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.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 formThe catalytic domain (or kinase domain) of eukaryotic protein kinases is highly conserved both in sequence and structure Kinase activity requires binding of the peptide substrate (to be phosphorylated) and Mg-ATP to the catalytic domain.N-lobe Kinase domains have a bilobal structure composed of an N-lobe (amino lobe) that • contains a 5-stranded beta sheet and an alpha helix (αC).N-lobe Kinase domains have a bilobal structure composed of an N-lobe (amino lobe) that • contains a 5-stranded beta sheet and an alpha helix (αC). • comprises residues 225-350 of Abl (shown here). • contributes to ATP binding.N-lobe Kinase domains have a bilobal structure composed of an N-lobe (amino lobe) that • contains a 5-stranded beta sheet and an alpha helix (αC). • comprises residues 225-350 of Abl (shown here). • contributes to ATP binding.C-lobe Kinase domains have a bilobal structure composed of an N-lobe and a C-lobe (carboxy lobe) that • is made up of multiple alpha helices. • comprises residues 354-498 of Abl (the larger lobe). • is the location of peptide substrate binding.C-lobe Kinase domains have a bilobal structure composed of an N-lobe and a C-lobe (carboxy lobe) that • is made up of multiple alpha helices. • comprises residues 354-498 of Abl (the larger lobe). • is the location of peptide substrate binding.The hinge region (between the two lobes) contains several conserved residues that provide the catalytic machinery and make up an essential part of the ATP binding pocket. hinge region Among all kinases, Mg-ATP binding is primarily in the N-lobe and hinge region.(conserved residues in magenta) ATP Binding (P) loop • 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 atoms oxygen- red nitrogen- blue carbon- black, grey, or background color sulfur- yellow phosphorus- orangeATP Binding (P) loop A ____-rich region in the N-lobe (typically a flexible loop between (conserved residues in magenta) • Gly strands of the beta sheet or between the beta sheet and an alpha helix) that is highly conserved among kinases. • 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 MKHKLGGGQYGE.Activation (A) loop Asp Phe Gly • a principal regulatory structure for modulating kinase activity. Inthe closed form (shown above), the A-loop can block substratebinding to the C-lobe. • The A-loop can vary significantly in sequence and size betweenkinase subfamilies. • A conserved Asp-Phe-Gly (DFG) motif implicated in ATP bindingis located at the N-terminus of the A-loop.In the Abl or Bcr-Abl kinase domains: • N-lobe: Abl residues 225-350 ⇒ P-loop: residues 244-255 • hinge region: interface of N and C lobes • C-lobe: 354-498 ⇒ A-loop: residues 381-402 ⇒ DFG motif: residues 381-383 Note that all Abl numbering is provided for isoform 1A of human Abl (swissprot accession number: P00519).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 formIn an active kinase, the activation (A) loop is in an “open” conformation. activation loop Abl kinase domain: active conformation Features of an open or extended 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 Asp side chain interacts with the Mg coordinated to the phophate groups of ATP. ATP binding the cAPK kinase domain (PDB: 1atp)The Asp side chain interacts with the Mg coordinated to the phophate groups of ATP. Asp Phe Gly ATP binding the cAPK kinase domain (PDB: 1atp)“Happy families are all alike; every unhappy family is unhappy in its own way.” Active domains are all alike; every _______ inactive“______ kinase kinase domain is _______ in its own way.”inactiveThe 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 ~180°. inactive Abl active Abl Asp Phe ATP-Mg2+ ATP-Mg2+ Asp Phe With the Asp side chain is flipped away from the ATP binding site, Mg coordination (with the Mg-ATP complex) is prevented.The inactive conformation of the Abl kinase domain ylic acid functional group binds the Mg2+ ve kinases. served among all protein kinases, the DFG y a few other kinase subfamilies. Recall that the Asp carboxcoordinated to ATP in actiATP-Mg2+ ATP-Mg2+ Asp Phe Asp Phe active inactive While the DFG motif is conflip is unique to Abl and onlThe inactive conformation of the Abl kinase domain Also, in the inactive form, the A-loop blocks the substrate active inactive binding region of the C-lobe.The inactive conformation of the Abl kinase domain Specifically, Tyr393 mimics the target Tyr (to be phosphorylated) on the substrate. active inactive Tyr393 is typically phosphorylated in the active form, and is not phosphorylated in the inactive form.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 formGleevec inhibition of the Abl (and Bcr-Abl) kinase domains: interdomain rinhibitors that bind in the kinase domain hinge reegion The vast majority of kinase inhibitors are ATP competitive