NalP: Difference between revisions

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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 Properties==

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=== Alpha 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. 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.

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.

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== ~~Mechanism of Action ==~~

== Similar Structure in Other Proteins ==

<Structure ~~load='2w3m' size='500' frame='true' align='right' caption~~=~~'Human DHFR' scene='Insert optional scene name here' />~~

Methotrexate, is an antifolate which plays an inhibiting role in the synthesis of thymidylate through the prevention of THF regeneration. Methotrexate is a slow and tight binding competitive inhibitor of <scene name='Sandbox_58/Dhfr/1'>DHFR</scene>, resulting in the prevention of important metabolites necessary in thymidylate synthesis and nucleotide metabolism. Specifically, methotrexate acts as a DHF analog and through competitive inhibition of the DHFR active site, prevents the regeneration reaction necessary for further nucleotide biosynthesis. Methotrexate’s antimetabolite function seen in the competitive inhibition mechanism affects the metabolism of folic acid. Methotrexate is phase specific to the S phase of the cell cycle inhibiting DNA synthesis and replication within the afflicted cell. Competitive inhibition of the DHFR active site is possible because of the close resemblance that methotrexate shares with the metabolite being interfered with, dihydrofolate<ref>Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn (2002). "Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics". Proceedings of the National Academy of Sciences 99 (21): 13481–6.</ref>.

[[Image:Methotrexate and folic acid compared.png|folic acid methotrexate]]<ref>Methotrexate and Folic Acid. (2006, September 3). Wikimedia Commons. Retrieved March 10, 2011, from commons.wikimedia.org/.png </ref>

~~Folic Acid (left) Methotrexate (right)~~

The nature of this binding has a 1000 fold increase in affinity relative to the natural folate affinity of DHFR , producing a practically irreversible inhibition of <scene name='Sandbox_58/N_to_c/1'>DHFR</scene> activity, (blue = N-terminal, red C-terminal). Methotrexate is a competitive inhibitor that can bind to and inhibit the <scene name='Sandbox_58/Dhf_reductase/1'>DHRF active site</scene>, residues displayed in red, and the flexible Met20 loop surrounding the active site displayed in blue. Specifically, methotrexate is able to competitively interact</scene> with the <scene name='Sandbox_58/Active_site_mxt/2'>active site </scene> residues of DHFR, specifically Asp27, Phe31, Arg57, and Tyr100, with associations with the Asn18, Leu28, and Ile50 residues. The active site is buried within the enzyme as is depicted by the <scene name='Sandbox_58/Solvent_accessable_surface/1'>solvent accessable surface</scene> shown in orange at the entrance to the active site. The

<scene name='Sandbox_58/Relative_temperature/1'>relative temperature</scene> are color depictions of each atom in regards to mobility or position uncertainty relative to the molecule, with increasing mobility as the color scheme goes from blue to red. The interactions of the rest of the protein are depicted through the <scene name='Sandbox_58/H_bonds/1'>hydrogen bonds</scene> displayed in red<ref>Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Xuong N, Kraut J, Poe M, Williams M, Hoogsteen K (July 1977). "Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate". Science 197 (4302): 452–455.</ref>.

~~[[Image:2011~~-~~03-10_0224~~.~~png|500|left|thumb| Methotrexate Residue Interaction ]]<ref>DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE~~. ~~(n.d~~.~~). RCSB Protein Database. Retrieved March 10~~, ~~2011, from www~~.~~rcsb~~.~~org/pdb/results </ref>[[Image:DHFR ligands~~.~~png|500|center|thumb| DHFR substrates ]]<ref>Enzymes. (n.d.). Oregon State University. Retrieved March 10~~, ~~2011, from http://oregonstate.edu/instruction/bb450/fall2010/lecture/enzymesoutline~~.~~html </ref>~~

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.

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@@ Line 1: / Line 1: @@
   <Structure load='1UYN' size='350' frame='true' align='right' caption='Methotrexate' scene='Insert optional scene name here' />
+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 Properties==
@@ Line 46: / Line 46: @@
 === Alpha 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. 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.
+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.
@@ Line 54: / Line 54: @@
-== Mechanism of Action ==
+== Similar Structure in Other Proteins ==
-<Structure load='2w3m' size='500' frame='true' align='right' caption='Human DHFR' scene='Insert optional scene name here' />
-Methotrexate, is an antifolate which plays an inhibiting role in the synthesis of thymidylate through the prevention of THF regeneration. Methotrexate is a slow and tight binding competitive inhibitor of <scene name='Sandbox_58/Dhfr/1'>DHFR</scene>, resulting in the prevention of important metabolites necessary in thymidylate synthesis and nucleotide metabolism. Specifically, methotrexate acts as a DHF analog and through competitive inhibition of the DHFR active site, prevents the regeneration reaction necessary for further nucleotide biosynthesis.  Methotrexate’s antimetabolite function seen in the competitive inhibition mechanism affects the metabolism of folic acid.  Methotrexate is phase specific to the S phase of the cell cycle inhibiting DNA synthesis and replication within the afflicted cell.  Competitive inhibition of the DHFR active site is possible because of the close resemblance that methotrexate shares with the metabolite being interfered with, dihydrofolate<ref>Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn (2002). "Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics". Proceedings of the National Academy of Sciences 99 (21): 13481–6.</ref>.
-[[Image:Methotrexate and folic acid compared.png|folic acid                methotrexate]]<ref>Methotrexate and Folic Acid. (2006, September 3). Wikimedia Commons. Retrieved March 10, 2011, from commons.wikimedia.org/.png </ref>
-Folic Acid (left)                           Methotrexate (right)
-The nature of this binding has a 1000 fold increase in affinity relative to the natural folate affinity of DHFR , producing a practically irreversible inhibition of <scene name='Sandbox_58/N_to_c/1'>DHFR</scene> activity, (blue = N-terminal, red C-terminal).  Methotrexate is a competitive inhibitor that can bind to and inhibit the <scene name='Sandbox_58/Dhf_reductase/1'>DHRF active site</scene>, residues displayed in red, and the flexible Met20 loop surrounding the active site displayed in blue. Specifically, methotrexate is able to competitively interact</scene> with the <scene name='Sandbox_58/Active_site_mxt/2'>active site </scene> residues of DHFR, specifically Asp27, Phe31, Arg57, and Tyr100, with associations with the Asn18, Leu28, and Ile50 residues. The active site is buried within the enzyme as is depicted by the <scene name='Sandbox_58/Solvent_accessable_surface/1'>solvent accessable surface</scene> shown in orange at the entrance to the active site.  The
-<scene name='Sandbox_58/Relative_temperature/1'>relative temperature</scene> are color depictions of each atom in regards to mobility or position uncertainty relative to the molecule, with increasing mobility as the color scheme goes from blue to red.  The interactions of the rest of the protein are depicted through the <scene name='Sandbox_58/H_bonds/1'>hydrogen bonds</scene> displayed in red<ref>Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Xuong N, Kraut J, Poe M, Williams M, Hoogsteen K (July 1977). "Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate". Science 197 (4302): 452–455.</ref>.
-[[Image:2011-03-10_0224.png|500|left|thumb| Methotrexate Residue Interaction ]]<ref>DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE. (n.d.). RCSB Protein Database. Retrieved March 10, 2011, from www.rcsb.org/pdb/results </ref>[[Image:DHFR ligands.png|500|center|thumb| DHFR substrates ]]<ref>Enzymes. (n.d.). Oregon State University. Retrieved March 10, 2011, from http://oregonstate.edu/instruction/bb450/fall2010/lecture/enzymesoutline.html </ref>
+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.