Student Projects for UMass Chemistry 423 Spring 2011
From Proteopedia
Instructions and signup page (1-6 below) were posted in Sandbox423 for all students to edit. Students created projects (7-8 below, and additional links in section 3) on separate sandbox pages.
Spring 2011 Chem423 Team Projects: Understanding Drug Mechanisms
Project Instructions1. Team & Structure selection, due 4/1/11: All projects & names must be posted on the list below, linking to sandbox pages displaying initial structure.
2. Project completion, due 4/22/11. Your proteopedia page should be organized into the following 5 required sections, with each team member responsible for one of these sections of the team project. a. Introduction
b. Overall structure
c. Drug binding site
d. Additional features
e. Credits -- at the end list who did which portion of the project:
f. References This will include the published paper that describes your structure (the reference associated with your pdb code). You will get much of your information about specific interactions to look for and highlight in the structure from this reference (which is much easier than trying to find these on your own with no guidance!). 3. In-class presentations, to be announced ExampleThis is a complex between a macromolecule and its ligand (but this ligand is not a drug) that illlustrates the use of green scenes:
Asp Receptor Ligand-binding domain Overall structure The ligand binding domain of the aspartate receptor () ) is a dimer of two 4-helix bundles that is shown here with the bound.[1] In this the N and C termini are at the bottom of the structure; this is where the connections to the transmembrane helices have been truncated.
Ligand binding site Interactions that stabilize ligand binding[2] include hydrogen bonding from Tyr149 and Gln152 backbone carbonyls and Thr154 sidechain OH to the and hydrogen bonding from the sidechain nitrogens of Arg64, Arg69, and Arg73 to the two .
References
Project Teams, Topics, Links, and Presentation DatesC See comment on your page. Example (but not a drug complex): Lynmarie Thompson, ..., ..., ... - Asp receptor in complex with Asp (above)
Monday 4/25/11 Nick DeGraan-Weber, Jackie Dorhout, Rachael Jayne, Mike Reardon - flu neuraminidase in complex with tamiflu John Hickey, Josh Drolet, Josephine Harrington, Andrea Simoni - influenza M2 proton channel
UMass Chem 423 Student Projects 2011-1: Wednesday 4/27/11 Brittany Forkus, Katie Geldart, Elizabeth Schutsky, Breanna Zerfas - Beta Adrenergic GPCR Lucia Tringali, Shaina Boyle, Jaclyn Somadelis , Dany Mbakop -- HIV Protease Andy Kim, Zach Brentzel, Tyler Vlass, Zach Hitzig -- Acetylcholinesterase
Friday 4/29/11 Varun Chalupadi, Anthony Laviola, Tiffany Brucker, Alan Stebbins - Cyclooxygenase UMass Chem 423 Student Projects 2011-2: Inna Brockman, Robert Nathan, Sarena Horava, Nick Cadirov - p38 kinase David Peltier, Donald Einck, Ethan Leighton, Chris Coakley - Rituximab Fab
Monday 5/2/11 Max Moulton, Sally Stras, Jordan Schleeweis, Anh Huynh -- HIV reverse transcription Chris Brueckner, Daniel Roy, John Clarkson, Justin Srodulski -- Ketamine in binding complex with NMDA receptor Lyes Khendek, Paul Breslin, William Rowley, Joe Perito, Ashley Rivera - G-Quadruplex Students looking for group membersEach group should contain at least one person from a different primary major (typically Chemistry or Chemical Engineering) than the rest. List yourself + your major, list partial groups looking for members, list your complex if you have chosen one. Contact others to form a group. 4/1/11 update by Prof Thompson: The remaining students can go ahead and form teams regardless of major. Luis Cristian, Chem major, lcristia@student.umass.edu - looking to be in a group with chem eng Help EditingHint: Ctl-click or right-click on links below and select "Open Link in New Window" Start with Help in the navigation box on the left. Some things I've found useful:
[General help with Wiki editing], plus more [Wiki Text examples] Some of the above are for help editing Wikipedia pages, but the syntax is mostly the same. Proteopedia ADDS protein stuff to the WikiMedia markup language, which powers both WEB sites. Questions & AnswersHere is a place to post questions and answers for each other about how to do things in Proteopedia: A very useful color scheme is "chain" which colors separate proteins or DNA strands in different colors (first select all protein or DNA). Anyone know what format we should be putting our references in? For references, follow the format used in the example on the Asp receptor and they will be put in automatically. You just find out the PMID code (listed in pubmed for example) and insert it into the following, at the place where you want the reference cited (click edit to see what is actually inserted here). [1] You also need to add the section: References
To highlight some interesting portion of your protein: Under the selections tab, you can "limit to residue numbers." So for example enter in 60-65, then click "replace selection" below. Then if you go to the colors tab you can pick a color for just the residues you have selected. If it is a loop or if they are hard to see you can go to the representation tab and set selection to ball and stick or spacefill. It is also useful to click the "selection halos:" box under the picture. That shows you what you have in your selection.
Flu Neuraminidase IntroductionNeuraminidase protein is one of two glycoproteins that coats the envelope of the influenza virus. Neuraminidase's particular function is the removal of sialic acid from the host cell, allowing the replicated influenza viruses to escape the host cell and spread to other cells. Tamiflu, a drug designed to combat influenza, binds to and inhibits the function of neuraminidase, disabling its function and consequently disallowing the influenza virus to spread between cells. Influenza is a disease caused by the various species and genera of the influenza RNA virus. The disease, commonly referred to as "the Flu," is highly contagious and travels around the world in seasonal epidemics. It is responsible for the deaths of between 250,000 and 500,000 people each year, and these numbers can sometimes reach the millions in particularly pandemic years. The virus is particularly lethal to people with weakened immune systems, since the virus can enable the spread of dangerous secondary infections such as pneumonias within the host. The typical symptoms of the Flu include chills, fever, a sore throat, muscle pains, severe headache, coughing, and weakness or fatigue, as well as general discomfort. Overall StructureInfluenza (flu) Neuraminidase is a homotetramer, with four identical subunits. Each subunit consists mostly of antiparallel beta sheets and three alpha helices, with a beta-propeller folding pattern (). As shown, the alpha helices lie toward the center of the protein. The is a pocket on the surface of the protein, lined with highly conserved residues. Shown is the ligand Oseltamivir (Tamiflu) bound to one of the four subunits. When Tamiflu is bound, it tightly blocks the virus release sites on nonresistant strains of flu neuraminidase because it binds with the hydrophilic residues exposed on the surface. Different conformations account for drug resistance of flu neuraminidase. 2hu4 is the “closed” conformation of the wild type 3hu0. For example, a mutation at the site H274Y, on the closed-form wild type, causes a change in the usual binding site for Tamiflu, possibly affecting resistance. Research on the structures of the different mutants of flu Neuraminidase suggests that the slight change in the binding site affects the drug resistance of the molecule. In a specific case presented by Wang , et al., the H5N1 mutant being observed has a hydrophobic Tyr347 compared to the hydrophilic Asn347 of the wild type and closed form. Since Tamiflu binds to hydrophilic regions of the pore, this change has the potential to greatly affect the binding affinity, hence the mutant’s resistance to oseltamivir. Drug Binding SiteFlu neuraminidase is a homotetramer, and each of the four protein chains has a catalytic site. The catalytic sites are shown with Tamiflu shown in purple. Tamiflu was designed specifically to fit to the binding site of neuraminidase by induced fit. The binding site contains a loop of residues 147-152. When it binds at the active site, Tamiflu pulls a loop made of Asp151 and Glu119 closer to the inhibitor, thereby enclosing the Tamiflu inhibitor. There are five specific residues that are in contact with the Tamiflu substrate - Arg292, Glu276, Arg152, Arg371, and Arg118. They are in the active site. Tamiflu does not work on all types of the flu virus, and some are becoming immune to it. However, it was recently discovered that one of the subunits has a larger surface-accessible cavity in the substrate binding region. The larger end of this cavity is not in contact with Tamiflu when the substrate is bound. This presents an opportunity to design another drug with greater specificity to this pocket. Additional FeaturesNeuraminidase breaks the groups from glycoproteins. from on the virus attaches to the sialic acid groups. The virus is from the cell and is released when neuraminidase breaks the sialic acid groups. This will allow flu replication to happen and a neuraminidase inhibitor, such as Tamiflu, is required to stop neuraminidase. The interaction of Tamiflu with N1 neuraminidase is shown with this (1). Tamiflu in this model is bound when it is visible and the loop in moved in towards the drug through induced fit. When neuraminidase breaks the sialic acid groups, the virus (virion) is released. This will allow replication of the virus. The inhibitor prevents the breaking of the sialic acid groups, so no virion is released and replicated.
CreditsIntroduction - Mike Reardon Overall Structure - Rachael Jayne Drug Binding Site - Jackie Dorhout Additional Features - Nick DeGraan-Weber References(1) Martz, E.; Hodis, E.; Canner, D.; Samish, I.; Prilusky, J.; Goodsell, D. S.; Strong, M. Avian Influenza Neuraminidase, Tamiflu and Relenza. http://www.proteopedia.org/wiki/index.php/Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza 2011. (2) Influenza (Seasonal). World Health Organization. http://www.who.int/mediacentre/factsheets/fs211/en/ 2011 (3) "Influenza: Viral Infections: Merck Manual Home Edition". http://www.merck.com/mmhe/sec17/ch198/ch198d.html 2011 (2) OCA 7nn9 - Native Influenza Virus Neuraminidase Subtype N9. http://www.proteopedia.org/wiki/index.php/7nn9 2011. (3) OCA 2hu4 - N1 Neuraminidase in Complex with Oseltamivir 2. http://www.proteopedia.org/wiki/index.php/2hu4 2011. (4) Goodsell, David. Molecule of the Month: Influenza Neuraminidase- RCSB PDB. http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb113_1.html 2009. (5) Russell, R.J.; Haire, L.F.; Stevens, D.J.; Collins, P.J.; Lin, Y.P.; Blackburn, G.M.; Hay, A.J.; Gamblin, S.J.; Skehel, J.J. N1 neuraminidase in complex with oseltamivir 2. http://www.rcsb.org/pdb/explore.do?structureId=2hu4. (6) Garcia-Sosa, Alfonso; Sild, Sulev; Maran, Uko. Design of Multi-Binding-Site Inhibitors, Ligand Efficiency, and Consensus Screening of Avian Influenza H5N1 Wild-Type Neuraminidase and of the Oseltamivir-Resistant H274Y Variant. Journal of Chemical Information and Modeling. http://pubs.acs.org/doi/full/10.1021/ci800242z. 2009. (7) Wang, Shu-Qing; Dub, Qi-Shi; Huang, Ri-Bo; Zhang, Da-Wei; Chou, Kuo-Chen. Insights from investigating the interaction of oseltamivir (Tamiflu) with neuraminidase of the 2009 H1N1 swine flu virus. http://www.scirp.org/kcchou/papers/BBRC_2009_H1N1_Tamiflu.pdf. 2009. (8) Neuraminidase Inhibitors. http://www.fluwiki.info/pmwiki.php?n=Consequences.NeuraminidaseInhibitors. 2009. (9) Investor Update. http://www.roche.com/investors/ir_update/inv-update-2009-09-13.htm. 2009.
Influenza M2 Proton ChannelIntroductionInfluenza A, better known as the flu, an infection of the nose, throat, and lungs caused by the influenza virus. Basic symptoms include aches, chills, fever, loss of energy and dizziness, but complications can include pneumonia, encephalitis, bronchitis, and death—about 36,000 people every year die of complications from the flu (CDC). The that plays an important role in the life cycle of the influenza A virus. The channel itself is a homotetramer with four identical M2 units, where each M2 protein is a helix stabilized by two disulfide bonds. At a low pH, the channel allows hydrogen ions to enter the viral particle form the endosome, effectively lowering the pH on the interior of the virus. This in turn causes the dissociation of the viral matrix protein M1 from the ribonucleoprotein, a critical step in “uncoating” the virus to introduce its contents to the cytoplasm of the host cell (Stouffer). The M2 protein itself consists of three major protein domains: a stretch of 24 amino acids on the N-terminal end that are exposed to the external environment, 22 (largely hydrophobic) amino acids in the transmembrane region, and 52 amino acids on the C-terminal end which are exposed to the inside of the viral particle (Schnell). The function of the M2 channel can be inhibited by the antiviral drug Amantadine, an inhibition that effectively blocks the virus from taking over the host cell. Amantadine inhibits the replication of influenza A viruses by interfering with the uncoating of the virus within the cell. Amantadine is an M2 inhibitor that blocks the ion channel formed by the M2 protein that spans the viral membrane. By blocking this channel, Amantadine effectively prevents the acidification and subsequent release of viral elements into the host cell. Unfortunately, the M2 gene is susceptible to mutations. When one of five (of the 22) amino acids in the transmembrane region is suitably substituted, Amantadine no longer binds in such a way that would block the motion of the protons into the virus. As of 2009, the CDC noted that a full 100% of Influenza A viruses of types H3N2 and 2009 pandemic flu samples cultured in the United States showed a resistance to Amantadine (CDC). Overall StructureThe M2 proton channel , made up of 97 residues, is a homotetramer transmembrane protein made up of 4 helices. The amino terminus of the protein is exposed to the outside environment, while the carboxy terminus is exposed to the internal environment. At pH 7.5, form the N-terminus. create a transmembrane helix which forms a channel. Furthermore, residues 47-50 create a ‘short flexible loop,’ and residues 51-59 form a C-terminal amphipathic helix (Schnell). The loop created by residues 47-50 at the C-terminus connects the amphipathic helices to the transmembrane domain. The amphipathic helices lie perpendicular to the transmembrane helices. These amphipathic helices form a base that is resistant to changes in pH and therefore acts to stabilize the protein. It should be noted that the transmembrane helices are left handed while the amphipathic helices forming the base are right handed. These amphipathic helicves are also arranged head to tail (Schnell). The pore created by the four tansmembrane helices is constricted at the N-terminus by the methyl groups on Val 27. On the other end of the pore, interactions between Trp41 also create a blockage. The four helices are packed so tightly that van der Waals forces are created between the indole rings of Trp41. This forms a gate. Together, the interactions between Trp41 and Val27 block the passage of water through the pore. Furthermore, hydrogen bonds between Asp44 and Trp41 stabilize the gate. When the pH is lowered, the imidazole rings of His37 are protonated and the helices undergo electrostatic repulsion. This in turn breaks the Asp44 and Trp41 interactions and the gate will open. As previously mentioned, the base created by the amphipathic helices prevents the protein from dissociating. However, cysteins at the N-terminus create disulphide bonds that also act to prevent dissociation (Schnell). Drug Binding SiteAmantadine binds with high affinity to a site in the M2 protein spanning five residues: Leu 26, Val 27, Ala 30, Ser 31, and Gly 34 (Cady). This high affinity is seen at pH’s closer to neutral. In lower pH’s the protein is only somewhat bound to amantadine. Therefore, when determining the mechanism by which amantadine blocks the channel experiments must be conducted at neutral pH. to the M2 protein is illustrated for viewing of the bonds. When amantadine isn't present, the created in the M2 complex is open, allowing viral particles to pass through. In the presence of amantadine, this pore is , which prevents entry of the viral particles. Additional Features– His37 has been found to exhibit significant proton selectivity. This suggests that His37 is involved in the opening of the conductive channel. The channel is non-conductive when His37 is not protonated, and conductive when it is in the protonated state (Pielak/Chou). – Replacing Trp41 with a substitute amino acid results in increased channel current in in both directions. As such, the Trp 41 site is important to directional selectivity, in that it regulates the direction of the proton channel flow. This is accomplished in part because it forms a ring that prevents water from entering the channel and reaching the His37, and thus deprotonating if the pH is high, from the C-terminus(Pielak/Chou). In addition, the proton transport channel is used to equalize the pH in the channel with that of the cytoplasm in the host cell. This prevents rearrangement of the haemmagglutanin during its transport to the host cell. (Schnell) ReferencesCady, S.D., Schmidt-Rohr, K., Wang, J., Soto, C., DeGrado, W.F., Hong, M. "Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers," (2010) Nature 463: 689-692. Stouffer AL, Acharya R, Salom D, Levine AS, Di Costanzo L, Soto CS, Tereshko V, Nanda V, Stayrook S, DeGrado WF (2008). "Structural basis for the function and inhibition of an influenza virus proton channel". Nature 451 (7178): 596-9 Schnell JR, Chou JJ (2008). "Structure and mechanism of the M2 proton channel of influenza A virus". Nature 451 (7178): 591–5. Pielak RM, Schnell JR, Chou JJ: Mechanism of drug inhibition and drug resistance of influenza A M2 channel. Proc Natl Acad Sci, 106(18):7379-84 (2009). "CDC Recommends against the Use of Amantadine and Rimantadine for the Treatment or Prophylaxis of Influenza in the United States during the 2005–06 Influenza Season". CDC Health Alert. Centers for Disease Control and Prevention Hay AJ, Wolstenholme AJ, Skehel JJ, Smith MH. The molecular basis of the specific anti-influenza action of amantadine. EMBO J 1985; 4: 3021-4. Stephenson I, Nicholson KG. Influenza: vaccination and treatment. Eur Respir J 2001; 17: 1282-93. Pielak RM, Chou JJ. "Infuenza M2 proton channels". BBAMEM-80262; No. of pages: 8; 4C: 2, 3, 5, 6. CreditsIntroduction -- Josephine Harrington Overall structure -- Andrea Simoni Drug binding site -- Joshua Drolet Additional features -- John Hickey | ||||||||||||
