Two Component Signal Transduction System and Bacterial Chemotaxis

Question 1

What system do Prokaryotes prefer?

Answer 1

One Component Signal Transduction Pathways

Question 2

Which transduction pathway is better with respect to extracellular signalling?

Answer 2

Two component pathway, 73% of two component pathways hold sensor histidine kinases were predicted to be membrane associated

Question 3

What is the prototypical structure of the two-component signal transduction system?

Answer 3

Sensor kinases are usually integral membrane proteins that autophosphorylate from ATP at a conserved histidine residue and then transfer the phosphoryl group to a conserved aspartate in the response regulator. Phosphorylation of a response regulator changes the biochemical properties of its output domain, which can participate in DNA binding and transcriptional control, perform enzymatic activities, bind RNA, or engage in protein–protein interactions (Gao et al. 2007)

Question 4

What are the two systems in a two component signal transduction pathway?

Answer 4

1. Histidine Kinase

2. Response Regulator

Question 5

What is the difference between phosphotransfer and phosphorelay?

Answer 5

Phosphotransfer is a direct transfer, whereas s phosphorelay: here a hybrid HK autophosphorylates and then transfers the phosphoryl group to an internal receiver domain, rather than to a separate RR protein. The phosphoryl group is then shuttled to histidine phosphotransferase (HPT) and subsequently to a terminal RR, which can evoke the desired response

Question 6

What is a hybrid HK

Answer 6

Hybrid HKs make up 25% of HKs. A hybrid HK holds a histidine kinase domain, the phosphorelay occurs between the HK and an internal reciever domain, which typically holds and Asp residue, the phosphate can then be transferred to a Histidine Phosphotransferase domain, which can either be attached or separate from the Hybrid HK.

Question 7

What is the structure of the kinase core

Answer 7

Either a homo or heterodimer, E.coli contain one CA domain, whilst T.martima contain 2 CA domains encircling a DHp domain.
Histidine kinases usually have an N-terminal ligand-binding domain and a C-terminal kinase domain, but other domains may also be present.

Question 8

What is the mechanism of stimulus perception?

Answer 8

1. The periplasmic aspartate-sensing domain of Salmonella enterica chemoreceptor Tar
2. The cytoplasmic HAMP domain of Af1503 from Archaeoglobus fulgidus
3. The cytoplasmic photoactive bacteriophytochrome sensory domains from Pseudomonas aeruginosa
4. The kinase core domains of Thermotoga maritima HK853
5. Signalling in LuxPQ
6. Two packing modes of the HAMP domain
7. Autophosphorylated kinase is relaxed to a conformation that allows the RR (violet) access to the phosphorylation site

Question 9

What is the structure of Response Regulators?

Answer 9

Either held in inactive or active conformation with a receiver domain. Substitution mutagenesis has proven the significance of the residues involved in these mechanism - this is because substitution mutations rarely confer the same active or inactive conformations (Smith et al. 2004)

Question 10

What is the inactive conformation of the RR?

Answer 10

Ser/Thr on the Beta4 and Phe/Tyr on B5 orientated away from the active site

Question 11

What is the active conformation of the RR?

Answer 11

Ser/Thr on β4 and Phe/Tyr on β5 oriented toward the active site Hydroxyl of the Ser/Thr forms a hydrogen bond with a phosphate oxygen and the aromatic ring of the Phe/Tyr becomes buried in the space vacated by the repositioned Ser/Thr

Question 12

What are the diverse regulatory mechanisms of RR?

Answer 12

1. Phosphorylation induced dimerisation. OmpR/PhoB family.
2. AAA+ ATPase. NtrC/DctD family
3. RRs containing GGDEF domains with diguanylate cyclase activity

Question 13

How is phosphotransfer specificity maintained?

Answer 13

1. The presence of multiple paralogous HK/RR proteins
2. High sequence and structure similarity of various DHp and REC domains
3. Multiple cognate pairs (CheA phosphorylate both CheB and CheY)

Question 14

Can you summarise the structural and signalling characteristics of the two signal transduction pathway?

Answer 14

1. HK and RR proteins have modular architectures – variations in arrangements
2. Conserved structural and functional features that are common to most HK and RR proteins
3. HKs and RRs exhibit specificity of intermolecular intermolecular interactions
4. Signalling, in general, involves stabilization of alternative conformation through the specific ligand binding, resulting in the specific protein-protein interactions and domain arrangements that modulate specific functions (on/off switch)
5. It is possible to engineer and change HK/RR specificity
6. Prediction of domain arrangements and regulatory mechanisms from sequence remains to be challenging

Question 15

What is an example of HK?

Answer 15

Examples of histidine kinases are EnvZ, which plays a central role in osmoregulation, and CheA, which plays a central role in the chemotaxis system.

Question 16

Whats an example of an orthodox HK?

Answer 16

Most orthodox HKs, typified by the E. coli EnvZ protein, function as periplasmic membrane receptors and have a signal peptide and transmembrane segment(s) that separate the protein into a periplasmic N-terminal sensing domain and a highly conserved cytoplasmic C-terminal kinase core.

Question 17

Why do bacteria need signal transduction?

Answer 17

Free-living organisms modulate their gene expression patterns in response to environmental cues. This modulation requires sensors to detect chemical and/or physical signals, and regulators to bring about changes in the levels of gene products.

Question 18

What is the significance of phosphorylation in these transduction pathways?

Answer 18

The vast majority of response regulators are active only when phosphorylated - thus the process of phosphorylation is exceedingly important to these systems (Hoch 2000; Gao et al. 2007). Therefore, any condition or product that affects the phosphorylated state of a response regulator will impact its ability to exert its biological functions. Consequently, the output of a response regulator is determined not only by the presence of the specific signals sensed by its cognate sensor kinase but also by gene products that stimulate or inhibit its phosphorylation. Additionally certain RRs will only be phosphorylated by specific phosphotransfer/phosphorelay reactions.

Question 19

How can the system be regulated?

Answer 19

Through regulating potential points of phosphoylation, thus Hybrid HKs offer more platforms for regulation.

Question 20

What is the function of the RR?

Answer 20

The RR gives rise to the appropriate cellular response, which is mediated by the C-terminal effector (or output) domain, via changes in its biochemical properties and strucutre, of the RR through protein-protein interaction (e.g. chemotaxis) or protein-DNA interactions leading to differential gene expression, such as change in expression of biosynthetic genes under nutrient stress.

Question 21

What are the phospho-related interactions?

Answer 21

1. The autophosphorylation of a conserved histidine in the transmitter domain of the sensor
2. The phosphotransfer to a conserved aspartate in the receiver domain of the RR (by the activity of the RR)
3. Dephosphorylation of the RR to set the system back to the prestimulus state

Question 22

How are they organised on a bacterial genome?

Answer 22

The structural genes for the HK and the cognate RR are organized in operons. However 5 out of 32 characterised cognate pairs are not located next to one and other in E.Coli (Mizuna 1997)

Question 23

What systems have been studied in great detail?

Answer 23

The paradigmatic systems EnvZ/OmpR, CheA/CheY, and NtrB/NtrC in Escherichia coli

Question 24

How have the HKs been assigned into groups?

Answer 24

Sequence of the H domain (autophosphorylation domain.

Question 25

What makes membrane bound HKs different?

Answer 25

The largest group, the periplasmic (or extracellular)-sensing HKs, includes proteins with an extracellular sensory domain which is framed by at least two TM helices.The kinase is localized in the cytoplasm (as for all other HKs). Thus, sensory and kinase domains are located in two different cellular compartments which are separated by a membrane, necessitating TM signal transduction. This type of membrane topology is typical for sensing solutes and nutrients. There is another group known as TMRs.

Question 26

What are TMRs and what do they sense?

Answer 26

Transmembrane Regions, therefore, the stimuli sensed either are membrane associated or occur directly within the membrane interface.

Question 27

What are HK TMRs characterised by?

Answer 27

Highly diverse group of sensor kinases is the presence of 2 to 20 transmembrane regions (TMR) implicated in signal perception. These TMR are connected by very short intra- or extracellular linkers; i.e., these sensors lack an obvious extracellular input domain

Question 28

What are the characteristics of cytoplasmic HKs?

Answer 28

The second-largest group of sensor kinases, the cytoplasmic-sensing HKs, includes either membrane-anchored or soluble proteins with their input domains inside the cytoplasm. This class of sensor proteins detects the presence of cytoplasmic solutes or of proteins signaling the metabolic or developmental state of the cell or of the cell cycle. Other cytoplasmic TCS respond to diffusible or internal stimuli, such as O2 or H2, or stimuli transmitted by TM sensors.

Question 29

Describe the transmitter domain of HKs

Answer 29

The transmitter domain comprises a sequence with the conserved histidine residue for autophosphorylation (the H box) and ends with the highly conserved kinase (or catalytic) domain. The domain with the conserved His residue typically contains two α-helices (X box), which serve as a dimerization domain (DHp [dimerization and histidine phosphotransfer] or HisKA domain)

Question 30

Describe the catalytic domain of HKs

Answer 30

The catalytic domain (HATPase) contains the conserved N, D, F, and G boxes with the respective highly conserved amino acid residues.

Question 31

Describe the prototypical periplasmic domain

Answer 31

Prototypical periplasmic-sensing histidine kinases are composed of two TM helices with an intervening extracytoplasmic domain of 50 to 300 amino acids, lacking large cytoplasmic linker regions. Some contain periplasmic binding proteins.

Question 32

What are the difference between TMRs and periplasmic HKs

Answer 32

most of TMRs have only short or no significant (extra)cytoplasmic linkers between them

Question 33

How does the common regulatory domain (20–30% sequence identity) control the activities of such structurally and functionally dissimilar effector domains?

Answer 33

An elegantly simple answer to this question emerged ∼10 years ago when the inactive and active states of isolated receiver domains were first characterized. Regulatory domains were observed to exist primarily in two conformations designated inactive and active, with the latter stabilized by phosphorylation. It was postulated that molecular surfaces that differed in the two states could be exploited for regulatory protein–protein interactions, enabling a variety of regulatory strategies.

Question 34

What is the common structure of the RRs?

Answer 34

The defining feature of RRs is the presence of a structurally conserved α/β domain, referred to as a regulatory or receiver domain. This consists of a five-stranded parallel β sheet surrounded by five amphipathic helices. he active site contains a cluster of conserved acidic residues that includes the aspartic acid at the C-terminal end of β3, which is the phosphorylation site. Two additional acidic residues in the β1–α1 loop position a divalent metal ion, commonly Mg2+, that is required for both phosphotransfer and phosphate hydrolysis

Question 35

What are the changes that occur in the RR during phosphorylation

Answer 35

Phosphorylation does not result in substantial changes in secondary structure; rather, it usually involves subtle displacements of the backbone (typically ∼1 Å) and perturbations of the molecular surface that are localized primarily to the α4–β5–α5 face.

Question 36

How sensitive is the chemotactic response?

Answer 36

Extremely. In E.coli alone cells will respond to 10nM steps in concentration of Aspartate.

Question 37

How is motion controlled in the chemotactic response?

Answer 37

The motion is controlled by the movement of the flagella, which moves in a rotary like fashion. The ratio between swimming and tumbling produces the direction and speed of the movement.

Question 38

What is flagella made out of?

Answer 38

• The MS ring in the inner membrane, surrounded by the motor complex. Protruding from the MS ring is the C ring which encapsulates the Protein Export System.
  1. MS ring – A subunit
  2. Motor Complex – W subunits
• Extending from the MS ring is the rod, which extends into the periplasmic space to the P ring
• The L ring is attached to the P ring and is held in the out membrane
• The Hook extends from the L ring, from which filaments extended, constructed by flagellin subunits

Question 39

What does the P ring associate with?

Answer 39

P ring associates with petidoglycan in the periplasmic space

Question 40

What does the L ring associate with?

Answer 40

The L ring associates with liposaccharides on the outer membrane

Question 41

Describe the rotary engine of the flagella.

Answer 41

It is driven by the Mot complex, associated with the inner cell membrane, the movement is driven by proton motive force. The steeper the gradient the stronger the locomotive force.

Question 42

Describe the swimming movement of flagella.

Answer 42

The nutrients/components present promote counter clockwise flagellar rotations, this movement bundles the flagella together producing stronger locomotive forces

Question 43

Describe the tumbling movement of flagella.

Answer 43

The lack of nutrients means the flagella’s tumbling movement isnt inhibited, it will therefore promote a clockwise movement, the flagella ‘unbundle’ and no forward movement is measured.

Question 44

What is the bacterial paradigm for the molecular basis of biological signalling mechanisms?

Answer 44

The paradigm for biological signaling mechanisms is the E.coli’s methyl-accepting chemotaxis protein (MCPs), via direct cell locomotion by regulating the histidine kinase CheA; which phosphorylates a response regulator, which in turn controls the rotational direction of the flagella motor.

Question 45

How high can E.coli’s chemotaxic pathway amplify signals?

Answer 45

50-fold, a 1% increase in occupancy in receptors elicits a 50% in rotation bias. Thus one receptor can control a network of kinases producing a functional network.

Question 46

How are movements controlled?

Answer 46

Shifts in the kinase-on–kinase-off equilibrium modulate the flux of CheA phosphoryl groups to two response regulators, CheY for motor control, and CheB for sensory adaptation. In motor control, phospho-CheY binds to the flagellar rotary motor, enhancing the probability of clockwise (CW) rotation, which causes random directional changes; counter-clockwise (CCW) rotation, the default behavior, produces forward swimming. Phosphatase CheZ hydrolyzes phospho-CheY, ensuring a short duration in CW flagellar rotation so that phospho-CheY levels closely track receptor-modulated CheA activity.

Question 47

Which two mediators control CheA?

Answer 47

1. CheY - motor control in CW

2. CheB - sensory adaption, default behavious

Question 48

What does CheZ ensure?

Answer 48

It is a phosphotase, ensuring the hydrolysis of CheY-P, ensuring only a short duration of CW movement in flagellar rotation so that the response is tightly regulated and sensitive

Question 49

What is a nanodisc?

Answer 49

A nanodisc is a synthetic model membrane system which assists in the study of membrane proteins. Nanodiscs are useful for membrane biology in the study of membrane proteins because they represent a more native environment than liposomes, detergent micelles or bicelles and allow the study of purified membrane proteins. Nanodiscs consist of a small portion of membrane that has been solubilized by the addition of two molecules of an amphipathic protein, dubbed the membrane scaffold protein (MSP). This protein wraps around the hydrophobic core of the lipids, effectively creating a soluble portion of membrane. When prepared properly, nanodiscs are uniform in size and allow a native local environment to be maintained within a larger in vitro study

Question 50

What is the typical spatial organisation?

Answer 50

Cluster together in the membrane - amplifying the effects of the stimulus, aiding integration of chemotaxis stimuli in an extremely co-operative manner.

Question 51

What happens when there is a lower conc. of attractant?

Answer 51

Increase in Kinase Activity, this is the Che-B dependent adaption. Results in tumbling.

Question 52

What happens when there is an increase in conc. of attractant?

Answer 52

Decrease in kinase activity, Che-B dependent adaption. This is the normal state - will result in CCW movement, flagella bundling –> swimming.

Question 53

Describe sensor adaption in E.coli.

Answer 53

Cells swimming through spatial chemical gradients monitor temporal changes in chemoeffector concentrations by means of a sensory adaptation system that records recently encountered chemical conditions in the form of reversible chemoreceptor methylation at four to six glutamyl residues in the adaptation region of the receptor’s kinase control module. Hence, chemoreceptors are methyl-accepting chemotaxis proteins (MCPs). The modifications are catalyzed by two MCP-specific enzymes, methyltransferase CheR and methylesterase CheB.

Question 54

Describe the Sensor Adaption feedback system

Answer 54

The adaptation enzymes continuously update the methylation record using two feedback mechanisms: (i) activation of CheB by CheA-mediated phosphorylation and (ii) opposite propensities for the two modifications in the two receptor conformations by CheR. For example, the ‘kinase-off’ signalling conformation has high attractant affinity, high propensity for methylation and low propensity for demethylation, thus it will be driven to the kinase on state via methylation - whereas the ‘kinase-on’ conformation has low attractant affinity, low methylation propensity and high demethylation propensity. Both chemoeffector binding or release and methylation or demethylation can shift receptor signaling complexes from one state to the other. For example, attractant ligands drive receptors toward the kinase-off state; subsequent addition of methyl groups shifts receptors toward the kinase-on state, reestablishing the steady-state (adapted) balance between the two states and restoring random walk movements.

Question 55

What three structures make up the flagellum?

Answer 55

The flagellum consists of three parts: the filament (helical propeller), the
hook (universal joint), and the basal structure (rotary motor) - forming a double tubular structure (inner and outer tubes) and the flagella filaments consist of 11 protofilaments

Question 56

What encourages rotational movement in the flagella?

Answer 56

The hook allows for the production of torque: The base of the filament is connected to the short tubular structure called the hook, which is thought to function as a universal joint to smoothly transmit the torque produced by the motor to the filament

Question 57

What does the Basal body in the flagellum consist of?

Answer 57

The MS ring, which is surrounded by the C ring (and its associated proteins form the switch complex), the MS ring binds to the rod, which is encapsulated by the P ring followed by the L ring, which are resistant to chemical components. Attached to the switch complex is the export apparatus - essential for flagellum assembly. The basal body is associated with the Mot complex which generates movemetn via a proton gradient - the greater the gradient the greater the movement

Question 58

How is direction maintained in E.coli?

Answer 58

Through the interplay between motor control and sensory adaption, which is maintained through the guanyl-methylation of chemoreceptors