Multistep Signaling in Nature: A Close-Up of Geobacter Chemotaxis Sensing

Abstract

Environmental changes trigger the continuous adaptation of bacteria to ensure their survival. This is possible through a variety of signal transduction pathways involving chemoreceptors known as methyl-accepting chemotaxis proteins (MCP) that allow the microorganisms to redirect their mobility towards favorable environments. MCP are two-component regulatory (or signal transduction) systems (TCS) formed by a sensor and a response regulator domain. These domains synchronize transient protein phosphorylation and dephosphorylation events to convert the stimuli into an appropriate cellular response. In this review, the variability of TCS domains and the most common signaling mechanisms are highlighted. This is followed by the description of the overall cellular topology, classification and mechanisms of MCP. Finally, the structural and functional properties of a new family of MCP found in Geobacter sulfurreducens are revisited. This bacterium has a diverse repertoire of chemosensory systems, which represents a striking example of a survival mechanism in challenging environments. Two G. sulfurreducens MCP-GSU0582 and GSU0935-are members of a new family of chemotaxis sensor proteins containing a periplasmic PAS-like sensor domain with a c-type heme. Interestingly, the cellular location of this domain opens new routes to the understanding of the redox potential sensing signaling transduction pathways.

Keywords: Geobacter; c-type heme sensor domains; methyl-accepting chemotaxis proteins; redox-sensing; signal transduction; two-component system.

Figure 1

Simplified scheme for a two-component system. The histidine kinase (HK), the receiver—containing a specific aspartate residue (D)—and the DNA-binding (DBD) domains are represented.

Figure 2

Phosphotransfer mechanisms in TCS. In both cases, the sensor domain detects the stimuli and regulates the histidine kinase domain (HK) activity. (A) Prototypical two-component pathway. The pathway consists of a dimeric transmembrane sensor HK and a cytoplasmic response regulator (RR) protein. A monomer of a representative HK is presented with transmembrane (TM) segments represented by TM₁ and TM₂. N, G₁, F and G₂ are conserved sequence motifs in the ATP-binding domain. HK catalyzes an ATP-dependent autophosphorylation of a specific conserved His residue within the HK dimerization domain. The phosphoryl group (P) is then transferred to a specific aspartate residue (D) at the conserved RR domain. Phosphorylation of this domain usually triggers an associated (or downstream) effector domain, which ultimately produces a specific cellular response. (B) A multi-component phosphorelay system often involves a hybrid HK with an additional C-terminal RR domain. In these complex systems, at least two His–Asp phosphoryl transfer events occurs, typically involving a His-containing phosphotransfer protein (HPT) operating as an His-phosphorylated intermediate.

Figure 3

Sensor kinase domain architecture obtained from the available structural information combined with the SMART annotation (adapted from [30]). Abbreviations: sCache—single calcium channels and chemotaxis receptor; PAS—period clock protein, aryl hydrocarbon receptor and single-minded protein; HAMP—domain found in histidine kinases, adenylyl cyclases, methyl-binding proteins and phosphatases; RRR—response regulator receiver domain; HPT—histidine containing phosphotransfer; GAF domain—non-catalytic cGMP phosphodiesterase/adenylyl cyclase/FhlA-binding domain; PHY—phytochrome.

Figure 4

Representative structures of PAS (A) and sCache (B) domains. The first corresponds to the redox-sensing PAS domains of Azotobacter vinelandii NifL (PDB ID: 2GJ3) bound to FAD (flavin adenine dinucleotide) and the second to the ligand-binding domain of the Klebsiella pneumoniae (K. pneumoniae) sensor kinase CitA protein (PDB: 1P0Z). The core of β-strands are labeled with Arabic numbers. The structures were generated with CHIMERA [38] and are colored as follows: N-terminal end—dark blue; the first α-helix region—green; the β-strands 1 and 2—orange; the inter-domain α-helix region—pink; the β-strands 3 to 5—yellow and the C-terminal—red. FAD and citrate molecules are shown in light blue.

Figure 5

Structure of the sCache domains of PhoQ (PDB: 1ID0) from Salmonella enterica, DcuS (PDB: 3BY8) from E. coli and CitA (PDB: 1P0Z) from K. pneumoniae. The ligands of each sCache domain are also represented: Mg²⁺ (red), malate (coloured by element) and citrate (coloured by element). The β-strands and α-helices are colored green and blue, respectively. The sensor domain architectures are shown at the bottom of figure.

Figure 6

Structure of FixL (A) and DosP (B) PAS sensor domains. β-strands and α-helices are represented in green and blue, respectively. The hemes are colored by element.

Figure 7

Prototypical TCS signaling pathway. A variety of extracellular signals are detected by the SD, which triggers the membrane-bound HK dimerization. The TM helices form four-helix bundles in the HK dimeric states. The signaling mechanism then involves the autophosphorylation of HK mediated by hydrolysis of ATP and concomitant phosphorylation of a conserved histidine by the catalytic (CA) domain. This then leads to the phosphotransfer of the HK phosphoryl group to a cytoplasmatic response regulator (RR) that is composed by two domains: the receiver domain (D)—that recognizes and binds to the HK domain—and the effector DNA-binding domain (DBD) that modulates the expression of target genes and hence the cellular response. Upon phosphorylation, the RR is activated and undergoes conformational changes that promote dimerization or more ordered oligomerization states that favor the interaction between the RR and bacterial DNA. Alternatively, the RR may also act as an enzyme, such as a methylesterase or an ATPase. TCS signaling is terminated by dephosphorylation of the RR, which can be auto induced or mediated by the HK or by auxiliary proteins. The CA and the dimerization domains are conserved and found in all HK, whereas the remaining signaling domains (HAMP, GAF, PAS and PHY) are variable.

Figure 8

(A) Structure of the heme pocket region of MCP GSU0935 sensor domain from G. sulfurreducens and (B) schematic representation of heme group (in orange) with its binding residues (axial ligands—Met⁶⁰ and His¹⁴⁴—and cysteine residues from the heme binding motif—Cys¹⁴⁰ and Cys¹⁴³). The β-strands and α-helices are represented in green and blue, respectively. The heme-binding site is represented in purple and the axial ligands in yellow.

Figure 9

Classification of MCP according to Lacal and co-workers [52].

Figure 10

Mechanism of chemotaxis in E. coli. The chemoreceptors function as trimers of dimers. The rotation of the flagellar motor is regulated by the ratio of the phosphorylated form of CheY (CheY~P) versus its dephosphorylated form (CheY). Steady dephosphorylation of CheY~P by the phosphatase CheZ maintains a constant ratio of CheY~P/CheY and a basal level of alternating counterclockwise (CCW, causing cells to swim more straight runs—moving upwards the attractant gradient) and clockwise (CW, causing cells to tumble) rotation. This equilibrium is affected either by attractants or repellents. In the first case, the phosphorylation of CheA is blocked and the high levels of CheY favor the CCW rotation (see dashed arrows in the upper panel). On the other hand, repellents decrease the CheZ activity and increase both the levels of CheA~P and CheY~P. As a consequence, CW rotation is favored. Both processes are called excitation. A methylation feedback loop on the MCP re-establishes the basal CheY~P/CheY ratio (adaptation).

Figure 11

Proposed sensing mechanism of G. sulfurreducens heme MCP sensors. The periplasmic domain senses the external signal, which might be related with the redox sensing and transfers the signal through the TM helices, activates the HAMP domain and then the MCP. Then, the highly conserved signaling domain (HCD) is activated and regulates the ratios of CheW/CheW~P and CheA/CheA~P (see also Figure 10). The response regulators involved in chemotaxis, CheY and CheB, compete for CheA binding. CheY interacts with the flagellar motors and controls the direction of the motor rotation, while CheB demethylates specific and highly conserved segments located in MCP (methylation sites), thus controlling the adaptation for a ligand-bound receptor complex.

Figure 12

Amino acid sequence alignment of sensor domains GSU0935 and GSU0582 from G. sulfurreducens. The conserved residues are indicated in bold face and the heme binding motif in a gray box. The alignment of the proteins was performed with the basic local alignment search tool (BLAST) [142]. Helical and strand segments are indicated according to PHYRE automatic fold recognition server for secondary structure prediction [143].

Figure 13

Structure of the sensor domains GSU0582 (PDB: 3B47) and GSU0935 (PDB: 3B42) in the oxidized form. α-helices and β-strands are represented in green and blue, respectively. The heme groups are represented in orange.

Figure 14

GSU0582 and GSU0935 predicted monomer models constructed using the program 3D-PSSM. The two helical segments at the N-terminal followed by four-stranded antiparallel β-sheets are represented in blue and green, respectively. The heme groups are represented in orange.

Figure 15

Model for the signal transduction mechanism mediated by MCP in G. sulfurreducens. Signal sensing promotes the phosphorylation of CheA coupled by an adaptor protein CheW. The cascade continues with the phosphorylation of CheY. CheY~P is responsible for clockwise flagellar rotation (CW) and tumbling movements dominate. On the other hand, signal saturation blocks the cascade and the reverse events occur. In this case instead of tumbling, running movements predominates because of the counterclockwise flagellar rotation (CCW). Once a directional flagellar motion is no longer necessary, the phosphatase CheZ oligomerizes with the phosphorylated CheY and increases the spontaneous dephosphorylation rate of CheY∼P. Then, adaptation follows: CheB causes demethylation of the methylated MCP, while CheR causes its methylation.