Signaling with structural detail!

Receptor Clustering!

Most receptors are clustered at the two poles of the bacterium Advantageous in allowing an amplified response to the signal Receptor clusters appear to regulate the activity of multiple CheA kinase molecules such that CheY-P production by the CheA kinase amplifies the signal up to 35 fold, with Hill coefficients as high as 10! Thus, the changes in the concentration of CheY-P vary much more steeply than the changes in concentration of the chemoattractant.

Cryoelectron tomography

A circular 250 nm patch could contain ~ 40% of the total of ~7500 receptor dimers present in the cell and would occupy ~1% of the cell surface.

CheW3 and CheW4 Different chemotaxis pathway components can localize to different regions in the cell : e.g. Rohodobacter

Overview of the chemotaxis pathway

Bacteria can follow more than one gradient at the same time, so they must have more than one type of detector. They can also respond to concentrations of attractants spanning five orders of magnitude. This requires some method to adjust the sensitivity of the detector systems so that they do not become swamped at very high ligand concentrations. The central components in the bacterial chemotaxis system are four varieties of methyl-accepting chemotaxis proteins or MCPs. All four proteins span the bacterial inner membrane. Their conformation changes from a clockwise signalling [CWS] to a counter-clockwise signalling [CCWS] state after attractant binding, either directly, or via one of the periplasmic receptor proteins. It should be noted that the effector ligand does not enter the bacterial cell cytoplasm in order to exert its effects. The ligand binding affinities of the MCPs can be adjusted by multiple methylation of glutamate residues within a cytoplasmic domain of these proteins.

Chemotaxis and Adaptation

Chemotactic response shows high sensitivity to small concentration differences and response can extend over 100,000-fold difference in concentrations Example of high signal gain and large dynamic range characteristic of other sensory systems where multi-step enzyme cascades provide these properties Where does the gain and sensitivity come from in the chemotaxis pathway?

Overview of chemotaxis

General Features
The kinase activity of receptorsignaling complexes in E. coli is generally maintained such that the level of phospho-CheY is at or near 50%. This optimizes the sensitivity of the system to both positive and negative fluctuations in kinase activation. The feedback methylation system acts to preserve this delicately balanced state. Attractant stimuli like aspartate and serine cause increases in CheR-dependent methylation and decreases in CheB-dependent demethylation and deamidation. The attractant-induced increases in methylation promote CheA activity precisely enough to counteract the inhibitory effect of attractant-sensory domain binding. Repellent stimuli have opposing effectscausing increased CheA activity which was inactivated by decreases in methylation associated with increased CheB and decreased CheR activities.

2-state model
The regulation of the autokinase activity of CheA has been described in terms of a two-state model in which a receptor is in either an active or an inactive state, either promoting or inhibiting the activity of CheA Binding of attractant increases the probability that a receptor is inactive, whereas methylation of a receptor on four specic glutamate residues increases the probability that it is active Receptors in lower modication states, although less active, have higher afnities to attractant Does not explain the high sensitivity/integration of many signals

E coli strains used

Levels of expression of Tar, Tsr, Tap, CheA and/or CheW were varied Strains lacking the enzymes required for adaptation: the methyltransferase CheR and the methylesterase CheB receptors encoded by wild-type genes remain in the half-modied state, QEQE, where Q (glutamine) mimics methylated glutamate

High abundant receptors Tar, Tsr Low abundant Tap, Trg, Aer

FRET assays with YFP-CheY and CFP-CheZ

Che A phosphorylates CheY at the same rate as it is dephophosphorylated by CheZ. Therefore, the FRET ratio is a reflection of the activation status of CheA

Tar +, Tsr +, Tap +

Tar +, Tap + Tar +

Effect of level of expression of Tar in Tsr+ cells

At low expression levels of Tar, the dominant receptor was Tsr, and the addition of serine reduced the kinase activity to zero. With Tar at six times the native level, Tar became the dominant receptor. In all cases, a saturating dose of both attractants reduced the kinase activity to zero (not shown). White bars, no attractant grey bars, MeASP (5mM) black bars, serine (10mM)

Only Tar at native levels Tar at twice the level Tar at 6 times the level

The larger the number of receptors of a particular type in a complex, the higher the sensitivity and co-operativity of the response of that receptor to the ligand. Receptors cluster together

Clusters of receptors of essentially one kind gave responses of high cooperativity, depending on the receptor to CheW to CheA ratio

Varying levels of CheW in the cell increased co-operativity 0.01 native levels of CheW 0.1 native levels of CheW 0.7 native levels of CheW

Varying levels of CheA in the cell decreased the co-operativity 0.25 native levels 0.3 native levels

8 native levels

High co-operativity reflects the interactions between receptors in a cluster

Critical concentration of components of the signaling pathway also important

Weber-Fechner Law
This law hails from the middle of the nineteenth century. Persons were given two nearly identical stimuli (for example, two similar weights) and tested whether they could notice a difference between them. Weber found that the smallest noticeable difference in weight (the least difference that the test person can still perceive as a difference), was proportional to the starting value of the weight. That is to say, if the weight is 1 kg, an increase of a few grams will not be noticed.

Weber-Fechner Law
The detectable change in stimulus intensity is proportional to the level of background stimulus over a wide range of stimuli. For example, if the addition of another receptor-bound molecule can be detected against a background of ten receptor-bound molecules, then a change of ten more could be distinguished in a pool of a hundred, a change of 100 in 1000, and so forth. To maintain this relationship, systems adapted to low backgrounds of stimulation operate with high gain, such as those acting in dark-adapted vision; and systems adapted to high backgrounds of stimulation operate with low gain, such as those acting in light-adapted vision. The bacterial chemotaxis system also follows the WeberFechner law, whereby increases in methylation function to reduce the gain of stimulusresponse coupling that occurs with increasing ambient stimulus intensities.

Adaptation
Detection of temporary changes in chemoeffectors requires a stored history of past environments In chemotaxis, memory is stored in the form of covalent modification of receptors by methylation When current conditions do not agree with the methylation state of the receptors, the cell responds and updates its memory state to the current chemoeffector concentration

CheB is a methyl esterase CheR is a methyl transferase Both are critical components of the adaptation pathway They target several specific glutamate, glutamine and g-methylglutamate residues in the cytoplasmic part of the receptor Attractants induce increases in methylation that feed back to counteract attractant-induced kinase inhibition. Repellants cause decreases in receptor methylation that feed back to inhibit repellant-induced kinase activity

Nature 400: 787 (1999)

Chemotaxis, receptors, methylation and memory

The activity of CheA is dependent on the methylation state of the MCPs with which it is associated Low levels of MCP methylation are associated with low CheA activity Elevated methylation levels are associated with high CheA activity

Adaptation
Increased concentrations of attractants act via their MCPs to cause an IMMEDIATE inhibition of CheA activity The same changes in MCP conformation that inhibit CheA lead to relatively slow increases in MCP methylation by CheR, so that despite the continued presence of attractant, CheA activity is eventually restored to the same value it had in the absence of attractant CheB acts to demethylate the MCPs under conditions that caused an elevated CheA activity Methylation and demethylation occur much more slowly than phosphorylation of CheA and CheY-thereby providing a memory mechanism that allows a cell to compare its present situation to its recent past.

Each MCP can have 16 possible patterns of methylation, and if this is seen for 10,000 receptors on the surface there is a huge number of possible states.
That is why no two bacteria respond in exactly the same way.

 Bacteria mounted under a coverslip and movement monitored by video imaging Fix bacteria by using anti-flagellin antibodies to cover slip and monitor rotation under a microscope. Tumbling frequency increased in a weakly sigmoidal fashion

DCheZ

WT

Nature 397:168 (1999)

CheR expression regulated by lac promoter. Concentrations varied 100-fold by altering IPTG levels

Unstimulated cells

Adaptation time

wild type cells

DCheR

Cells treated with aspartate Adaptation precision defined as ratio of steady state tumbling frequency of unstimulated and stimulated cells

 CheB deletion: decrease in tumbling frequency, increase in adaptation time CheZ deletion: increase in tumbling frequency CheBc: activated CheB that cannot be phosphorylated by CheA but can still demethylate receptor : here again adaptation precise Exact adaptation maintained despite variation in concentration of different proteins in the network Many bacteria that show deficient adaptation are poorly chemotactic In contrast, bacteria that show low tumbling frequency and precise adaptation can still show perfect chemotaxis

Take home message!

Exact adaptation is maintained despite substantial variations in the network-protein concentrations. Properties that are critical to the functioning of the network are selected to be robust so that they can withstand natural variations. Exact adaptation is critical for chemotaxis. Chemotaxis ability is not dependent on the precise value of the steady-state tumbling frequency and the adaptation time.

Scatchard plot for serine binding to receptor One serine/TSr dimer

No difference in serine affinity for receptor whether receptor was alone or in complex with CheA and CheW

Receptor sensitivity varies in relation to background stimulus intensity Weber-Fechner relationship true of most vertebrate sensory systems

Mutant phenotypes
protein MCP I (tsr) location Inner membrane Inner membrane function binds serine (attractant), or leucine (repellant) directly binds aspartate, the maltose receptor protein (attractants) and Ni, Co (repellants) binds the galactose and ribose receptor proteins binds the dipeptide receptor protein binds maltose to MCP II effect of gene deletion no response to either serine or leucine, remainder OK no response to aspartate, maltose, Ni or Co, but all other systems operate OK no response to galactose or ribose, all other systems OK no response to dipeptides, but all other systems are OK no response to maltose, but all other systems are OK no response to galactose, but all other systems are OK no response to ribose, but all other systems are OK no response to dipeptides, but all other systems are OK MCP II (tar)

MCP III (trg)

Inner membrane Inner membrane periplasm

MCP IV (tap)

maltose receptor galactose receptor ribose receptor dipeptide receptor

periplasm

binds galactose to MCP III

periplasm periplasm

binds ribose to MCP III binds dipeptides to MCP IV

More mutant phenotypes

Che A cytoplasm phosphorylates itself if MCP empty, then Che Y & Che B counterclockwise bias: swim smoothly, cannot tumble Che B cytoplasm MCP methyl esterase when phosphorylated insensitive, no adaptation, no memory

Che R Che W Che Y

cytoplasm cytoplasm cytoplasm

MCP methylator (uses SAM) connects MCPs to Che A motor turns clockwise if this protein is phosphorylated

hypersensitive, no adaptation counterclockwise bias counterclockwise bias

Che Z

cytoplasm

Che Y phosphatase

incessant tumbling

The bacterial nanobrain

Two-component systems

Senses glycopeptide antibiotic; histidine kinase

Response regulator; increases expression of VanH, VanA, VanX

Plasmid borne genes

Evolutionary origins of the chemotaxis pathway organization

The important step in the emergence of the chemotaxis system from the canonical two-component pathway was separation of the sensor into multiple proteins. The primary evolutionary pressure for this process is likely to have come from the need to sense a large variety of ligands while feeding all the signals into the same signalling output, the task that is not easily accomplished by a single sensor.

The resulting separation of receptors and kinase further simplified the evolution of new ligand specificities by receptor duplication and mutations without affecting the functionality of the existing system.
On the other hand, it raised the problem of coupling multiple receptors to the same kinase, which was apparently solved by the appearance of the adaptor protein CheWevolutionary related to the receptor-binding C-terminal domain of CheAand led to the emergence of receptor clustering. Subsequently, the allosteric interactions between receptors in clusters may have evolved giving rise to exquisite sensitivity.

A final tribute!
Bacterial chemotaxis receptor complexes seem to function as rudimentary brains If a brain is an organ that uses sensory information to control motor activity, then the bacterial nanobrain would fit the definition Are bacteria really primitive? Do they have intelligence? Do we worry about individual versus population behaviour? Non-genetic variability? It seems prudent at this time to leave open the question of how smart bacteria really are until we have a more thorough understanding of what they might be thinking and how much they might be talking to each other Webre et al (2003) Current Biology 13: R47-R49