Cache Domains That are Homologous to, but Different from PAS Domains Comprise the Largest Superfamily of Extracellular Sensors in Prokaryotes

<div><p>Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly built computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms. Furthermore, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes.</p></div

Relationship between Cache (red), PAS (blue) and GAF (green) superfamilies.

<p>(<b>A</b>) HMM-to-HMM comparisons. The nodes represent domain families. Links represent reciprocal hits in hhsearch. Hits with an E-value <1e-3 are shown as thick lines, those with E-value <1e-1 are shown as thin lines and dotted lines represent hits with >90 probability score. Filled circles represent PAS and GAF domain families that were identified in HHpred search using new Cache models. Families that were not identified in these searches are depicted by empty circles (<b>B</b>) Sequence-to-sequence comparisons. The outer circle represents domain families. Links between individual sequences represent reciprocal BLAST hits with an E-value threshold of 1e-8, the lowest E-value at which no links between superfamilies were found. However, the overall relationships shown here remain at less stringent E-values.</p

Relative abundance of known extracellular sensory domains in prokaryotes.

<p>Domain counts were obtained by running Pfamscan against a dataset of non-redundant prokaryotic extracellular sequences, which was also used for HMM construction (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004862#sec010" target="_blank">Methods</a>).</p

Length distribution of Cache domains identified using the new domain models.

<p>Results for searches of the Pfam 27.0 associated UniProt database (June 2012 release) using the newly built single and double Cache models and the unchanged YkuI_C model are shown. Shaded areas show the upper and lower boundaries of known single and double Cache domain structures. Outliers represent partial protein sequences as well as partial matches to models (very short sequences) and sequences with large insertions within the Cache domain (very long sequences). See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004862#pcbi.1004862.s018" target="_blank">S2 Data</a> for details.</p

Newly defined Cache superfamily.

<p>Newly defined Cache superfamily.</p

Superfamily assignment of PAS domains in sequence and structure classification databases.

<p>(A) Extracellular PAS-like domains; (B) intracellular PAS domains. Assignments of PDB structures by Pfam [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004862#pcbi.1004862.ref026" target="_blank">26</a>] (red), SCOP [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004862#pcbi.1004862.ref027" target="_blank">27</a>] (green) and CATH [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004862#pcbi.1004862.ref028" target="_blank">28</a>] (blue) are shown as Venn diagrams to scale.</p

Comparison of sequence- and structure-based definitions for extracellular PAS-like domains.

<p>(A) <i>Vibrio parahaemolyticus</i> chemoreceptor (PDB: 2QHK); (B) <i>Vibrio cholerae</i> chemoreceptor (PDB: 3C8C). Domains are visualized on sequences with corresponding amino acid positions (top) and structures (bottom). Cache (cyan) domains are defined by Pfam; PAS domains (green and magenta) were defined by visual inspection of corresponding structures.</p

Examples of newly identified and better defined Cache domains in diverse signal transduction proteins from bacteria, archaea and eukaryotes.

<p>Domain architectures for representative sequences from model organisms are shown along with their UniProt accession numbers. Newly defined Cache domains are shown in red. Cache boundaries defined by the previous Pfam models are shown in pink (Cache) and green (MCP_N). HAMP domains are shown as grey circles, PAS domains as cyan circles, and HisKA domains as white circles. Other Pfam domains are abbreviated as follows: MCP, MCPsignal; GGDEF, GGDEF; GC, guanylate cyclase; HK, the histidine kinase HATPase_c domian; RR, response regulator; VWA, a combination of VWA_N and VWA domains; VGCC, VGCC_alpha2.</p

Phyletic distribution of PAS (blue), GAF (green) and Cache (red) domains.

<p>Flags at the outer three layers represent the domain presence in a corresponding genome. The tree was built using taxonomic ranks retrieved from NCBI.</p

The 3.2 Å Resolution Structure of a Receptor:CheA:CheW Signaling Complex Defines Overlapping Binding Sites and Key Residue Interactions within Bacterial Chemosensory Arrays

Bacterial chemosensory arrays are composed of extended networks of chemoreceptors (also known as methyl-accepting chemotaxis proteins, MCPs), the histidine kinase CheA, and the adaptor protein CheW. Models of these arrays have been developed from cryoelectron microscopy, crystal structures of binary and ternary complexes, NMR spectroscopy, mutational, data and biochemical studies. A new 3.2 Å resolution crystal structure of a <i>Thermotoga maritima</i> MCP protein interaction region in complex with the CheA kinase-regulatory module (P4–P5) and adaptor protein CheW provides sufficient detail to define residue contacts at the interfaces formed among the three proteins. As in a previous 4.5 Å resolution structure, CheA-P5 and CheW interact through conserved hydrophobic surfaces at the ends of their β-barrels to form pseudo 6-fold symmetric rings in which the two proteins alternate around the circumference. The interface between P5 subdomain 1 and CheW subdomain 2 was anticipated from previous studies, whereas the related interface between CheW subdomain 1 and P5 subdomain 2 has only been observed in these ring assemblies. The receptor forms an unexpected structure in that the helical hairpin tip of each subunit has “unzipped” into a continuous α-helix; four such helices associate into a bundle, and the tetramers bridge adjacent P5-CheW rings in the lattice through interactions with both P5 and CheW. P5 and CheW each bind a receptor helix with a groove of conserved hydrophobic residues between subdomains 1 and 2. P5 binds the receptor helix N-terminal to the tip region (lower site), whereas CheW binds the same helix with inverted polarity near the bundle end (upper site). Sequence comparisons among different evolutionary classes of chemotaxis proteins show that the binding partners undergo correlated changes at key residue positions that involve the lower site. Such evolutionary analyses argue that both CheW and P5 bind to the receptor tip at overlapping positions. Computational genomics further reveal that two distinct CheW proteins in Thermotogae utilize the analogous recognition motifs to couple different receptor classes to the same CheA kinase. Important residues for function previously identified by mutagenesis, chemical modification and biophysical approaches also map to these same interfaces. Thus, although the native CheW–receptor interaction is not observed in the present crystal structure, the bioinformatics and previous data predict key features of this interface. The companion study of the P5-receptor interface in native arrays (accompanying paper Piasta et al. (2013) <i>Biochemistry</i>, DOI: 10.1021/bi400385c) shows that, despite the non-native receptor fold in the present crystal structure, the local helix-in-groove contacts of the crystallographic P5-receptor interaction are present in native arrays and are essential for receptor regulation of kinase activity