The signaling helix: a common functional theme in diverse signaling proteins

Abstract

Background: The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood.

Results: Using sensitive sequence analysis methods we identify a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S)-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins, such as the Dimerization-Histidine phosphotransfer module of histidine kinases, the intra-cellular domains of the chemotaxis receptors, inter-GAF domain helical linkers and the alpha-helical HAMP module. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains such as histidine kinases, cNMP cyclase, PP2C phosphatases, NtrC-like AAA+ ATPases and diguanylate cyclases. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. The sequence conservation pattern of the S-helix revealed the presence of a unique constellation of polar residues in the dimer-interface positions within the central heptad of the coiled-coil formed by the S-helix.

Conclusion: Combining these observations with previously reported mutagenesis studies on different S-helix-containing proteins we suggest that it functions as a switch that prevents constitutive activation of linked downstream signaling domains. However, upon occurrence of specific conformational changes due to binding of ligand or other sensory inputs in a linked upstream domain it transmits the signal to the downstream domain. Thus, the S-helix represents one of the most prevalent functional themes involved in the flow of signals between modules in diverse prokaryote-type multi-domain signaling proteins.

Reviewers: This article was reviewed by Frank Eisenhaber, Arcady Mushegian and Sandor Pongor.

Figure 1

"Cross-hit" plots for the S-helix vis-à-vis examples of various parallel and anti-parallel CCs. The axes indicate the negative log of E-values from RPS-BLAST searches as a measure of significance. Typical S-helix and b-ZIP, Myosin tail domain or DHp domains are evident as two separated clusters, with no sequences having significant scores with both profiles. The blue dots in all the three graphs are plots of negative log of e-values from RPS-BLAST of S-helix database with S-Helix profile (x1) and of negative log of e-values from RPS-BLAST of S-helix database with bZIP, Myosin Tail domain or DHp profiles (y1). A) The pink dots are plots of negative log of e-values from RPS-BLAST of bZIP database with S-Helix profile (x2) and of negative log of e-values from RPS-BLAST of bZIP database with bZIP profiles (y2). B) The orange dots are plots of negative log of e-values from RPS-BLAST of Myosin Tail domain database with S-Helix profile (x2) and of negative log of e-values from RPS-BLAST of Myosin Tail domain database with Myosin Tail domain profiles (y2). C) The red dots are plots of negative log of e-values from RPS-BLAST of DHp domain database with S-Helix profile (x2) and of negative log of e-values from RPS-BLAST of DHp domain database with DHp domain profiles (y2).

Figure 2

Multiple alignment of representative examples of the S-helix. Representatives from a multiple alignment of the S-Helix domain, generated using the MUSCLE program [49] and corrected using PSI-BLAST [47] search results, are shown. The logo and the heptad notations are shown. The 80% consensus shown below the alignment was derived from an alignment of all the members using the following amino acid classes: consensus from the logo is also shown and colored using the following amino acid classes: hydrophobic (h: ACFILMVWY, yellow shading); aliphatic subset of the hydrophobic class (l; ILV, yellow shading); the aromatic subset of the hydrophobic class (a; FHWY, yellow shading); small (s: ACDGNPSTV, green); the tiny subclass of small (u; GAS, Green shading); polar (p: CDEHKNQRST, blue); the charged subclass of polar (c: DEHKR, pink); the positive subclass of charged (+: HKR, pink); the negative subclass of charged (-: DE, pink); alcohol (o: ST, Blue); and big (b: KFILMQRWYE, grey). A 'L', or 'T' show the completely conserved amino acid in that group. The limits of the domains are indicated by the residue positions, on each side. The domain architecture is shown to the right. The domain abbreviations are as in section 2 Materials and Methods and legend to Fig. 4. The mutations discussed in the paper are marked with boxes. The sequences are denoted by their gene name followed by the species abbreviation and GeneBank Identifier. The species abbreviations are: Ana: Nostoc sp.; Atum: Agrobacterium tumefaciens; Bant: Bacillus anthracis; Cele: Caenorhabditis elegans; Dmel: Drosophila melanogaster; Ecol: Escherichia coli; Hsap: Homo sapiens; Iloi: Idiomarina loihiensis; Lint: Leptospira interrogans; Mace: Methanosarcina acetivorans; Paer: Pseudomonas aeruginosa; Scer: Saccharomyces cerevisiae; Vvul: Vibrio vulnificus; Xaxo: Xanthomonas axonopodis; Ypes: Yersinia pestis

Figure 3

Sequence logo and interaction models for theS-helix. A) The sequence logo generated using the Weblogo program [50] is shown. The 'a' and 'd' positions of each of the 5 heptads, as per the notation of MacLachlan and Stewart [23], is also shown below the logo. B) the heptad interaction between two parallel helices is shown. The dotted red arrow indicates the 'g'-'e' interaction. The red negative sign ("-") indicates that most prevalent residues at the b and c positions are negatively charged. C) The most prevalent residue of each position on the S-Helix is shown as a table with the rows showing the positions in each heptad, and the columns showing the five heptads. The residues in red indicate the highly conserved positions.

Figure 4

Domain architecture graph for the S-helix. The ordered graph for the contextual information contained in domain fusions, drawn using Pajek [71] and modified with CorelDraw, is shown. The direction of the edge denotes the order of the fusion of domain in the polypeptide. If a domain is found on either side of another domain in different architectures, the edge points in both the direction. Domains with tandem repeats have loops pointing to themselves. The loop on TM includes bacterial 7TM receptors, 9TM receptors and 12TM Na+/proline symporters (found fused to bacterial histidine kinases in proteobacteria) with multiple successive TM segments separated by short hydrophilic loops. All connections to the S-helix are shown in red, while the other connections are in black. Domain abbreviations are as shown in section 2 Materials and Method or: SH – S-helix, Cyclase-NMP cyclase, HisKin – Histidine Kinase (including the DHp module), DISMED1 (for 7TMR-DISM extracellular domains 1); STYKIN – S/T/Y Kinase; NarQ – Extracellular nitrate sensing domain domain found in NarQ family of proteins; Glo – Globin domain and Hem – Hemerythrin.

Figure 5

Domain neighborhoods for the S-helix. The architectural context in which the S-helix occurs along with its immediately adjacent domains is shown here. The lower bound of numbers of such contexts in different proteins from 255 completely sequenced domains is shown to the right. The two contexts with the numbers in the green boxes are seen in animals. The domain abbreviations are as in section 2 Materials and Methods and Figure 4. The grey boxes are uncharacterized domains.

Figure 6

Approximate structural model for the S-helix. A model of the S-Helix domain was constructed using other parallel CCs as templates (e.g. PDB: 1YSA). The sequence that was model was derived from the the logo shown in Fig. 2 and represents an idealized S-helix. The hydrophobic residues at position 'd' are shown as yellow spheres. The surface view is shown and the negatively charged ridges on the surface formed by 'b' and 'c' positions are shown in red. The hydrogen bonds, red dots, show the 'a'-'a' interaction of N10-N10; the R20-T21 interaction; the 'g'-'e' interactions of R16-E14 (Top) and E23-N28 (bottom).