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536lecntwthbnk_4 11.45.23 PM

536lecntwthbnk_4 11.45.23 PM - MIT OpenCourseWare...

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Unformatted text preview: MIT OpenCourseWare http://ocw.mit.edu 5.36 Biochemistry Laboratory Spring 2009 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms. 5.36 Lecture Summary #4 Thursday, February 26, 2009 CI-M assignment: The first draft of your minireview is due on Thursday, March 5th at noon on the MIT class website. Next Laboratory Session: #7 and 8 ________________________________________________________________________________ Topics: Kinase domains: structure and inhibition I. Conserved and variable features of kinase domains A. Structural similarities B. Active and inactive forms II. Abl and Bcr-Abl inhibition by Gleevec III. Gleevec resistance in Bcr-Abl mutants A. Direct interference with Gleevec binding B. Destabilization of the inactive conformation ________________________________________________________________________________ I. CONSERVED AND VARIABLE FEATURES OF KINASE DOMAINS A) STRUCTURAL SIMILARITIES The catalytic domain (or _____________ domain) of eukaryotic protein kinases is highly ______________ both in sequence and structure. Kinase activity requires binding of the peptide substrate (to be phosphorylated) and _____________ to the catalytic domain. Kinase domains have a _______________ structure composed of an N-lobe (amino lobe) that • contains a 5-stranded beta sheet and an alpha helix (_______). • comprises residues _______ to _______ of Abl (shown here). • contributes to ATP binding. ____ -lobe __________ region and a C-lobe (carboxy lobe) that • is made up of multiple alpha helices. • comprises residues _______ to _______ of Abl (the larger lobe). • is the location of peptide _____________ binding. ____ -lobe The hinge region (between the two lobes) contains several conserved residues that provide the catalytic machinery and make up an essential part of the _______ binding pocket. Among all kinases, Mg-ATP binding is primarily in the _____-lobe and hinge region. 1 ATP Binding ( ___ ) Loop (shown in orange) • A ______-rich region in the N-lobe (typically a flexible loop between strands of the beta sheet or between the beta sheet and an alpha helix) that is highly conserved among kinases. Color scheme for individual atoms: oxygen (red), nitrogen (blue), carbon (background color), sulfur (yellow), P (orange) • The backbone atoms of the conserved P-loop sequence, GXGXXG, interact with the non-transferred phosphate atoms of ATP. • In Abl, the P-loop sequence is MKHKL___G___QY___E. P-loop (orange) Abl kinase domain A-loop (green) (conserved residues in magenta) Activation (A) Loop (shown in green) • a principal ________________ structure for modulating kinase activity. In the closed form (above), the A-loop can block substrate binding to the C-lobe. • The A-loop can vary significantly in sequence and size between kinase subfamilies. • A conserved _______-_______-_______ (DFG) motif implicated in ATP binding is located at the N-terminus of the A-loop. Numbering in the Abl and Bcr-Abl kinase domain: • N-lobe: Abl residues 225-350 ⇒ P-loop: residues ______-______ • hinge region: interface of N and C lobes • C-lobe: 354-498 ⇒ A-loop: residues ______-______ ⇒ DFG motif: residues ______-______ Note that all Abl numbering is provided for isoform 1A of human Abl (swissprot accession number: P00519). 2 A) ACTIVE AND INACTIVE FORMS OF PROTEIN KINASES In an active kinase, the activation (A) loop is in an ___________ conformation. Abl kinase domain ACTIVE conformation ATP binding orientation with the A loop (shown with cAPK) Asp-Phe-Gly motif Features of an open or ________________ A loop conformation: • The body of the A loop does not block the C-lobe, enabling the C-lobe to be available for binding the substrate. • The Asp within the DFG conserved motif (381 in Abl) is oriented toward the ATP binding pocket. The ______ side chain interacts with the ______ coordinated to the phophate groups of ATP. “Happy families are all alike; every unhappy family is unhappy in its own way.” -Tolstoy = activation loop Nagar, B. et al. Cancer Res 2002;62:4236-4243 __________ kinase domains are all alike; every _____________ kinase domain is _____________ in its own way. 3 The inactive conformation of the Abl kinase domain. The Abl kinase domain switch from an active to an inactive form results in a conformation change at the start of the A loop. This flips the orientation of the DFG motif by ~______°. Abl kinase domain ACTIVE conformation Abl kinase domain INACTIVE conformation DFG orientation ACTIVE conformation ATPMg2+ INACTIVE conformation ATPMg2+ Recall that the Asp carboxylic acid functional group binds the Mg2+ coordinated to ATP in active kinases. While the DFG motif is conserved among all protein kinases, the DFG _________ is unique to Abl and only a few other kinase subfamilies. Also, in the inactive form, the A-loop blocks the substrate binding region of the Clobe. Specifically, Tyr393 mimics the _________ Tyr (to be phosphorylated) on the substrate. Tyr393 is typically phosphorylated in the active form, and it is not phosphorylated in the inactive form. II. ABL AND BCR-ABL INHIBITION BY GLEEVEC The vast majority of kinase inhibitors are ATP competitive inhibitors that bind in the kinase domain __________ region. As with most kinase inhibitors, Gleevec competes with _______ to bind in the hinge region of the kinase domain. In contrast to most kinase inhibitors, only part of the Gleevec molecule blocks ATP binding. 4 Specifically, only the ________________ and ____________________ rings of Gleevec interfere directly with ATP binding, blocking the adenine base. Gleevec-Abl complex AT P Gleevec N N NH2 HN N N N HO N N NH O O HO O O P O P O- O N N O- O O P O- -O In active Abl, the adenine base of ATP forms two hydrogen bonds with the protein ___________________ in the hinge region. Small molecule inhibitors of numerous kinases form H-bonds with the corresponding residues in the ATP binding pocket of the target kinase. Although Gleevec forms similar hydrogen bonds, there is no H-bond formed with ____________. Gleevec has a unique position in the binding pocket. (Note: You will identify the additional Abl-Gleevec H-bonds using PyMol in lab session 15.) There is ______________ overlap in ATP and the Gleevec binding to the Abl kinase domain. ATP-kinase complex (shown with cAPK) Gleevec-Abl complex Hydrogen bonds and hydrophobic “cage” Tyr315 Met318 Leu 370 Phe382 Tyr253 The Gleevec molecule penetrates deeper into the __________________ core of the 5 ATP binding site compared to ATP. The majority of the Gleevec binding energy comes from van der Waals and hydrophobic interactions (NOT just H-bonds). For example, a hydrophobic “cage” around Gleevec’s pyridine and pyrimidine rings is formed by Leu 370 and residues from the P-loop (________) and A-loop (________). Phe382 is part of the conserved _________ motif. The Phe382 orientation toward the pyrimidine ring is critical for Gleevec binding. In the active form the Asp381 side chain is oriented toward the ATP binding pocket. In the the inactive form the Phe side chain is oriented toward the binding pocket (see figures on page 4). Gleevec binds Abl in the __________________ conformation! The _____________ of Gleevec for Abl relies on the binding of Gleevec to the inactive form and the differences between the inactive forms of Abl and other protein kinases. Another look at the binding pocket in the inactive form of the Abl kinase domain: Side note: Piperazine rings are often included in drugs to increase solubility. While the ring may participate in H-bonds with the target protein, it is often solvent exposed and in many cases does not contribute to the drug binding. If Bcr-Abl is constitutively active, how can Gleevec bind to the Bcr-Abl kinase domain in CML cells? Possibilities include: 1. The orientation of the activation loop is ______________, transiently passing through an inactive conformation that can bind Gleevec. 2. The Gleevec “_________” the Bcr-Abl protein as it is _______________, prior to taking on the active conformation 6 III. GLEEVEC RESISTANCE IN BCR-ABL MUTANTS Our class selected target mutants that include some of the most prevalent mutations found in CML patients. Common mutations in patients with chronic phase (__________) CML: M244, L248, F317, ____396, ____417 Common mutations in patients with ________________ phase CML: Q252, Y253, _____255, T315, E459, F486 A 2006 study comparing the kinase activity of 5 common mutations with wild type (wt) Bcr-Abl found: T315I, ____351____, and H396P < wt E255K comparable to wt Y253F > wt Apperley, J. F. Lancet Onco l 8, 1018-1029 (2007) How can single amino acid mutations in Bcr-Abl confer Gleevec resistence? • Directly interfere with Gleevec binding (ie. sterics) • Destabilize the inactive (Gleevec binding) conformation of Abl A) DIRECT INTERFERENCE WITH GLEEVEC BINDING Kinase domains contain a ___________________ residue that partially or fully blocks a hydrophobic region deep in the ATP binding pocket. The gatekeeper residue contributes to the _______________ of kinases for small molecule inhibitors. A small gatekeeper residue allow an inhibitor to access the “gated” hydrophobic regions of the binding pocket. A larger residue _______________ blocks inhibitor binding. ATP binding is not affected because ATP does not access the “gated” part of the binding pocket. 7 The gatekeeper residue is a conserved _______ in _____% of all human kinases. This is residue ________ in Abl Hydrophobic pocket gatekeeper (Thr315) gatekeeper (Thr315) In some kinases, the gatekeeper residue has a bulkier side chain compared to Thr, and this precludes the binding of small molecule inhibitors in the hydrophobic pocket. Abl mutations at the gatekeeper position (315) from a Thr to a bulkier residue block inhibitor penetration past the gatekeeper and and confer Gleevec resistance. Question: what residues are bulkier than Thr and can be accessed with a single base pair substitution? Thr 315 is coded by ACT Ala/A Arg/R Asn/N Asp/D Cys/C Gln/Q Glu/E Gly/G His/H Ile/I START GCU, GCC, GCA, GCG CGU, CGC, CGA, CGG, AGA, AGG AAU, AAC GAU, GAC UGU, UGC CAA, CAG GAA, GAG GGU, GGC, GGA, GGG CAU, CAC AUU, AUC, AUA AUG Leu/L Lys/K Met/M Phe/F Pro/P Ser/S Thr/T Trp/W Tyr/Y Val/V STOP UUA, UUG, CUU, CUC, CUA, CUG AAA, AAG AUG UUU, UUC CCU, CCC, CCA, CCG UCU, UCC, UCA, UCG, AGU, AGC ACU, ACC, ACA, ACG UGG UAU, UAC GUU, GUC, GUA, GUG UAG, UGA, UAA O NH2 The Thr315_______ Abl mutant demonstrates high kinase activity even in the presence of 10 µM Gleevec (STI571). 8 The T315I mutation makes up ~13% of reported Bcr-Abl mutations. Other mutants that interact directly with Gleevec (but not ATP) include F317 and F359. Those two mutants make up a combined total of 14% of all reported Bcr-Abl mutations. B) DESTABILIZATION OF THE INACTIVE CONFORMATION The majority of mutations result in a __________________ of the _________________ (Gleevec-binding) form of the Abl kinase domain. A-loop mutations Mutations found within the A-loop (381-402) of the C-lobe can destabilize or prevent rearrangement to the inactive conformation of that loop. This includes the ___________ mutant that you are working with in lab. P-loop mutations P-loop mutants may destabilize the inactive conformation of the P-loop (residues 244-255). Mutants have been identified for every X residue in the P loop consensus sequence, GXGXXGX: ______250, Gln(Q)252, Tyr253, ______(E)255. Ie. Tyr253 mutations result in the loss of a loop-stabilizing Hbond with the carboxy group of Asn322. In addition, the Tyr253 forms part of the hydrophobic cage for Gleevec (see additional figure on p. 5). 9 ...
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