋白质晶体结构的孔域（S5-P-S6）。以这个模型为起点，我们开发了静止构象和中间构象的模型。我们的模型具有“传统的”跨膜拓扑结构，其中每个S1-S4片段以所有构象横贯整个跨膜区域。S4的大部分运动是通过螺旋螺旋机制发生的。在80年代中期确定了第一个电压门控Na+通道序列后不久，我们是第一个提出这种拓扑结构和门控机制的小组。我们对嵌入脂质双分子层的蛋白质进行的分子动力学模拟表明，我们的模型比完整的KvAP蛋白质的晶体结构要稳定得多。我们的KvAP通道模型与其他Kv通道（主要是激振器通道）的实验结果一致。由于这些蛋白质在进化上距离较远，并且从KvAP序列中删除或可能处于不同构象的Shaker残基位置获得了大量数据，这些限制因素变得复杂。为了更好地解决这些问题，我们开发了与KvAP模型相似的振动器通道模型，但在结构上有一些重要的差异。这些调整使激振器模型与许多与麦金农的桨门模型不一致的实验观察结果一致。我们使用不同构象的KvAP和Shaker通道模型的一般结构来模拟另一类重要的K+通道——Herg通道的一般主干折叠和门控机制。我们对Herg， KvAP和Shaker序列的比对是基于对所有电压门控和环核苷酸门控6TM通道的非常大的多序列比对，以及对不同蛋白质家族之间相关突变的分析。我们利用Herg和密切相关的EAG通道的诱变实验结果、核磁共振研究结果以及基本的建模原理来调整这些模型的特征和模型区域，例如Herg通道中的长S5-P环，其中类似的残基在KvAP中被删除。我们也在模拟BeKm-1蝎子毒素是如何在Herg的外前庭结合的。Herg项目是与曾兆妮博士合作进行的，曾兆妮博士的实验室进行诱变实验，对我们的模型进行约束和测试。我们还开发了原核钠选择通道NaChBac的结构和门控机制模型。之所以选择NaChBac，是因为几个实验室正试图通过实验解决它的结构问题。此外，它的序列介于我们所模拟的电压门控K+通道和真核Ca2+和Na+通道之间。因此，NaChBac模型是我们努力模拟这些重要的Na+和Ca2+通道超家族的合乎逻辑的第一步。我们正在对嵌入脂质双分子层的膜蛋白结构进行分子动力学模拟，每侧都有水。这些模拟包含许多原子，计算强度很大。通过将模型的能量稳定性与实验确定的晶体结构的稳定性进行比较，它们帮助我们评估结构的可行性。项目2：机械敏感通道模型（mscl）该项目举例说明了我们对膜蛋白结构和功能机制建模的一般方法。我们模拟了原核生物机械敏感通道MscL的结构，因为它经历了一个非常大的