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  Topic: Evolution of prokaryote flagella, Links to discussions, webpages, refs< Next Oldest | Next Newest >  

Posts: 319
Joined: May 2002

(Permalink) Posted: May 06 2003,16:00   

Yet another reply for the archive:

[A brief aside: Nelson, your sentences often run-on and seem rather breathless.  This makes them difficult to interpret sometimes.  Just a heads-up...]

Regarding the mechanism of flagellar rotation, Nelson really should have read Kojima and Blair's 2001 paper before citing older papers in favor of Nelson/Mike Gene's favored model:


With respect to electrostatic interaction, I don't think we can say that the channel is internal to ExbBD. TonB might function in opening the channel or causing some kind of structural change. ExbB has only 3 membrane segments as opposed to MotA which has four.

The electrostatic model of the bacterial flagellar motor seems to be a bit more then a hunch, in fact, that electrostatic model behind flagella rotation seems to be near certainty that it is correct:

Bren, Anat and Michael Eisenbach. "How Signals Are Heard during Bacterial Chemotaxis: Protein-Protein Interactions in Sensory Signal Propagation." Journal of Bacteriology. 182:6865-6873 (2000).

I think even Blair, the author of the paper you cite for the ExbBD complex has done some work on this:

Zhou, J.D., Lloyd, S.A., and Blair. D.F. (1998) Electrostatic interactions between rotor and stator in the bacterial flagellar motor. Proc. Natl. Acad. Sci. USA 95, 6436-6441

Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator.

So it may not even matter that MotAB is homologous to ExbBD, if the electrostatic interactions are completely essential. Thus, I think Mike's objection should be taken seriously here with respect to the logistical problems with random mutations and ion channels.

LOL!  Virtually everything you argue ("near certainty" that the electrostatic model is correct, the channel is not internal to MotAB) is contradicted by the very Kojima and Blair article I've been citing.

Kojima and Blair write that based on the results of their 2001 paper, they think that the proton channel is internal and that the MotAB conformational-change model is probably correct:


In the years since the discovery of flagellar rotation, many hypotheses for the mechanism have been proposed (reviewed in ref 43). The models are diverse, but can be classified according to whether the proton pathway includes elements of both the rotor and stator or is confined to just the stator (Figure 1). Because the mutational studies found no critical titratable residues on the rotor, we currently favor models in which protons remain within the stator. In this case, proton flow must be coupled to rotation by some means other than direct proton-rotor contact. Our hypothesis is that protonation of Asp32 in MotB drives conformational changes in the stator, which work on the rotor to drive rotation.


Here, we test for conformational changes in the MotA/MotB complex by using limited proteolysis. Patterns of proteolysis of MotA were compared in wild-type MotA/MotB complexes and complexes with mutations in key residues of one or both of the proteins. The results support a mechanism in which the stator undergoes significant changes in conformation. [bold added]
Indeed, the separability of the motor as an independent subunit appears to be what led them to look this is what led them to look for independently-functioning homologs in simpler contexts:


The occurrence of significant conformational change in the stator has implications not only for the present-day mechanism but also for the evolution of the flagellar motor. A membrane complex that undergoes proton-driven conformational changes could perform useful work in contexts other than (and simpler than) the flagellar motor, and ancestral forms of the MotA/MotB complex might have arisen independently of any part of the rotor. We queried the sequence database using the sequence of the best-conserved part of MotA (the segment containing membrane segments 3 and 4) from Aquifex aeolicus, a species whose lineage is deeply branched from other bacteria. In addition to the expected MotA homologues, the search returned a protein sequence from the archaeal species Methanobacterium thermoautotrophicum (protein MTH1022) that shows significant sequence similarity not only to MotA but also to the protein ExbB (Figure 9).

As for mutational flexibility:


Residues of MotA that are important for function include two charged residues in the cytoplasmic domain, Arg90 and Glu98, that interact with the functionally important charged residues of FliG (39, 40). Like the charged residues of FliG, Arg90 and Glu98 of MotA function redundantly, and charge appears to be their key property. Two Pro residues of MotA located at the cytoplasmic ends of membrane segments, Pro173 and Pro222, are also important for rotation and might function to regulate conformational changes occurring during the torque-generating cycle (41). In MotB, an aspartic acid residue near the cytoplasmic end of the membrane segment, Asp32, is conserved and critical for motor rotation. A survey of conserved residues in MotA, MotB, FliG, FliM, and FliN found that no other titratable residue is critical for motor rotation (42). Asp32 is likely to have a direct role in the conduction of protons.
It appears that, basically, you need the Asp32 to transfer the proton through the channel internal to MotA/B, and then a significant variety of charged residues on the C-ring (FliG/M/N) and MotA can transfer the conformational change in MotA to "push on" the C-ring.  This seems rather less daunting than Mike Gene's characterization which you quoted:


Of all the ways to mutate an ion channel, the number of ways that would result in its interacting with the base of some filament is surely in the distinct minority. And of all the ways to mutate an ion channel that gloms onto a filament, the number of ways to mutate it such that rotation does not occur is probably much higher than the number of ways to elicit some rotation...This [mutation] allows some ion channel to glom onto the base of a filament and open its channel and expose the ion flow to the proto-rotor in such a way that a set of electrostatic interactions just happen to form and elicit significant rotation. Suffice it to say that such an improbable mutation has never been observed in nature or the lab.
This is not the only place where Kojima and Blair tripped up Mike Gene, indeed, he was originally skeptical of the existence of nonflagellar MotAB homologs:

MotA/MotB, on the other hand, could plausibly exist as some ion channel prior to the existence of the flagella, but there is no evidence of this.

I'll address the other points when I have time.  Like I said, why should we take your probability calculation seriously, when it is based on incorrect assumptions about the mechanism of rotation, the independence of the MotAB complex homologs, etc.?  Why doesn't your calculation include the possibility of cooption of MotAB from a pre-existing, simpler system?  Why does your calculation assume that proteins are drawn at random from a mythical "protein supermarket" when we have reasonable hypotheses for the prior functions of many of these proteins, and we have a large body of empirical evidence for the efficacy of natural processes to produce new genes by cooption and modification of old genes?

Edited by niiicholas on May 06 2003,16:03

  46 replies since Nov. 28 2002,22:50 < Next Oldest | Next Newest >  

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