Finding Protein Sequence Motifs--Methods and Application

在过去的几年中，基因组序列和蛋白质结构的快速积累与序列数据库搜索方法的重大进展是并行的。NCBI开发的功能强大的位置特异性迭代BLAST （PSI-BLAST）方法为我们的蛋白质基序分析工作奠定了基础。在过去的一年里，我们在详细分析几类蛋白质的分类、进化和功能方面取得了进一步的进展。特别地，描述了一类新的主要的p环NTPases，即所谓的STAND类，在信号转导之后具有许多结构域的atp酶。STAND类包括ap - atp酶（动物凋亡调节因子CED4/Apaf-1、植物抗病蛋白和细菌afsr样转录调节因子）和NACHT NTPases（如NAIP、TLP1、et- e -1），它们在动物和植物的凋亡、病原体反应和细菌的转录调节方面得到了广泛的研究。我们发现，除了这些特征明确的蛋白家族外，STAND类还包括来自细菌和真核生物的不同信号和转录调节蛋白的其他几组（预测的）NTPase结构域，以及三个古细菌特异性家族。我们在几个生物学特征良好的蛋白质中发现了STAND结构域，这些蛋白质没有被怀疑具有NTPase活性，包括可溶性腺苷酸环化酶、肾囊素3（与多囊肾病有关）和Rolling pebble（肌肉发育的调节剂）；这些发现将有助于阐明这些蛋白的功能。STAND类与AAA+ ATPases、RecA/解旋酶相关的ATPases、ABC-ATPases和VirD4/PilT-like ATPases一起属于p环NTPases的附加链，催化E分裂。STAND蛋白与其他p环NTPases的区别在于，它存在与n端螺旋和核心链4相关的独特序列基序，以及与NTPase结构域融合的c端螺旋束。该螺旋模块在两个远端螺旋之间的环路中包含一个签名GxP motif。除了古细菌家族外，几乎所有的STAND NTPases都是包含三个或更多结构域的多结构域蛋白。除了NTPase结构域外，这些蛋白质通常还包含dna结合或蛋白质结合结构域，上层结构形成重复序列，如WD40和TPR，以及参与信号转导的酶结构域，包括腺苷酸环化酶和激酶。与AAA+ ATPases类似，可以预测STAND NTPases利用c端螺旋束作为“杠杆”，将NTP水解带来的构象变化传递到效应域。STAND NTPases代表了信号转导的一种新范式，其中适配器，调节开关，支架，在某些情况下，信号产生部分被组合成一个单一的多肽。STAND类由14个不同的家族组成，其中大多数家族的进化史充满了戏剧性的谱系特异性扩张和明显的水平基因转移。STAND NTPases在发育和组织复杂的原核生物和真核生物中最为丰富。STAND NTPases基因从细菌向真核生物的转移可能在真核生物信号系统的进化中发挥了重要作用。

In the last few years, rapid accumulation of genome sequences and protein structures has been paralleled by major advances in sequence database search methods. The powerful Position-Specific Iterating BLAST (PSI-BLAST) method developed at the NCBI formed the basis of our work on protein motif analysis. During last year, we made further progress in detailed analysis of the classification, evolution, and functions of several classes of proteins. In particular, a new major class of P-loop NTPases was described, the so-called STAND class, after signal transduction ATPases with numerous domains. The STAND class includes the AP-ATPases (animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators) and NACHT NTPases (e.g. NAIP, TLP1, Het-E-1) that have been studied extensively in the context of apoptosis, pathogen response in animals and plants, and transcriptional regulation in bacteria. We show that, in addition to these well-characterized protein families, the STAND class includes several other groups of (predicted) NTPase domains from diverse signaling and transcription regulatory proteins from bacteria and eukaryotes, and three Archaea-specific families. We identified the STAND domain in several biologically well-characterized proteins that have not been suspected to have NTPase activity, including soluble adenylyl cyclases, nephrocystin 3 (implicated in polycystic kidney disease), and Rolling pebble (a regulator of muscle development); these findings are expected to facilitate elucidation of the functions of these proteins. The STAND class belongs to the additional strand, catalytic E division of P-loop NTPases together with the AAA+ ATPases, RecA/helicase-related ATPases, ABC-ATPases, and VirD4/PilT-like ATPases. The STAND proteins are distinguished from other P-loop NTPases by the presence of unique sequence motifs associated with the N-terminal helix and the core strand-4, as well as a C-terminal helical bundle that is fused to the NTPase domain. This helical module contains a signature GxP motif in the loop between the two distal helices. With the exception of the archaeal families, almost all STAND NTPases are multidomain proteins containing three or more domains. In addition to the NTPase domain, these proteins typically contain DNA-binding or protein-binding domains, superstructure-forming repeats, such as WD40 and TPR, and enzymatic domains involved in signal transduction, including adenylate cyclases and kinases. By analogy to the AAA+ ATPases, it can be predicted that STAND NTPases use the C-terminal helical bundle as a "lever" to transmit the conformational changes brought about by NTP hydrolysis to effector domains. STAND NTPases represent a novel paradigm in signal transduction, whereby adaptor, regulatory switch, scaffolding, and, in some cases, signal-generating moieties are combined into a single polypeptide. The STAND class consists of 14 distinct families, and the evolutionary history of most of these families is riddled with dramatic instances of lineage-specific expansion and apparent horizontal gene transfer. The STAND NTPases are most abundant in developmentally and organizationally complex prokaryotes and eukaryotes. Transfer of genes for STAND NTPases from bacteria to eukaryotes on several occasions might have played a significant role in the evolution of eukaryotic signaling systems. Additionally, we identified a previously uncharacterized family of P-loop NTPases, which includes the neuronal membrane protein and receptor tyrosine kinase substrate Kidins220/ARMS, which is conserved in animals, the F-plasmid PifA protein involved in phage T7 exclusion, and several uncharacterized bacterial proteins. We refer to these (predicted) NTPases as the KAP family, after Kidins220/ARMS and PifA. The KAP family NTPases are sporadically distributed across a wide phylogenetic range in bacteria but ammong the eukaryotes are represented only in animals. Many of the prokaryotic KAP NTPases are encoded in plasmids and tend to undergo disruption to form pseudogenes. A unique feature of all eukaryotic and certain bacterial KAP NTPases is the presence of two or four transmembrane helices inserted into the P-loop NTPase domain. These transmembrane helices anchor KAP NTPases in the membrane such that the P-loop domain is located on the intracellular side. We show that the KAP family belongs to the same major division of the P-loop NTPase fold with the AAA+, ABC, RecA-like, VirD4-like, PilT-like, and AP/NACHT-like NTPase classes. In addition to the KAP family, we identified another small family of predicted bacterial NTPases, with two transmembrane helices inserted into the P-loop domain. This family is not specifically related to the KAP NTPases, suggesting independent acquisition of the transmembrane helices. CONCLUSIONS: We predict that KAP family NTPases function principally in the NTP-dependent dynamics of protein complexes, especially those associated with the intracellular surface of cell membranes. Animal KAP NTPases, including Kidins220/ARMS, are likely to function as NTP-dependent regulators of the assembly of membrane-associated signaling complexes involved in neurite growth and development. One possible function of the prokaryotic KAP NTPases might be in the exclusion of selfish replicons, such as viruses, from the host cells. Phylogenetic analysis and phyletic patterns suggest that the common ancestor of the animals acquired a KAP NTPase via lateral transfer from bacteria. However, an earlier transfer into eukaryotes followed by multiple losses in several eukaryotic lineages cannot be ruled out.