NalP

spinqualitylabelsall models Methotrexate Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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

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.

spinqualitylabelsall models Human DHFR complexed with NADPH and folate Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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).

NADPH Residue Interaction

Folate Residue Interaction

Thymidine Synthesis Mechanism

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.

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.

Experimental MutationExperimental Mutation

spinqualitylabelsall models DHFR methotrexate Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The features of DHFR ligand binding, specifically to methotrexate can be observed and analyzed through various molecular docking and mutation experiments. The a structurally engineered variant of the altered the F 31 residue of the protein to R, and the Q 35 residue of the protein to E in an attempt to explore the specifics of the methotrexate affinity for DHFR active site residues, resulting in varied active site residues from phenylalanine and glutamine to . This mutated enzyme featured a 650x decrease in affinity for the ligand, methotrexate, but retained an amount of methotrexate interaction similar to the enzyme in its native state with native substrates. Crystal structure analysis revealed that the lack of cooperative action and presence of residue disorder lead to the significant decrease in methotrexate activity with the resulting . The arginine residue at place 31, was specifically observed in numerous conformations, a characteristic unique to the mutated enzyme, and the probable cause of the loss of polar contacts and binding affinity between methotrexate and DHFR. A loss of van der Waal forces due to the conformations of the side chains along with an unfavorable placement of Glu-35 causing an “unfavorable electrostatic contact” with methotrexate’s “glutamate portion.” Interestingly this variant was found to display a greater decrease in methotrexate affinity than the decrease in affinity of Dihydrofolate, found to be 9x, evident of catalytic efficiency retention which hold many drug binding resistance implications.

Methotrexate Variant Residue Interaction

Pharmaceutical ImplicationsPharmaceutical Implications

Methotrexate’s inhibition of cellular replication causes it to have an increased toxic response on cells performing DNA replication especially rapidly proliferating cells. These cells display decreased growth and division due to a lack of nucleoside biosynthesis metabolites, resulting in decreased dTMP. Methotrexate is able to interfere with rapid cell growth in this manner, specifically infecting cells including skin cells, bone marrow cells, and often cancer cells, making methotrexate an effective cancer treatment drug. Other DHF analogs exist which can be useful as anticancer agents or antibacterial agents, through inhibition of DHFR .

Drug TreatmentDrug Treatment

Methotrexate is often used in a regimental approach to the chemotherapeutic treatment of cancers and other diseases involving replicating tissue. A variety of specific cancer types have been treated with methotrexate including head, lung, skin, or breast cancer. Methotrexate has also been used in the treatment of various autoimmune diseases. Rheumatoid arthritis and psoriasis have also utilized methotrexate as a treatment method, presumably to diminish immune function. The mechanism of methotrexate in these instances varies from the inhibition of DHFR, but involves inhibition of enzymes involved with purine metabolism resulting in various types of immune suppression including inhibition of T cell activation. Because of this altered mechanism, treated patients are often administered folate to offset the antifolate characteristics of methotrexate. The targeting of rapidly replicating cells allows methotrexate to function as an abortifacient as well. These uses of methotrexate need to be carefully monitored with proper dosage because methotrexate is embryotoxic, carcinogenic, and teratogenic.

Trexall is a drug, methotrexate tablet, used as an antimetabolite for treatment of neoplastic diseases, severe rheumatoid arthritis and psoriasis.

TreatmentTreatment

Methotrexate can be administered orally as well as intravenous, intramuscular, subcutaneous, or intrathecal injection. Dosage amount is a crucial aspect of any methyltrexate treatment because of the serious side affects, and often results in dosages being taken rarely more than once or twice a week. The immune system, blood cells, and other rapidly replicating cells including liver, lungs, kidneys are often succeptable to damage which requires regular tests. Side effects from this drug can be common and sever including neutropenia, hair loss, nausea, dermatitis, and anemia, often representative of the antimetabolite function of methotrexate. Stomatitis is not commonly seen with weekly doses, but daily doses for 5 consecutive days often results in these symptoms including renal impairment, toxicity, and possible failure. Myelosuppression may develop with increased dosages, enhancing tissue damage resulting most commonly from radiation of cancer patients. Additional drugs including antibiotics can often result in adverse side effects, and increased methotrexate retention due to additional drugs can often lead to a dangerous increase in concentration of methotrexate in the blood .

The nature of this treatment type can requires the “rescue” of a patient, through withdrawal of the inhibitor and possible administering of thymidine or folic acid based drugs to prevent the toxicity sometimes seen in beneficial rapidly replicating cells. Leucovorin is often administered for this rescuing effect. Leucovorin or folinic acid is a derivative of THF, and can be readily converted to tetrahydrofolate overcoming the effect of methotrexate because it bypasses the dihydrofolate reductase mechanism to produce THF.

PharmacokineticsPharmacokinetics

Dosage size of methotrexate is extremely important because of the antimetabolic function of the drug, therefore many pharmacokinetic properties must be considered prior to treatment. Methotrexate is a dicarboxylic acid, although with a pKa of 4.8 and 5.5 is weak and often ionized in physiological conditions. Bioavailability following oral absorption is dose dependent, with 60 percent at doses lower than 30 mg/m2, and at concentrations above 80 mg/m2, there is only 20 percent bioavailability, percentages that can be increased with intramuscular administering of the drug. Only about 5 percent of the total loss of the oral dose is due to bacterial degradation. The kidney, spleen, liver, gallbladder, as well as the skin display the highest levels of methotrexate upon treatment. This drug does not cross the blood brain barrier efficiently, but the distribution to the kidney and liver may be prolonged with higher doses extending drug clearance time. Methotrexate can be metabolized through the liver and intracellular mechanisms, and the kidneys are capable of excreting from 80 to 90 percent of the drug without metabolizing methotrexate.

Additional InformationAdditional Information

For additional information see: Pharmaceutical Drugs