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Transcript
ICE 6 Review Marty and Aaron November 23, 2014 A quick review of kinases • A kinase is an enzyme that utilizes ATP to transfer a phosphate onto a serine, threonine, or tyrosine amino acid. • This reaction is thermodynamically favorable, but slow. • Kinases are specific – only certain amino acids are phosphorylated. • Phosphorylation changes protein shape and function. • Kinases are often used in signaling. ATP Protein substrate Kinase regulation: The activation loop Src-family kinases have another level of regulation A particular phosphatase (an enzyme that dephosphorylates an amino acid side chain) acts on Src and increases its activity. On which tyrosine does this phosphatase exert its enzymatic activity, Y527 or Y416? Briefly explain your answer. Closed, inactive Y527 Y416 Phosphatase Kinase Kinase Open, inactive Phosphatase Open, active This phosphatase dephosphorylates Y527. Dephosphorylation of Y527 would destabilize the closed conformation since binding of Y527 to the SH2 domain is dependent upon the phosphorylation of Y527. Abl is a Src-family kinase A particular mutation decreases the Kd for binding of the kinase C-lobe to the myristate. How would this mutation affect the activity of Abl? Mutation lowers the energy of closed, inactive Lower Kd, stronger interaction Phosphorylation of Y412 Myristate detaches from C-lobe Closed, inactive Myristate attaches to C-lobe Open, inactive This equilibrium constant decreases Dephosphorylation of Y412 Open, active The amount of active enzyme decreases This equilibrium shifts left Abl activity would decrease. The lower Kd would stabilize the energy of the “closed” conformation of Abl, which would lead to a lower concentration of active enzyme. CML is caused by a misregulated kinase: Bcr-Abl Bcr-Abl leads to increased Abl activity Myristate detaches from C-lobe Closed, inactive Myristate attaches to C-lobe Open, inactive There is no myristate! Keq increases Open, active This equilibrium shifts right The amount of active enzyme increases Gleevec decreases the concentration of active Bcr-Abl Which decreases closed, inactive… Which decreases open, inactive… Which shifts this equilibrium left… Gleevec Kd = 10 nM + Closed, inactive Which shifts this equilibrium left… Open, inactive This equilibrium heavily favors bound state… Closed, inactive, bound to Gleevec Which decreases open, active… Open, active Dasatinib and Gleevec bind to the same location, but to different Abl conformations Dasatinib • Both drugs bind to the ATP binding site. • The drugs bind to different Abl conformations. Gleevec binds closed, inactive, while Dasatinib binds open, active. Gleevec Dasatinib vs. Gleevec Binds the open, active conformation Treats some Drug-resistant mutants Inhibit Bcr-Abl Bind to the ATP binding site Gleevec Closed, inactive Binds the closed, inactive conformation Dasatinib Open, inactive Open, active Lots of terms… Latch Dasatinib Clamp SH2 Src Closed Myristate Abl Gleevec Kinase Myr Phosphorylation Bcr-Abl CML Open Inactive Linker SH3 Activation loop Active Y412/Y416 Y527 An example concept map… Src kinases have an additional level of regulation in which they can be either open or Open closed. Phosphorylation Enables… Catalyzes… Active Inactive Determines whether a kinase is… Activation loop Kinase Src Regulated by the position of the… Determines the position of the… Y412/Y416 Closed Stabilized by the… Abl Is regulated by… Misregulated by a translocation that creates a fusion protein called… Gleevec Is a particular type of… Interacts with the Clobe to form a… Myristate (“Myr”) Inhibits by binding to its closed, inactive form… Is missing its… Latch Interact to form the… Clamp Interact to form the… Bcr-Abl Causes… Dasatinib Inhibits Bcr-Abl and is capable of binding to Gleevec-resistant mutants CML Y527 + SH2 Linker + SH3 The HIV Viral Replication Cycle (Membrane Fusion) HIV viral replication cycle The Viral Life Cycle Arts EJ and Hazuda DJ. “HIV-1 antiretroviral drug therapy.” Cold Spring Harb Perspect Med. 2012 Apr; 2 (4): 1-23. Membrane Fusion We’ve learned two examples, which work analogously to one another • gp41 – gp41 extends into the pre-hairpin conformation after gp120 recognizes CD4 and CCR5 or CXCR4 – N- and C-terminal domains of gp41 convert from a pre-hairpin to a hairpin conformation – N-terminal domain begins as an alpha helix, C-terminal domain gets ordered into a helix as the two domains favorably wind together • SNAREs – v-SNARE and t-SNARE are both pre-formed alpha helices – Cognate v-SNAREs and t-SNAREs recognize one another – Cognate v-SNARE and t-SNARE favorably wind around another • In both, as alpha helical domains wind together, the two membranes that the alpha helices are embedded in are brought closer together gp41 facilitates membrane fusion between the viral envelope and the host cell plasma membrane SNAREs facilitate membrane fusion between vesicles and their target membranes (secretory pathway) SNAREs gp41 fusion peptide cytosol t-SNARE TM target organelle membrane cell membrane N-terminal gp41 extracellular space gp41 pre-hairpin C-terminal gp41 target organelle lumen t-SNARE v-SNARE vesicle membrane viral envelope v-SNARE TM gp41 TM domain viral particle interior vesicle lumen gp41 SNAREs Hemifusion Hemifusion gp41 SNAREs target organelle lumen fusion peptide t-SNARE TM cytosol cell membrane target organelle membrane t-SNARE N-terminal gp41 extracellular space C-terminal gp41 v-SNARE vesicle membrane gp41 TM domain viral envelope viral particle interior v-SNARE TM vesicle lumen Structure of the N- and C-domains of gp41 (same structure, one image rotate 90 degrees from the other) Fusion peptide C-terminal N-terminal • Which conformation is this structure of gp41: pre-hairpin or hairpin? Hairpin • Which set of helices are the Nterminus of gp41? Which are the Cterminus? • Where would the fusion peptide be? • Which set of helices would Fuzeon replace? C-terminal, cyan Viral Protein Trafficking After integration into the host genome, an HIV-infected cell will start to synthesize gp120, which travels through the endomembrane system until it reaches the plasma membrane. Once the virus starts synthesizing gp120, it has to begin degrading the cell’s supply of CD4. In addition to decreasing the expression of CD4 at the plasma membrane to prevent other strains of HIV from entering the cell, why else must the cell begin decreasing its cellular concentration of CD4 once synthesis of gp120 begins? Why is it less necessary for the cell to also decrease its cellular concentration of CCR5 or CXCR4? Newly synthesized cellular CD4 would bind newly synthesized gp120 in the endomembrane system and render it nonfunctional (it actually stalls it in the ER). In order for gp120 to be expressed on the viral envelope and available to bind with the next cell’s CD4, it must be free of CD4 to begin with. It is less necessary to downregulated CCR5/CXCR4 since gp120 can only interact with them AFTER having interacted with CD4, first.