Department of Biochemistry & Molecular Biology, Institute for Genome Sciences, University of Maryland School of Medicine, BioPark II, Room 617, 801 West Baltimore St, Baltimore, MD 21201, USA.
Biol Direct. 2010 Dec 3;5:66. doi: 10.1186/1745-6150-5-66.
Certain residues within proteins are highly conserved across very distantly related organisms, yet their (presumably critical) structural or mechanistic roles are completely unknown. To obtain clues regarding such residues within Arf and Arf-like (Arf/Arl) GTPases--which function as on/off switches regulating vesicle trafficking, phospholipid metabolism and cytoskeletal remodeling--I apply a new sampling procedure for comparative sequence analysis, termed multiple category Bayesian Partitioning with Pattern Selection (mcBPPS).
The mcBPPS sampler classified sequences within the entire P-loop GTPase class into multiple categories by identifying those evolutionarily-divergent residues most likely to be responsible for functional specialization. Here I focus on categories of residues that most distinguish various Arf/Arl GTPases from other GTPases. This identified residues whose specific roles have been previously proposed (and in some cases corroborated experimentally and that thus serve as positive controls), as well as several categories of co-conserved residues whose possible roles are first hinted at here. For example, Arf/Arl/Sar GTPases are most distinguished from other GTPases by a conserved aspartate residue within the phosphate binding loop (P-loop) and by co-conserved residues nearby that, together, can form a network of salt-bridge and hydrogen bond interactions centered on the GTPase active site. Residues corresponding to an N-[VI] motif that is conserved within Arf/Arl GTPases may play a role in the interswitch toggle characteristic of the Arf family, whereas other, co-conserved residues may modulate the flexibility of the guanine binding loop. Arl8 GTPases conserve residues that strikingly diverge from those typically found in other Arf/Arl GTPases and that form structural interactions suggestive of a novel interswitch toggle mechanism.
This analysis suggests specific mutagenesis experiments to explore mechanisms underlying GTP hydrolysis, nucleotide exchange and interswitch toggling within Arf/Arl GTPases. More generally, it illustrates how the mcBPPS sampler can complement traditional evolutionary analyses by providing an objective, quantitative and statistically rigorous way to explore protein functional-divergence in molecular detail. Because the sampler classifies the input sequences at the same time, it can be used to generate subgroup profiles, in which functionally-divergent categories of residues are annotated automatically.
蛋白质中的某些残基在非常远缘的生物中高度保守，但它们（推测是关键的）结构或机制作用完全未知。为了获得在 Arf 和 Arf 样（Arf/Arl）GTP 酶中的此类残基的线索——它们作为调节囊泡运输、磷脂代谢和细胞骨架重塑的开/关开关——我应用了一种新的比较序列分析采样程序，称为多类别贝叶斯分区与模式选择（mcBPPS）。
mcBPPS 采样器通过识别那些最有可能负责功能特化的进化分歧残基，将整个 P 环 GTP 酶类中的序列分类为多个类别。在这里，我专注于最能区分各种 Arf/Arl GTP 酶与其他 GTP 酶的残基类别。这确定了以前提出过特定作用的残基（在某些情况下，实验也得到了证实，因此作为阳性对照），以及几个共同保守残基类别，其可能的作用在这里首次暗示。例如，Arf/Arl/Sar GTP 酶与其他 GTP 酶的区别最大的是在磷酸盐结合环（P 环）内的一个保守天冬氨酸残基，以及附近的共同保守残基，它们共同形成一个以 GTP 酶活性位点为中心的盐桥和氢键相互作用网络。与 Arf/Arl GTP 酶中保守的 N-[VI] 基序相对应的残基可能在 Arf 家族特征的开关切换中起作用，而其他共同保守的残基可能调节鸟嘌呤结合环的灵活性。Arl8 GTP 酶保守的残基与其他 Arf/Arl GTP 酶中通常发现的残基明显不同，并形成结构相互作用，暗示了一种新的开关切换机制。
该分析提出了具体的诱变实验，以探索 Arf/Arl GTP 酶中 GTP 水解、核苷酸交换和开关切换的机制。更一般地说，它说明了 mcBPPS 采样器如何通过提供一种客观、定量和统计学上严格的方法来探索分子细节中的蛋白质功能分化，从而补充传统的进化分析。因为采样器同时对输入序列进行分类，所以它可以用于生成子组配置文件，其中自动注释功能分化的残基类别。
