NalP
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The Translocator Domain for the Autotransporter NaIP within Neisseria meningitidis provides a novel protein pore that contains an alpha helix running axially through its hydrophobic center. Classically many outer membrane pores contain a 12 member beta barrel which is able to allow for different conditions than the peptidoglycan layer that would typically stop many types of proteins and ions from passing through. This alpha helix blocks the pore from being totally open and allows for more regulation of what enters and leaves the cell.
Chemical PropertiesChemical Properties
Chemical Formula: C20H22N8O5
Molecular Weight: 454.44 g/mol
Half-life: 3–15 hours[1]
HistoryHistory
Methotrexate has provided as a treatment option in clinical setting since the year 1948. Leukemia patients whom received folic acid, were observed to decline, while patients with restricted folic acid consumption improved, prompting experiments with analogs of folic acids. Methotrexate was originally developed from these observations suggesting that an analog of folic acid was able to cause a remission in symptoms of acute lymphoblastic leukemia in 1947. The subsequent derivation of a mechanism of action for methotrexate was developed and methotrexate was used for treatment of various cancerous even non-cancerous cases[2].
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Structural Features DHFRStructural Features DHFR
Human DHFR can be visualized as an as well as its . DHFR contains 4 alpha helical regions and 8 beta sheets as can be seen in its . The can also be seen. Human DHFR catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid, with NADPH serving as the electron donor in this reaction. The can be seen with the residues that facilitate substrate binding and reaction process. The red residues represent the active site amino acid side chains interacting with the substrate, and the blue amino acid side chains help bind NADPH, with both folate and NADPH represented in white. can both be seen interacting with the DHFR enzyme (folate surrounded by red sidechains, and NADPH surrounded by blue sidechains)[3].
StructureStructure
Beta BarrelBeta Barrel
The unique structure that makes this pore is able to allow for transportation in and out of the gram-negative cell is what is called a beta barrel. This beta barrel is created with 12 anti-parellel beta-pleated sheets that have wrapped around creating anti-parellel interaction between sheet one and sheet 12. This creates a tube structure that transcends through the membrane of a cell creating a new environment that allows for polar molecules to move through the cell membrane and cell wall when they would have otherwise been stopped by the hydrophobic center of peptidoglycan. The start and end of the beta barrel is on the periplasm side of the membrane and after a short tight turn T0 becomes the alpha-helix which has its n-terminus side facing outward toward extracellular material.
Alpha HelixAlpha Helix
The Alpha Helix within the Beta Barrel is a major obstruction which allows for regulated channel. The Alpha Helix corresponds to the .15nS opening that is observed and without this obstruction a 1.3nS open pore is created which allows for a much more free flowing pore. This is found to be infrequent occurrence which could be caused by a detergent and high salt concentration. Due to this being the more infrequent type of pore it is able to be deduced that the internal alpha helix is what is found in vivo. The alpha helix is found internally on the N-terminus side of the protein and extends from n-terminus facing the extracellular space leading inward toward the cytoplasm which turns then into a beta pleated sheet that creates the barrel shape. This structure is consistent with the final stage of translocation which allows for proteins to be released in to the extracellular space. The alpha helix is charged almost solely on one side. This charged side is able to interact with an axial line of charged side chains that point inward from the beta barrel. Through seven salt bridges as well as through 16 hydrogen bonds and van der Waals contacts the alpha helix is able to interact with one side of the beta barrel.
Function of Alpha HelixFunction of Alpha Helix
In order to test the function of the alpha helix within the domain a test was done in order to compare results of uptake within the domain with alpha helix and without the helix. An easy way to test this was a antibiotic assay. By isolating colonies strictly of gram negative bacteria with the alpha helix, NalPβ, and isolating colonies strictly of gram negative bacteria without the alpha helix, NalPβΔhelix, they could plate these separately. From there it was possible to compare susceptibility to antibiotics by placing the small circular tabs of antibiotics on the plate and measuring the difference in how effect the antibiotics was in penetrating the cell. The more penetration would show less growth inhibition or more sensitivity to the antibiotics. When the alpha helix was removed there was much more sensitivity to antibiotics showing that the removal of it leads to a more open pore.
Protein Transportation MechanismProtein Transportation Mechanism
Picture 1Picture 1
Interesting questions were raised on how the alpha helix in the center if the beta barrel effected the mechanism of protein transportation out of the cell. The first step into understanding what shapes of proteins can move though the pore was figured by trying to move a disulfide bind through the pore. This was unsuccessful and led to part of the understanding that the only way that proteins can move though this pore was by being completely unfolded. Yet once inside of the extracellular material the protein much be folded. Knowing these tow crucial pieces of data it was clear that as the protein passes through the pore it is folded, and due to the c-terminal end's placement on the periplasm side of the pore it was highly unlikely that was the participating portion that effected the change in conformation of the protein as it passes through. Oppositely the n-terminal side of the pore lies on the alpha helix facing the extracellular matter placing it in prime location the change the conformation of the passing protein. Another possible place where interaction could occur between the passing protein and the pore would be at a large hairpin loop that is on the extracellular side of the pore. This would also provide a prime placement for initiation of protein folding.
Threading ModelThreading Model
The Threading Model could be one possible model that would allow of transportation of passenger proteins out of the cell and into extracellular material. The threading model is a possible explanation for how a protein would able to fit through the narrow gap that the beta barrel and alpha helix provide. The threading model allows for one strand of the DNA to pass
Similar Structure in Other ProteinsSimilar Structure in Other Proteins
Recent research has showed that there are possible conserved features to the this pore within other pores in other types of gram-negative bacteria. Proteins include; AidaI of E. coli, BrkA of B. pertussis, Hap of Hemophilus influenzae and IgA protease and App of N. meningitidis. Much of the these proteins show low conservation within their alpha helix's yet they all have a long transversing alpha helix that leads into the 12 sheeted beta barrel. Due to much of the research that is being done within Neisseria meningitidis' NalPβ protein, its crystal structure is being used in order to compare against other autotransporter secreting proteins.