[Hplusroadmap] Bacteria with 'brain' proteins?

[Bryan Bishop]

On Wednesday 25 June 2008, Dan Bolser wrote:
> Just for your interest...
> http://www.rcsb.org/pdb/explore/pubmed.do?structureId=2A65

Actual PDF:
http://www.ministerosalute.it/imgs/C_17_orizzonti_639_fileabstract.pdf

> Correspondence to: Eric Gouaux1,2 Correspondence and requests for
> materials should be addressed to E.G. (Email: jeg52 at columbia.edu).
> The coordinates for the structure have been deposited in the Protein
> Data Bank under the accession code 2A65.

So I guess the real question is what sort of searches could we do over 
the FASTA sequence data, and what can we relate it to in the 
neurotransmitter protein/gene databases? Here's the AA seq:

MEVKREHWATRLGLILAMAGNAVGLGNFLRFPVQAAENGGGAFMIPYIIAFLLVGIPLMWIEWAMGRYGGAQGHGTTPAI
FYLLWRNRFAKILGVFGLWIPLVVAIYYVYIESWTLGFAIKFLVGLVPEPPPNATDPDSILRPFKEFLYSYIGVPKGDEP
ILKPSLFAYIVFLITMFINVSILIRGISKGIERFAKIAMPTLFILAVFLVIRVFLLETPNGTAADGLNFLWTPDFEKLKD
PGVWIAAVGQIFFTLSLGFGAIITYASYVRKDQDIVLSGLTAATLNEKAEVILGGSISIPAAVAFFGVANAVAIAKAGAF
NLGFITLPAIFSQTAGGTFLGFLWFFLLFFAGLTSSIAIMQPMIAFLEDELKLSRKHAVLWTAAIVFFSAHLVMFLNKSL
DEMDFWAGTIGVVFFGLTELIIFFWIFGADKAWEEINRGGIIKVPRIYYYVMRYITPAFLAVLLVVWAREYIPKIMEETH
WTVWITRFYIIGLFLFLTFLVFLAERRRNHESAGTLVPR

Better:
http://www.rcsb.org/pdb/download/downloadFile.do?fileFormat=FASTA&compression=NO&structureId=2A65

> Article Title           
> Crystal structure of a bacterial homologue of Na+/Cl--dependent
> neurotransmitter transporters.

http://heybryan.org/mediawiki/index.php/Neurochem_kit
http://heybryan.org/new_exp.html
http://biohack.sf.net/

I wonder what the extraction protocol is for leucine extraction? In 
fact, it doesn't look like that's what they are doing here.

> Abstract
> Na+/Cl--dependent transporters terminate synaptic transmission by
> using electrochemical gradients to drive the uptake of
> neurotransmitters, including the biogenic amines, from the synapse to
> the cytoplasm of neurons and glia. These transporters are the targets
> of therapeutic and illicit compounds, and their dysfunction has been
> implicated in multiple diseases of the nervous system. Here we
> present the crystal structure of a bacterial homologue of these
> transporters from Aquifex aeolicus, in complex with its substrate,

Hm. Aquifex aeolicus?

http://microbewiki.kenyon.edu/index.php/Aquifex
> Deep-branching bacteria of the genus Aquifex, such as Aquifex
> pyrophilus and Aquifex aeolicus, can grow at 95 degrees C; they are
> among the most extreme thermophilic bacteria known. A. aeolicus was
> first obtained by R. Huber and K.O. Stetter at the Aeolic Islands
> (north of Sicily, Italy), whereas A. pyrophilus was obtained at the
> Kolbensey Ridge, North of Iceland..
>
> Because of this, Aquifex is thought to be one of the earliest
> bacteria to diverge from eubacteria. Hyperthermophilic bacteria such
> as Aquifex are important for industrial processes and its genes can
> be used in a variety of biotechnological applications.
>
> Even though Aquifex has the ability to survive at extreme
> temperatures, there are only a few specific heat-resistance
> indicators evident in the A. aeolicusgenome. The genome, which is
> 1,551,335 bp in length, is densely packed and contains genes that
> overlap others. In addition, no introns or protein splicing elements
> have been found.. This, along with a reduced metabolic flexability,
> is probably due to the limited genome size; the genome of this
> complex organism is ony one-third of the E. coli genome. The
> calculated sequence redundancy for the A. aeolicus genome is 4.83
> (Deckert et al. 1998). Comparison of the Aquifex genome to other
> organisms showed that 16% of it genes originated from archaea
> bacteria. Visit TIGR for more information on the genome and
> chromosomal structure of A. aeolicus VF5.
>
> Aquifex are nonsporeforming, gram-negative, generally rod-shaped
> organisms. They are about 2.0-6.0 micrometers in length and have a
> diameter of 0.4-0.5 micrometers. As autotrophic organisms, Aquifex
> fix carbon dioxide from the environment to get the carbon that they
> need. They are chemolithotrophic, which means that they draw energy
> for biosynthesis from inorganic chemical sources. The enzymes this
> organism uses for aerobic respiration are similar to the enzymes
> found in other aerobic bacteria (Deckert et al. 1998). A. aeolicus
> requires oxygen from the air as an electron acceptor to oxidize
> hydrogen gas (Prokaryotes):
>
> 2 H2 + O2 → 2 H2O
>
> Aquifex, meaning "water-maker," got its name because the the final
> product of this reaction is water. Even so, most Aquificales can use
> thiosulfate or sulfur as an energy source (much like chlorobium and
> other green sulfur bacteria) and produce sulfuric acid and H2S
> instead of water. Although most Aquifcales are strictly aerobic, A.
> pyrophilus was shown to be able to grow anaerobically by reducing
> nitrogen instead of oxygen (forming an end product of N2 instead of
> water). For more information and diagrams of known A. aeolicus
> metabolic pathways, visit Systems Biology Inc.
>
> One recent study has found that Non-N-methylated APT lipids can be
> observed as a common phospholipid headgroup in Aquifex. The study
> concluded that "this apparent evidence for a more widespread presence
> of the APT headgroup among the deeply branching bacteria
> provideshurther evidence that certain deeply branching bacteria share
> structural features other than the well-described presence of
> ether-bound alkyl chains withthe archaeal domain."
>> As a hyperthermophilic bactertium, Aquifex aeolicus grows in
> extremely hot tempuratures such as near volcanoes or hot springs.
> They grow optimally at temperatures around 85 degrees but can grow at
> temperatures up to 95 degrees. It needs oxygen to carry on its
> metabolic machinery, but it can function in relatively low levels of
> oxygen (A. pyrophilus can grow in levels of oxygen as low as 7.5
> ppm). A. aeolicus can grow on hydrogen, oxygen, carbon dioxide, and
> mineral salts (Deckert et al. 1998). Aquifex species generally form
> large cell aggregates, which can be comprised of up to 100 individual
> cells.

Deckert, Gerard, et al. 1998. "The complete genome of the 
hyperthermophilic bacterium Aquifex aeolicus." Nature, Vol. 392. 
Macmillan Publishers. 353-358. 

Huber, R., et al. 1998 Thermocrinus ruber, gen. nov., sp. nov., a 
pink-filament-forming hyperthemophilic bacterium isolated from 
Yellowstone National Park. Appl. Env. Microbiol. 64:3576-3583. 

Prescott, Harley, Klein. 21Bacteria: The Deinococci and 
Nonproteobacteria: Gram-Negatives. McGraw Hill Higher Education. 

Sturt, Helen F.; Roger E. Summons, Kristin Smith, Marcus Elvert, and 
Kai-Uwe Hinrichs. "Intact polar membrane lipids in prokaryotes and 
sediments deciphered by high-performance liquid 
chromatography/electrospray ionization multistage mass spectrometry—new 
biomarkers for biogeochemistry and microbial ecology." Rapid Commun. 
Mass Spectrom. 2004; 18: 617–628. 

The Prokaryotes: An Evolving Electronic Resource for the Mircobiological 
Community. 2004. Springer-Verlag New York, LLC.

http://en.wikipedia.org/wiki/Aquifex_aeolicus
> Aquifex aeolicus is a rod-shaped bacterium with a length of 2 to 6
> micrometers and a diameter of around half a micrometer. It is one of
> a handful of species in the Aquificae phylum, an unusual group of
> thermophilic bacteria that are thought to be some of the oldest
> species of bacteria.
>
> A. aeolicus grows best in water between 85 to 95 °C, and can be found
> near underwater volcanoes or hot springs. It requires oxygen to
> survive (though it can grow in levels of oxygen as low as 7.5 ppm),
> and its method of respiration produces water as a byproduct.
> ("Aquifex" means "water-maker.") Members of the species tend to form
> large cell conglomerations, comprised of up to 100 individual cells.
> It was discovered around islands north of Sicily.
>
> The genome of A. aeolicus has been successfully mapped. This was made
> easier by the fact that the length of the genome is only about a
> third of the length of the genome for E. coli. Comparison of the
> Aquifex aeolicus genome to other organisms showed that around 16% of
> its genes originated from the Archaea domain.

Back to the abstract:
> leucine, and two sodium ions. The protein core consists of the first

http://en.wikipedia.org/wiki/Leucine

> Leucine (abbreviated as Leu or L)[1] is an α-amino acid with the
> chemical formula HO2CCH(NH2)CH2CH(CH3)2. It is an essential amino
> acid, which means that humans cannot synthesise it. Its codons are
> UUA, UUG, CUU, CUC, CUA, and CUG. With a hydrocarbon side chain,
> leucine is classified as a hydrophobic amino acid.
>
> As an essential amino acid, leucine is not synthesized in animals,
> hence it must be ingested, usually as a component of proteins. It is
> synthesized in plants and microorganisms via several steps starting
> from pyruvic acid. The initial part of the pathway also leads to
> valine. The intermediate α-ketovalerate is converted to
> α-isopropylmalate and then β-isopropylmalate, which is dehydrogenated
> to α-ketoisocaproate, which in the final step undergoes reductive
> amination. Enzymes involved in a typical leucine biosynthesis
> include[2] Acetolactate synthase,
> Acetohydroxy acid isomeroreductase,
> Dihydroxyacid dehydratase,
> α-Isopropylmalate synthase,
> α-Isopropylmalate isomerase,
> Leucine aminotransferase.
>
> As a dietary supplement, leucine has been found to slow the
> degradation of muscle tissue by increasing the synthesis of muscle
> proteins.[3] Leucine is utilized in the liver, adipose tissue, and
> muscle tissue. In adipose and muscle tissue, leucine is used in the
> formation of sterols, and the combined usage of leucine in these two
> tissues is seven times greater than its use in the liver.[4]

An interesting side-note:
http://en.wikipedia.org/wiki/Photo-reactive_amino_acid_analog
> Photo-reactive amino acid analogs for in-vivo crosslinking of protein
> complexes were introduced in 2005 by researchers from the Max Planck
> Institute [1] In this method, cells are grown with photoreactive
> diazirine analogs to leucine and methionine, which are incorporated
> into proteins. Upon exposure to ultraviolet light, the diazirines are
> activated and bind to interacting proteins that are within a few
> angstroms of the photo-reactive amino acid analog.
>
> L-Photo-Leucine and L-Photo-Methionine are analogs of the naturally
> occurring L-Leucine and L-Methionine amino acids that are
> endogenously incorporated into the primary sequence of proteins
> during synthesis using the normal translation machinery. They are
> then ultraviolet light (UV)-activated to covalently crosslink
> proteins within protein-protein interaction domains in their native
> in-vivo environment. The method enables the determination and
> characterization of both stable and transient protein interactions in
> cells without the addition of chemical crosslinkers and associated
> solvents that can adversely affect the cell biology being studied in
> the experiment.
>
> When used in combination with limiting media that is devoid of
> leucine and methionine, the photo-activatable derivatives are treated
> like naturally occurring amino acids by the cellular protein
> synthesis machinery. As a result, they can be substituted for leucine
> or methionine in the primary structure of proteins. Photo-leucine and
> photo-methionine derivatives contain diazirine rings that are
> activated when exposed to UV light to become reactive intermediates
> that form covalent bonds with nearby protein side chains and
> backbones. Naturally interacting proteins within the cell can be
> instantly trapped by photoactivation of the diazirine-containing
> proteins in the cultured cells. Crosslinked protein complexes can be
> detected by decreased mobility on SDS-PAGE followed by Western
> blotting, size exclusion chromatography, sucrose density gradient
> sedimentation or mass spectrometry.

External links on leucine:

Leucine biosynthesis
http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/AminoAcid/Leu.html

Leucine content in food
http://www.dietaryfiberfood.com/leucine-rich.php

Computational Chemistry Wiki
http://www.compchemwiki.org/index.php?title=Leucine

Leucine prevents muscle loss in rats
http://news.bbc.co.uk/1/hi/health/4502862.stm

Leucine helps regulate appetite in rats
http://www.newscientist.com/article/dn9147-why-highprotein-meat-may-curb-appetite.html

Combined ingestion of protein and free leucine with carbohydrate 
increases postexercise muscle protein synthesis in vivo in male 
subjects
http://ajpendo.physiology.org/cgi/content/full/288/4/E645

> ten of twelve transmembrane segments, with segments 1-5 related to
> 6-10 by a pseudo-two-fold axis in the membrane plane. Leucine and the
> sodium ions are bound within the protein core, halfway across the

Odd how they don't say what the actual protein is. The abstract mentions 
leucine, but not what the name or gene id of the protein core.

> membrane bilayer, in an occluded site devoid of water. The leucine
> and ion binding sites are defined by partially unwound transmembrane
> helices, with main-chain atoms and helix dipoles having key roles in
> substrate and ion binding. The structure reveals the architecture of
> this important class of transporter, illuminates the determinants of
> substrate binding and ion selectivity, and defines the external and
> internal gates.

I don't get it. What is it a homologue _of_ ? Neurotransmitter 
transporters are quite varied. For instance:

http://en.wikipedia.org/wiki/Neurotransmitter_transporters

Iversen L. 2000. Neurotransmitter transporters: fruitful targets for CNS 
drug discovery Mol Psychiatry Volume 5, Issue 4, Pages 357-362. 
Accessed January 8, 2007.

There's more than 20 types.

Looks like the Blakey Lab is exploring the molecular dynamics behind 
these transporters. 

http://web.mac.com/rdblakely/iWeb/BlakelyLab/Home.html

Fun stuff. So what are these thermophiles doing with these proteins? I 
was going to ask this earlier but since I don't see the actual 
homologue I'm not entirely sure what's going on, however it's 
interesting to ponder in which direction the transporter came from. Was 
it sourced from the bacteria, or did the bacteria later incorporate it 
from mammalian genomes?

It would be interesting if there's a pattern of aggregation of various 
bacterial molecules in the animalean brains, since it would be 
evolutionarily advantageous to make the bacteria do all of the hard 
work ... but that doesn't explain how the framework would come 
together. 

Is it a case of coincidence?

- Bryan
________________________________________
http://heybryan.org/