MIT OpenCourseWare http://ocw.mit.edu5.36 Biochemistry LaboratorySpring 2009For 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 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 ishighly ______________ both in sequence and structure. Kinase activity requires binding of the peptide substrate (to be phosphorylated) and _____________ to the catalytic domain. 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. 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. Kinase domains have a _______________ structure composed of an ____ -lobe ____ -lobe __________ region The hinge region (between the two lobes) contains several conserved residues thatprovide the catalytic machinery and make up an essential part of the _______ bindingpocket. Among all kinases, Mg-ATP binding is primarily in the _____-lobe and hinge region. 1 MITclassATP Binding ( ___ ) Loop (shown in orange) • A ______-rich region in the N-lobe (typically a flexible loop between strands ofthe beta sheet or between the beta sheet and an alpha helix) that is highlyconserved 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. Abl kinase domain P-loop (orange) (conserved residues inmagenta) A-loop (green) 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). 2A) 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 orientationwith 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 theATP 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. 3The 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 DFGmotif by ~______°. Abl kinase domain ACTIVE conformation Abl kinase domain INACTIVE conformation DFG orientation ACTIVE conformation INACTIVE conformation Recall that the Asp carboxylic acid functional group binds the Mg2+ coordinated to ATP in active kinases. ATP-Mg2+ ATP-Mg2+ 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 C-lobe. 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 thekinase domain __________ region. As with most kinase inhibitors, Gleevec competes with _______ to bind in the hingeregion of the kinase domain. In contrast to most kinase inhibitors, only part of the Gleevec molecule blocks ATPbinding. 4Specifically, only the ________________ and ____________________ rings of Gleevec interfere directly with ATP binding, blocking the adenine base. Gleevec-Abl complex ATP Gleevec ONNNNNH2HOHOOPOO-OPOP-OOO-OO-NNHNNNHONNIn 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 andhydrophobic “cage” Tyr315