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The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain

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The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain

Postgraduate Seminar in Biochemistry 1/2016
Presented by: Bundit Boonyarit
M.S. student in Biochemistry, Kasetsart University

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The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain

  1. 1. The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain COMPUTATIONAL BIOLOGY Presented by: Bundit Boonyarit 5814400587 Dept. Biochemistry, Fac. Science, Kasetsart University Huber R. G., et.al (2015) IMAGE CREDIT: ANSGAR PHILIPPSEN Nov 8, 2016 11.30 am - 12.00 pm
  2. 2. STRUCTURAL BIOLOGY HAS THE POTENTIAL TO ACCELERATE DRUG DISCOVERY 2
  3. 3. What is structural biology? 3 Structural biology is the field of science seeking to visualize the structure of biological molecules, such as proteins, in order to understand their functions and properties. Structural biology accelerating drug discovery http://mva.org/wp-content/uploads/2015/01/StructuralBiology_onepager.pdf Why focus on structural biology? All companies involved in drug discovery today have a high interest in structural biology. Knowing the structure of both the target related to a disease and the structure of the possible new compound to treat, it is essential to drug design.
  4. 4. Fig 1. Overview of ZAP–70 kinase domain (PDB 1U59 [11]): Ligands staurosporine or ATP are l in the hinge region between the C-lobe (top) and N-lobe (bottom) of the protein (cartoons forma are depicted in gray/CPK wireframe format. The αC helix, depicted in red, contains the salt bridge K369-E386 indicated in magenta/CPK wireframe. Phosphorylation sites Y492 and Y493 are indicated 4 ZAP-70 (Zeta-chain-associated protein kinase 70) TYROSINE KINASE PROTEIN
  5. 5. 5 TYROSINE KINASE PROTEIN A tyrosine kinase is an enzyme that can transfer a phosphate group from ATP to a protein in a cell at tyrosine residue. Protein tyrosine kinase Protein tyrosine phosphatase P P Phosphorylated substrate Signal in Signal out ATP PTK PTP Substrate They play an important role in regulating cell division, cellular differentiation, and morphogenesis.
  6. 6. 6 TYROSINE KINASE PROTEIN Extracellular domain Cytoplasm Plasma membrane Activation loop N-lobe C-lobe C-terminal tail Kinase domainThe extracellular domain serves as the ligand-binding part of the molecule. The same mechanism can be used to bind two receptors together to form a homo- or heterodimer. The intracellular or cytoplasmic domain is responsible for the kinase activity, as well as several regulatory functions.
  7. 7. TYROSINE KINASE PROTEIN Plasma membrane Cytoplasm ATP ATP P ATP ATP P C N C N N N C C N N C C P P P P 1. Inactive receptor monomer 2. Trans-autophosphorylation 3. Active phosphorylated receptor dimer Ligand binding is caused by two reactions: 1. Dimerization of two monomeric receptor kinases or stabilization of a loose dimer. Many ligands of receptor tyrosine kinases are multivalent. 2. Trans-autophosphorylation (phosphorylation by the other kinase in the dimer or higher order multimer complex) of the kinase. 7
  8. 8. TYROSINE KINASE PROTEIN dimerization P P SOS RAS RAF MEK MAPK P PI3K AKTP LPA thrombin ET, etc. TGF-α epiregulin amphi- regulin NRG-1 cytokineEGF β–cellulin HB-EGF NRG-2 NRG-3 NRG-4 SRC CBL PLC PKC BAD S6K AKT PI3K SHP2 GRB7 GRB2GAP SHC SOS MAPK RAF RAC MEK PAK ABL JNK NCK VAV CRK STATERG1MYCSP1 FOS JUN ELK JAKRAS-GDP RAS-GTP JNKK plasma membrane cytoplasm nucleus Nucleus INPUTLAYERSIGNALPROCESSING growth factor ligands trans- membrane receptors adaptors and enzymes signaling cascades transcription factors Cytoplasm Plasma membrane Signal transduction: The active tyrosine kinase phosphorylates specific target proteins, which are often enzymes themselves. An important target is the ras protein signal-transduction chain. 8
  9. 9. TYROSINE KINASE PROTEIN Fer FesAbl Arg CSK CTK BMX BTK TXK TEC ITK BLK Lck HCK Lyn Src Yes Fyn Fgr FRK Srm Brk EphA4 EphA3 EphA5 EphA6 EphA7 EphA8 EphA2 EphA1 EphB6 EphA10 EphB4 EphB3 EphB1 EphB2 STYK1 FAK PYK2 Syk ZAP70 PTK7 LTK ALK Ros RYK Ron Met InsR IGF1R DDR1 DDR2 MuSK TRKA TRKB TRKC ROR2 ROR1 Axl Mer Ret Tyro3 FGFR1 FGFR2 FGFR3 FGFR4 FLT1 FLT4 KDR FLT3 TIE2 TIE1 CSF1R Kit Ack HER2EGFR HER3 HER4 Tnk1 Tyk2 JAK1 JAK2 JAK3 Lmr1 Lmr2 Lmr3 PDGFRβ PDGFRα TK PHYLOGENY 90 Proteins 9
  10. 10. Fig 1. Overview of ZAP–70 kinase domain (PDB 1U59 [11]): Ligands staurosporine or ATP are located in the hinge region between the C-lobe (top) and N-lobe (bottom) of the protein (cartoons format) and Regulation of ZAP-70 Kinase Domain ZAP-70 N-lobe C-lobe αC helix The helix αC plays a major role in catalysis and in the transition of the kinase between inactive and active states. Ligand DFG motif Y492 & Y493 ZAP-70 play an important role in the adaptive immune system. It is expressed in T cells and natural killer cells. Inactivating mutations of ZAP-70 cause selective T cell deficiency in humans such as severe combined immunodeficiency (SCID). The N-terminal portion of the activation loop contains a region spanning a highly conserved sequence Asp-Phe-Gly, called the DFG motif, that participates in coordination of metal cations. Activation loop 10
  11. 11. inhibited, ANP-bound crystal structures of ZAP–70 revealed that the αC helix is displaced out- wards leading to a loss of this key salt bridge. Available crystal structures presently offer an inconclusive picture of the relation of the ZAP–70 KD conformation to the various intermediate mechanistic states. As observed by Jin et al., the staurosporine-bound ZAP–70 KD surprisingly appears to adopt an active-like state when compared to other members of the Syk kinase family. Moreover, experimental data from Fig 1. Overview of ZAP–70 kinase domain (PDB 1U59 [11]): Ligands staurosporine or ATP are located in the hinge region between the C-lobe (top) and N-lobe (bottom) of the protein (cartoons format) and are depicted in gray/CPK wireframe format. The αC helix, depicted in red, contains the salt bridge K369-E386 indicated in magenta/CPK wireframe. Phosphorylation sites Y492 and Y493 are indicated in cyan/CPK wireframe. The DFG motif, D479, F480 and G481, is indicated in blue/CPK wireframe. The activation loop comprises all residues from the DFG motif to seven residues beyond the phosphorylation sites. The N-lobal region of residues 537–569 (green) exhibits significant changes in flexibility depending upon phosphorylation state. doi:10.1371/journal.pcbi.1004560.g001 Regulation of ZAP-70 Kinase Domain PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 3 / 16 N-lobe C-lobe αC helix Ligand DFG motif Y492 & Y493 Activation loop ZAP-70 From previous research: Mutations of residues Y492 and Y493 in the activation loop reveal distinct results for the adjacent phosphorylation sites. The Y492F mutation increases ZAP–70 activity, suggesting that phosphorylation of Y492 is not necessary for the biological function. However, the Y493F mutation impairs kinase activation. The salt bridge formed between K369 and E386, located in the αC helix is a conserved motif of active kinase conformations that is normally broken in inactive kinase states, but was formed in the staurosporine ligand ZAP–70 complex. To investigated the activation and inhibition mechanisms of ZAP–70 by using molecular dynamics simulations (MD) of its KD 11
  12. 12. WHAT IS MOLECULAR DYNAMICS SIMULATION (MD) ? 12
  13. 13. The Nobel Prize in Chemistry 2013 "for the development of multiscale models for complex chemical systems" Martin Karplus Michael Levitt Arieh Warshel 2013 Nobel Prize recognized early developments underlying modern biomolecular computation 13
  14. 14. 14 http://www.slideshare.net/qchemforqespresso/lecture5-46436732 MD MD is a computer simulation technique that allows one to predict the time evolution of a system of interacting particles (atoms, molecules, granules, etc.) using forcefield for calculation. Simulations can provide the ultimate detail concerning individual particle motions as a function of time. For example, by what pathways does oxygen enter into and exit from the heme pocket in myoglobin? The first protein simulations appeared in 1977 with the simulation of the bovine pancreatic trypsin inhibitor (BPTI) (McCammon, et al., 1977). (uses thermal energy to move smoothly over surface)
  15. 15. MD The forcefield is a collection of equations and associated constants designed to reproduce molecular geometry and selected properties of tested structures. Classical Dynamics (Newton’s Equation) properties of molecules directly from the underling interactions between the molecules. Classical Dynamics • Newton’s Equations of motion • Position, speed and acceleration are functions of time ri(t); vi(t); ai(t) • The force is related to the acceleration and, in turn, to the potential energy • Integration of the equations of motion => initial structure : ri(t=0); initial distribution of velocities: vi(t=0) Fi = mi !ai = mi ! dvi dt = m! d2 ri dt2 Fi = !"i E forcefield The idea • Mimic what atoms do in real life, assumin given potential energy function – The energy function allows us to calculate the experienced by any atom given the positions other atoms – Newton’s laws tell us how those forces will aff motions of the atoms Energy(U) PositionPosition where; F is force on an atom m is mass of the atom a is the atom’s acceleration R represents coordinates of all atoms U is the potential energy function Velocity (v) is the derivative of position (r), and acceleration (a) is the derivative of velocity (v). 15 Force field properties of molecules directly from the underling interactions between the molecules. Classical Dynamics • Newton’s Equations of motion • Position, speed and acceleration are functions of time ri(t); vi(t); ai(t) • The force is related to the acceleration and, in turn, to the potential energy • Integration of the equations of motion => initial structure : ri(t=0); initial distribution of velocities: vi(t=0) Fi = mi !ai = mi ! dvi dt = m! d2 ri dt2 Fi = !"i E
  16. 16. MD forcefield MOLECULAR POTENTI Simple sum over many terms UUU All Bonds All Angles All partial charges All Nonbonded pairs All Torsion Angles Hooke 1635 Fourier 1768 Van der Waals 1837 Coulomb 1736 All Bonds = oscillations about the equilibrium bond length All Angles = oscillations of 3 atoms about an equilibrium angle All Torsion Angles = torsional rotation of 4 atoms about a central bond All Nonbonded pairs = non-bonded energy terms (electrostatics and Lenard-Jones) Birth and Future of Multiscale Modeling for Macromolecular Systems: Nobel Lecture Slides Michael Levitt 16 Force field
  17. 17. MD Time scales 10-15 femto 10-12 pico 10-9 nano 10-6 micro 10-3 milli 100 seconds Bond vibration Bond Isomerization Water dynamics Helix forms Fast conformational change long MD run where we need to be MD step where we’d love to be Slow conformational change lipid rotation transport in ion channel lipid diffusion rapid protein folding normal protein folding ribosome synthesis http://www.slideshare.net/qchemforqespresso/lecture5-46436732 17 Events such as protein folding, protein–drug binding, and major conformational changes essential to protein function typically take place on timescales of microseconds to milliseconds. Simulations of even a few microseconds required months on the most powerful supercomputers available, and longer simulations had never been performed.
  18. 18. METHODS System Preparation Initial coordinates for ZAP–70 KD were obtained from the PDB structure 1U59. This structural template was used to construct five distinct complexes. Non- phosphoryla- tion Phosphoryla- tion (1) Staurosporine-bound complex (STA) (2) ATP-bound complex (Y0Y0) (3) ATP-bound complex with phosphorylated variant Y492 (YPY0) (4) ATP-bound complex with phosphorylated variant Y493 (Y0YP) (5) ATP-bound complex with di-phosphorylated variant Y492+Y493 (YPYP)18
  19. 19. METHODS System Preparation All ATP-bound complexes were constructed by superimposing ATP and the Mg2+ ion from PDB 4K2R onto PDB 1U59. Protein interactions were modeled using the CHARMM22/CMAP force field. All systems were solvated in a 0.1 M sodium chloride solution containing TIP3P water molecules. 19
  20. 20. METHODS Simulation Setup All simulations were performed using GROMACS 4.5.5. Energy minimization Heating Equilibration Production Energy minimization Structure that including small atomic clashes, strained bonds and angles is performed with clearly. Using Isothermal–isobaric (NpT) ensemble in the system - amount of substance (N), pressure (P) and temperature (T) are conserved. Heating Assign initial velocities, temperature is increased from 0 K to 298 K 20
  21. 21. METHODS Simulation Setup All simulations were performed using GROMACS 4.5.5. Energy minimization Heating Equilibration Production This process is performed by slow perturb system, usually using 1000 steps for 500 ps. Once reached desired temperature, need to allow the system to equilibrate to remove any artifacts. This process start the production run, all systems were simulated for 1.5 microseconds in the NpT ensemble. Equilibration Production 21
  22. 22. METHODS Analysis Analysis of the trajectories was performed using module from the AmberTools 14 package. All trajectories were aligned by their Cα atoms, subsequently, backbone RMSD and B-factors. Regions of particular interest comprise the activation loop, the conserved DFG motif containing D479, F480 and G481, and the αC helix with the associated salt bridge K369-E386. RMSD is the measure of the average distance between the atoms (usually the backbone atoms) of superimposed proteins that taken over a group of particles at a single time. B-factors is the measure of the average distance between the atoms (usually the backbone atoms) of superimposed proteins that taken for a single particle over an interval of time. 22
  23. 23. METHODS 23 Fragment Screening and Pharmacophore Model The three frames of the non- phosphorylated trajectory that show the largest volume for the cryptic pocket were selected for fragment screening. This process was performed using Schrodinger Maestro 2015-2. The 100 highest scoring fragments from each structure were pooled and used as a basis to create a pharmacophore model of the cryptic pocket. Fig 4. Specific structural features o bonding between N348-S497 (yello
  24. 24. RESULTS Non-phosphorylated staurosporine-bound complex (STA) This complex remained stable throughout the course of the simulation, by the constant Cα-RMSD, which rapidly plateaued at ~2–3 Å, as well as the low B-factors. The DFG motif was observed to form a stable at ~3-4 Å. 24 resulting from analysis of the individual states, yielding a coherent picture for the mechanistic steps involved in catalytic (auto)inhibition and activation. Staurosporine Complex STA The staurosporine-bound complex remained stable throughout the course of the simulation. This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation . Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential pendent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited he ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569. .1371/journal.pcbi.1004560.g002 S Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 4 / 16 This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation Fig 2. Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569. doi:10.1371/journal.pcbi.1004560.g002 PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 4 / 16 STA STA loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent- exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop Fig 3. Structural motifs of ZAP–70 kinase domain as observed in the different states. Time-dependent distances are shown for: (a) the DFG motif D479 carboxylate carbon to the G481 backbone amide hydrogen; (b) the αC helix to the C-lobe center of mass; and (c) the salt bridge K369-E386. doi:10.1371/journal.pcbi.1004560.g003 Regulation of ZAP-70 Kinase Domain DFG motif (D479 to G481) αC helix salt bridge K369-E386 STA STA STA
  25. 25. Non-phosphorylated staurosporine-bound complex (STA) The distance of the αC helix from the center of mass of the C-lobe initially increased and remained stable at this level for the remainder of the trajectory. The salt bridge K369-E386 was expressed during the entire course of the simulation. RESULTS 25 loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent- exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop structure through a hydrogen bond from the side chain carboxylate-oxygen on D479 through the backbone amide hydrogen on G481 (Fig 3a). This closed loop structure is also present in the staurosporine X-ray structure, 1U59. The distance of the αC helix from the center of mass of the C-lobe initially increased from a value of 14 Å (in the crystallographic state) to 15 Å within ~150 ns and remained stable at this level for the remainder of the trajectory (Fig 3b). Moreover, the phenyl ring of F480 of the DFG motif forms a close contact with the backbone amide of M390 within the αC helix (S1 Fig). The salt bridge K369-E386 was nevertheless pres- ent during the entire course of the simulation (Fig 3c). It is present in the staurosporine com- plex structure 1U59 but absent in the ANP complexes 2OZO and 4K2R. Structural features specific for the staurosporine complex during our simulations comprise an additional salt bridge (D379-R496) formed across the catalytic cleft, and an adjacent inter- mittent hydrogen bond between the S497 hydroxyl and the N348 side chain amide group. This interaction is not observed in any X-ray structures of ZAP–70. Moreover, this behavior was Fig 3. Structural motifs of ZAP–70 kinase domain as observed in the different states. Time-dependent distances are shown for: (a) the DFG motif D479 carboxylate carbon to the G481 backbone amide hydrogen; (b) the αC helix to the C-lobe center of mass; and (c) the salt bridge K369-E386. doi:10.1371/journal.pcbi.1004560.g003 Regulation of ZAP-70 Kinase Domain DFG motif (D479 to G481) αC helix salt bridge K369-E386 STA STA STA
  26. 26. Non-phosphorylated staurosporine-bound complex (STA) Fig 4. Specific structural features of the various states of ZAP70 kinase domain: (a) The salt bridge D379-R496 an bonding between N348-S497 (yellow) connect the C- and N-lobes and restrict access to the catalytic cleft. Addition loop positioning. (b) The cryptic pocket adjacent to the activation loop spontaneously opened during simulation and was u state. Key residues lining the pocket are indicated in gray. Four consensus pharmacophore features were identified by fra the acceptor A, donor D, ring R, and a hydrophobic feature H, indicated inset. (c) The cryptic pocket repeatedly opened an simulation time, reaching a maximum volume of ~1400 Å 3 R Structural features specific for the STA complex during simulations comprise an additional salt bridge formed across the catalytic cleft, and an adjacent intermittent hydrogen bond between the S497 hydroxyl and the N348 side chain amide group. Moreover, these interactions span the catalytic cleft and connect the N- and C-lobes, thereby significantly restricting substrate access. RESULTS 26
  27. 27. Non-phosphorylated ATP-bound complex (Y0Y0) This structure did not present a closed loop within the DFG motif during simulation. RESULTS Thiscomplex remained stable and rigid, by an RMSD and low B-factors. 27 resulting from analysis of the individual states, yielding a coherent picture for the mechanistic steps involved in catalytic (auto)inhibition and activation. Staurosporine Complex STA The staurosporine-bound complex remained stable throughout the course of the simulation. This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation . Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential pendent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited he ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569. .1371/journal.pcbi.1004560.g002 S Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 4 / 16 This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation Fig 2. Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569. doi:10.1371/journal.pcbi.1004560.g002 PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 4 / 16 Y0Y0 Y0Y0 loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent- exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop Fig 3. Structural motifs of ZAP–70 kinase domain as observed in the different states. Time-dependent distances are shown for: (a) the DFG motif D479 carboxylate carbon to the G481 backbone amide hydrogen; (b) the αC helix to the C-lobe center of mass; and (c) the salt bridge K369-E386. doi:10.1371/journal.pcbi.1004560.g003 Regulation of ZAP-70 Kinase Domain DFG motif (D479 to G481) αC helix salt bridge K369-E386 Y0Y0 Y0Y0 Y0Y0
  28. 28. The distance of the αC helix from the center of mass of the C-lobe is analogous to the behavior of the STA complex. The salt bridge K369-E386 was present intermittently, around 50% of the total simulation time. RESULTS Non-phosphorylated ATP-bound complex (Y0Y0) 28 loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent- exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop structure through a hydrogen bond from the side chain carboxylate-oxygen on D479 through the backbone amide hydrogen on G481 (Fig 3a). This closed loop structure is also present in the staurosporine X-ray structure, 1U59. The distance of the αC helix from the center of mass of the C-lobe initially increased from a value of 14 Å (in the crystallographic state) to 15 Å within ~150 ns and remained stable at this level for the remainder of the trajectory (Fig 3b). Moreover, the phenyl ring of F480 of the DFG motif forms a close contact with the backbone amide of M390 within the αC helix (S1 Fig). The salt bridge K369-E386 was nevertheless pres- ent during the entire course of the simulation (Fig 3c). It is present in the staurosporine com- plex structure 1U59 but absent in the ANP complexes 2OZO and 4K2R. Structural features specific for the staurosporine complex during our simulations comprise an additional salt bridge (D379-R496) formed across the catalytic cleft, and an adjacent inter- mittent hydrogen bond between the S497 hydroxyl and the N348 side chain amide group. This Fig 3. Structural motifs of ZAP–70 kinase domain as observed in the different states. Time-dependent distances are shown for: (a) the DFG motif D479 carboxylate carbon to the G481 backbone amide hydrogen; (b) the αC helix to the C-lobe center of mass; and (c) the salt bridge K369-E386. doi:10.1371/journal.pcbi.1004560.g003 Regulation of ZAP-70 Kinase Domain DFG motif (D479 to G481) αC helix salt bridge K369-E386 Y0Y0 Y0Y0 Y0Y0
  29. 29. RESULTS Non-phosphorylated ATP-bound complex (Y0Y0) pecific structural features of the various states of ZAP70 kinase domain: (a) The salt bridge D379-R496 and the interm g between N348-S497 (yellow) connect the C- and N-lobes and restrict access to the catalytic cleft. Additionally these bo itioning. (b) The cryptic pocket adjacent to the activation loop spontaneously opened during simulation and was unique to the ey residues lining the pocket are indicated in gray. Four consensus pharmacophore features were identified by fragment scree ptor A, donor D, ring R, and a hydrophobic feature H, indicated inset. (c) The cryptic pocket repeatedly opened and closed ove on time, reaching a maximum volume of ~1400 Å 3 1/journal.pcbi.1004560.g004 The scanning the surfaces of the KD in the thiscomplex revealed the spontaneous formation of a cryptic pocket adjacent to the activation loop. In the initial structure, W505 forms the core of a hydrophobic cluster. 29
  30. 30. This cryptic pocket repeatedly opened and closed from ~700 ns. The protein backbone geometry of the maximum open states encountered at 1050 ns, 1257 ns and 1378 ns is identical. Coupled to the motion of the activation loop, the W505 ring underwent conformational switching within the hydrophobic core which increasing the distance of separation by up to 8 Å over the final ~700 ns. RESULTS Non-phosphorylated ATP-bound complex (Y0Y0) ~700 ns the αC helix and the center of mass of the C-lobe increased from ~100 ns and subsequently remained constant for the duration of is analogous to the behavior of the staurosporine complex (Fig e DFG motif with M390 in the αC helix was significantly less pro- plex or any phosphorylated state (S1 Fig). The salt bridge mittently, around 50% of the total simulation time (Fig 3c). and projections of the four lowest-frequency modes during the ain: (a) The salt bridge D379-R496 and the intermittent hydrogen access to the catalytic cleft. Additionally these bonds restrict activation sly opened during simulation and was unique to the non-phosphorylated cophore features were identified by fragment screening. These comprise he cryptic pocket repeatedly opened and closed over the final ~1μs of Regulation of ZAP-70 Kinase Domain 1050 ns 1257 ns 1378 ns 30
  31. 31. RESULTS Non-phosphorylated ATP-bound complex (Y0Y0) W505 The cryptic pocket is primarily formed by residues R460, D461, L462, A463, K500, W501, P502, W505, Y506 and S524. Overlaid snapshots of the cryptic pocket site are shown, prior to its formation at 0 ns (grey), and along the pathway of partial to complete pocket formation at 1059 ns, 1257 ns, and 1377 ns (dark to light orange colors, respectively). 31
  32. 32. Normal modes projection was showed a movement of the αC helix towards the remainder of the C-lobe. RESULTS Non-phosphorylated ATP-bound complex (Y0Y0) 32d with state of the complex. Normal mode (a) is (b) is associated with Y493 phosphorylation Y0 YP .
  33. 33. RESULTS ATP-bound complex with phosphorylated variant Y492 (YPY0) Simulations revealed elevated flexibility across the entire protein compared to both the STA and YPY0 complexes. The Cα-RMSD increased gradually over the first microsecond, before plateauing at ~4 Å. Consistently, the B-factor was higher in several regions of the protein. 33 analysis was performed for the Cα atoms of all trajectories in order to identify state-specific global motions. These motions were then correlated with structural and dynamic information resulting from analysis of the individual states, yielding a coherent picture for the mechanistic steps involved in catalytic (auto)inhibition and activation. Staurosporine Complex STA The staurosporine-bound complex remained stable throughout the course of the simulation. This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation Fig 2. Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569. doi:10.1371/journal.pcbi.1004560.g002 PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 4 / 16 This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued a well as the low B-factors (Fig 2b) across the entire domain and associated varian the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, th Fig 2. Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurospo and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues doi:10.1371/journal.pcbi.1004560.g002 PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 YPY0 YPY0
  34. 34. RESULTS ATP-bound complex with phosphorylated variant Y492 (YPY0) The DFG motif began in a conformation associated with a distance of 5 Å, this distance rapidly increased at the ~600 ns mark to 8 Å. The αC helix initially adopts a close conformation from the center of mass of the C- lobe after around 500 ns. After around 500 ns, it underwent a rapid shift, increasing to a distance of 14.5 Å as observed in the STA and Y0Y0 complexes. The K369-E386 salt bridge was absent for the majority of the simulation. 34 loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent- exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop structure through a hydrogen bond from the side chain carboxylate-oxygen on D479 through the backbone amide hydrogen on G481 (Fig 3a). This closed loop structure is also present in the staurosporine X-ray structure, 1U59. The distance of the αC helix from the center of mass of the C-lobe initially increased from a value of 14 Å (in the crystallographic state) to 15 Å within ~150 ns and remained stable at this level for the remainder of the trajectory (Fig 3b). Moreover, the phenyl ring of F480 of the DFG motif forms a close contact with the backbone amide of M390 within the αC helix (S1 Fig). The salt bridge K369-E386 was nevertheless pres- ent during the entire course of the simulation (Fig 3c). It is present in the staurosporine com- plex structure 1U59 but absent in the ANP complexes 2OZO and 4K2R. Structural features specific for the staurosporine complex during our simulations comprise an additional salt bridge (D379-R496) formed across the catalytic cleft, and an adjacent inter- mittent hydrogen bond between the S497 hydroxyl and the N348 side chain amide group. This interaction is not observed in any X-ray structures of ZAP–70. Moreover, this behavior was not observed in any other complex. These interactions span the catalytic cleft and connect the Fig 3. Structural motifs of ZAP–70 kinase domain as observed in the different states. Time-dependent distances are shown for: (a) the DFG motif D479 carboxylate carbon to the G481 backbone amide hydrogen; (b) the αC helix to the C-lobe center of mass; and (c) the salt bridge K369-E386. doi:10.1371/journal.pcbi.1004560.g003 Regulation of ZAP-70 Kinase Domain DFG motif (D479 to G481) αC helix salt bridge K369-E386 YPY0 YPY0 YPY0
  35. 35. Normal modes projection was showed a extension of the activation loop but no associated movement of the αC helix. RESULTS 35 ATP-bound complex with phosphorylated variant Y492 (YPY0) frequency normal modes correlated with state of the complex. Normal mode (a) is P Y0 and the Y0 YP state. Normal mode (b) is associated with Y493 phosphorylation Y0 YP . concurrent extension of the activation loop. The tertiary mode (c) is characteristic for YP Y0 .
  36. 36. RESULTS ATP-bound complex with phosphorylated variant Y493 (Y0YP) Simulations revealed elevated flexibility of RMSD and B-factor caused primarily by increased flexibility of the activation loop. 36 analysis was performed for the Cα atoms of all trajectories in order to identify state-specific global motions. These motions were then correlated with structural and dynamic information resulting from analysis of the individual states, yielding a coherent picture for the mechanistic steps involved in catalytic (auto)inhibition and activation. Staurosporine Complex STA The staurosporine-bound complex remained stable throughout the course of the simulation. This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation Fig 2. Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569. doi:10.1371/journal.pcbi.1004560.g002 PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 4 / 16 This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued a well as the low B-factors (Fig 2b) across the entire domain and associated varian the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, th Fig 2. Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurospo and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues doi:10.1371/journal.pcbi.1004560.g002 PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 Y0YP Y0YP
  37. 37. RESULTS The αC helix adopted a distinct open conformation, indicated by an increased distance from the center of mass of the C-lobe to ~15.5 Å within the first ~100 ns. The salt bridge K369-E386 was absent for the duration of the simulation. ATP-bound complex with phosphorylated variant Y493 (Y0YP) 37 loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent- exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop structure through a hydrogen bond from the side chain carboxylate-oxygen on D479 through the backbone amide hydrogen on G481 (Fig 3a). This closed loop structure is also present in the staurosporine X-ray structure, 1U59. The distance of the αC helix from the center of mass of the C-lobe initially increased from a value of 14 Å (in the crystallographic state) to 15 Å within ~150 ns and remained stable at this level for the remainder of the trajectory (Fig 3b). Moreover, the phenyl ring of F480 of the DFG motif forms a close contact with the backbone amide of M390 within the αC helix (S1 Fig). The salt bridge K369-E386 was nevertheless pres- ent during the entire course of the simulation (Fig 3c). It is present in the staurosporine com- plex structure 1U59 but absent in the ANP complexes 2OZO and 4K2R. Structural features specific for the staurosporine complex during our simulations comprise an additional salt bridge (D379-R496) formed across the catalytic cleft, and an adjacent inter- mittent hydrogen bond between the S497 hydroxyl and the N348 side chain amide group. This interaction is not observed in any X-ray structures of ZAP–70. Moreover, this behavior was not observed in any other complex. These interactions span the catalytic cleft and connect the Fig 3. Structural motifs of ZAP–70 kinase domain as observed in the different states. Time-dependent distances are shown for: (a) the DFG motif D479 carboxylate carbon to the G481 backbone amide hydrogen; (b) the αC helix to the C-lobe center of mass; and (c) the salt bridge K369-E386. doi:10.1371/journal.pcbi.1004560.g003 Regulation of ZAP-70 Kinase Domain DFG motif (D479 to G481) αC helix salt bridge K369-E386 Y0YP Y0YP Y0YP
  38. 38. Normal modes projection was showed extension of the activation loop and concurrent rearrangement of the αC helix. Strikingly, a general opening motion at the hinge region likely allows for easier substrate access to the catalytic cleft. RESULTS 38 ATP-bound complex with phosphorylated variant Y493 (Y0YP) n amplitudes for the four lowest-frequency normal modes correlated with state of the complex. Normal mode ( orylation as it is concurrent in the YP Y0 and the Y0 YP state. Normal mode (b) is associated with Y493 phosphorylation the movement of the αC helix with concurrent extension of the activation loop. The tertiary mode (c) is characteristic fo
  39. 39. RESULTS ATP-bound complex with di-phosphorylated variant Y492+Y493 (YPYP) This complex is characterized by the highest flexibility of all investigated states. Cα RMSD indicates high conformational variability. Analysis of the B-factors profile revealed particularly pronounced dynamics within the activation loop and the N-lobal region. 39 analysis was performed for the Cα atoms of all trajectories in order to identify state-specific global motions. These motions were then correlated with structural and dynamic information resulting from analysis of the individual states, yielding a coherent picture for the mechanistic steps involved in catalytic (auto)inhibition and activation. Staurosporine Complex STA The staurosporine-bound complex remained stable throughout the course of the simulation. This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued at ~2–3 Å, as well as the low B-factors (Fig 2b) across the entire domain and associated variance throughout the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, the activation Fig 2. Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over 10 sequential independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurosporine-inhibited and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues 537–569. doi:10.1371/journal.pcbi.1004560.g002 PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 4 / 16 This is illustrated by the constant Cα-RMSD (Fig 2a), which rapidly plateaued a well as the low B-factors (Fig 2b) across the entire domain and associated varian the trajectory (Fig 2c). Residual flexibility was observed within the DFG motif, th Fig 2. Conformational dynamics of ZAP–70 kinase domain. (a) Cα RMSD, (b) B-factors over final 500 ns, and (c) B-factor variance over independent trajectory segments of all investigated systems. All phosphorylated states exhibit elevated flexibility compared to the staurospo and the ATP-bound non-phosphorylated complexes. Regions of interest comprise the N-lobal residues 500–519 and the “flap-like” residues doi:10.1371/journal.pcbi.1004560.g002 PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004560 October 16, 2015 Y0YP Y0YP
  40. 40. RESULTS The DFG motif was observed to rapidly lose its structure, and spontaneously adopted an extended conformation. The conformation of the αC helix observed in the present state was analogous to the conformation in the Y0YP state, while the salt bridge K369-E386 was not present for the duration of the trajectory. ATP-bound complex with di-phosphorylated variant Y492+Y493 (YPYP) 40 loop downstream of the unmodified phosphorylation sites Y492 and Y493 and the solvent- exposed regions of the αC helix. The DFG motif was observed to form a stable, closed loop structure through a hydrogen bond from the side chain carboxylate-oxygen on D479 through the backbone amide hydrogen on G481 (Fig 3a). This closed loop structure is also present in the staurosporine X-ray structure, 1U59. The distance of the αC helix from the center of mass of the C-lobe initially increased from a value of 14 Å (in the crystallographic state) to 15 Å within ~150 ns and remained stable at this level for the remainder of the trajectory (Fig 3b). Moreover, the phenyl ring of F480 of the DFG motif forms a close contact with the backbone amide of M390 within the αC helix (S1 Fig). The salt bridge K369-E386 was nevertheless pres- ent during the entire course of the simulation (Fig 3c). It is present in the staurosporine com- plex structure 1U59 but absent in the ANP complexes 2OZO and 4K2R. Structural features specific for the staurosporine complex during our simulations comprise an additional salt bridge (D379-R496) formed across the catalytic cleft, and an adjacent inter- mittent hydrogen bond between the S497 hydroxyl and the N348 side chain amide group. This interaction is not observed in any X-ray structures of ZAP–70. Moreover, this behavior was Fig 3. Structural motifs of ZAP–70 kinase domain as observed in the different states. Time-dependent distances are shown for: (a) the DFG motif D479 carboxylate carbon to the G481 backbone amide hydrogen; (b) the αC helix to the C-lobe center of mass; and (c) the salt bridge K369-E386. doi:10.1371/journal.pcbi.1004560.g003 Regulation of ZAP-70 Kinase Domain DFG motif (D479 to G481) αC helix salt bridge K369-E386 Y0YP Y0YPY0YP
  41. 41. DISCUSSION & CONCLUSIONS Patterns of conformational plasticity associated with ZAP-70 kinase domain Common patterns observed in all simulated states were characterized by two distinct, comparatively rigid cores of the C-lobe and N-lobe, as well as flexible segments of the activation loop. 41 The phosphorylation caused a global increase in protein flexibility, specific changes were localized around the activation loop as well as the αC helix region. The conserved DFG motif at the N-terminal side of the activation loop adopted three distinct states across the different simulation systems, characterized by a cyclization through hydrogen bonding.
  42. 42. DISCUSSION & CONCLUSIONS Patterns of conformational plasticity associated with ZAP-70 kinase domain 42 D479 observed in the staurosporine-bound X-ray structure 1U59 versus the ANP-bound com- plexes 2OZO and 4K2R. Generally, our observations signify that residue D479 located close to the catalytic center is highly sensitive to its local electrostatic environment. It should be noted that all simulations started from the staurosporine-bound protein conformation represented by 1U59, as it is the only structure in which all residues of the activation loop are resolved. While the choice of this starting state may introduce a bias towards active-like states in the remaining simulations, the reorientation of the DFG motif is consistent with the 2OZO and 4K2R struc- Fig 6. Conformations of the DFG motif: Closed (gray), semi-closed (green) and open conformation (cyan). Staurosporine causes the DFG motif in ZAP–70 to adopt the closed conformation exclusively, presumably because D479 is unable to interact with the Mg2+ ion coordinated by ATP. The Y0 Y0 and Y0 YP variants predominantly adopt the semi-closed form. The open extended geometry is only observed in the YP Y0 and YP YP states. Labels within the figure indicate measured distances, in Angstroms. doi:10.1371/journal.pcbi.1004560.g006 Regulation of ZAP-70 Kinase Domain Closed (gray) (STA) Semi-closed (green) (Y0YP,Y0Y0) Open (blue) (YPYP,YPY0) By considering on DFG motif, the distance between the carboxylate carbon of D479 and the backbone amide hydrogen of G481, it’s identified by a cyclic. Closed state at approximately 3 Å for STA state Semi-closed state at a distance of ~6 Å for Y0YP,Y0Y0 states Open state at a distance of ~8 Å for YPYP,YPY0 states.
  43. 43. DISCUSSION & CONCLUSIONS Patterns of conformational plasticity associated with ZAP-70 kinase domain 43 D479 observed in the staurosporine-bound X-ray structure 1U59 versus the ANP-bound com- plexes 2OZO and 4K2R. Generally, our observations signify that residue D479 located close to the catalytic center is highly sensitive to its local electrostatic environment. It should be noted that all simulations started from the staurosporine-bound protein conformation represented by 1U59, as it is the only structure in which all residues of the activation loop are resolved. While the choice of this starting state may introduce a bias towards active-like states in the remaining simulations, the reorientation of the DFG motif is consistent with the 2OZO and 4K2R struc- Fig 6. Conformations of the DFG motif: Closed (gray), semi-closed (green) and open conformation (cyan). Staurosporine causes the DFG motif in ZAP–70 to adopt the closed conformation exclusively, presumably because D479 is unable to interact with the Mg2+ ion coordinated by ATP. The Y0 Y0 and Y0 YP variants predominantly adopt the semi-closed form. The open extended geometry is only observed in the YP Y0 and YP YP states. Labels within the figure indicate measured distances, in Angstroms. doi:10.1371/journal.pcbi.1004560.g006 Regulation of ZAP-70 Kinase Domain YPY0 causes a repulsive charge-charge interaction with D479, thereby promoting the open state. Staurosporine is bereft of a negative charge proximal to the DFG motif and misses an Mg2+ ion, D479 is free to position itself in a hydrogen bonding orientation towards the back- bone N-H of G481. Closed (gray) (STA) Semi-closed (green) (Y0YP,Y0Y0) Open (blue) (YPYP,YPY0)
  44. 44. DISCUSSION & CONCLUSIONS Patterns of conformational plasticity associated with ZAP-70 kinase domain 44 Inhibition by staurosporine caused this salt bridge to adopt a permanently closed position. All phosphorylated states revealed an increased average distance between the guadinium group of R369 and the E386 carboxylate function, as well as elevated variance of this distance compared to the non-phosphorylated state. Characteristics DFG motif αC helix Salt bridge STA Closed Close position Strong Y0Y0 Semi-closed Close position - YPY0 Open Close position Weak Y0YP Semi-closed Reposition - YPYP Open Reposition - Inhibited, non-phosphorylated and phosphorylation of Y492 states show a closer positioning to the protein center of mass of the αC helix. Only phosphorylation of Y493 or di-phosphorylation induced repositioning of the αC helix, consistent with kinase activation.
  45. 45. DISCUSSION & CONCLUSIONS Patterns of conformational plasticity associated with ZAP-70 kinase domain 45 From these observations, conclude that the strength of the salt bridge R369-E386 is weakened by phosphorylation and is a necessary step for subsequent repositioning of the αC helix towards an active conformation. This repositioning is solely observed if Y493 is phosphorylated. Mono- phosphorylation of Y492 weakens the salt bridge, but does not induce a conformational shift in the αC helix. Characteristics DFG motif αC helix Salt bridge STA Closed Close position Strong Y0Y0 Semi-closed Close position - YPY0 Open Close position Weak Y0YP Semi-closed Reposition - YPYP Open Reposition -
  46. 46. DISCUSSION & CONCLUSIONS Mechanisms of inhibition 46 All investigated structural features comprising the DFG motif, the αC helix, the R369-E386 salt bridge, as well as patterns of normal modes and B- factors, indicate lowered dynamics for the staurosporine complex. The binding of staurosporine traps the ZAP–70 KD in a compact and rigid state. The observed rigidity would limit both substrate access and the likelihood of competitive binding by co-factor at the catalytic center.
  47. 47. DISCUSSION & CONCLUSIONS Cryptic Pocket 47 L1 3WE, United Kingdom ion pockets, that is, sites on protein targets parent when drugs bind, provide a to classical binding sites for drug investigate the nature and dynamical tes in four pharmacologically relevant ng the efficacy of various simulation- discovering them. We find that the not correspond to local minima in the onal free energy landscape of the hey thus promptly close in all of the mulations performed, irrespective of the force-field used. Temperature-based enhanced sampling allel Tempering, do not improve the situation, as the entropic term does not help in the opening of the nt probes helps, as in long simulations occasionally it leads to the opening and binding to the cryptic sites. sm of cryptic site formation is suggestive of an interplay between two classical mechanisms: induced-fit ection. Employing this insight, we developed a novel Hamiltonian Replica Exchange-based method Water Interfaces through Scaled Hamiltonians), which combined with probes resulted in a promising yptic site discovery. We also addressed the issue of “false-positives” and propose a simple approach to druggable cryptic pockets. Our simulations, whose cumulative sampling time was more than 200 μs, help lar mechanism of pocket formation, providing a solid basis for the choice of an efficient computational easingly expensive endeavor as many ects fail in clinical trials.1−5 Late-stage ostly, and lack of efficacy of lead researchers had tried and failed to develop K-RAS inhibitors, to the point it was thought to be undruggable. Yet very recently, a new cryptic pocket was found by tethered compounds and successfully targeted.12 The molecular mechanism by which cryptic sites are formed is not clear. Although ligands seem to From analyses have allowed to identify a novel pocket adjacent to the active site and the activation loop. This pocket is neither a classical type I or type II kinase pocket and offers potential possibilities for design of new specific inhibitory ligands and pocket only occurs in the non-phosphorylated state. Cryptic pockets is sites on protein targets that only become apparent when drugs bind. binding through interactions with this region. A flexible activation loop starting with a conserved amino acid sequence Asp-Phe-Gly (DFG) controls access to the active site [22] (Figure 1A). Categorized by binding modes, kinase inhibitors can be grouped into two classes: irreversible and reversible. The former tend to covalently bind with a reactive nucleophilic cysteine residue proximal to the ATP-binding site, resulting in the blockage of the ATP site exclusively in an allosteric pocket adjacent to ATP without making any interaction with the ATP-binding pocket. Type IV inhibitors bind to an allosteric site remote from the ATP-binding pocket [25]. Some kinase inhibitors, such as bisubstrate and bivalent inhibitors (type V) [26], exhibit more than one of these binding modes. Small-molecule kinase inhibitors are useful reagents to investigate and elucidate kinase functions in various Type I (B) Ala 162 Ser 160 Asp 223 Ser 94 Ser 92 Glu 166 (A) N-lobe C-lobe Hinge Type II Type III Type IV TRENDS in Pharmacological Sciences Type I kinase pocket is active conformation of the kinase with the aspartate residue (white backbone) of the DFG motif pointing into the ATP-binding pocket. binding through interactions with this region. A flexible activation loop starting with a conserved amino acid sequence Asp-Phe-Gly (DFG) controls access to the active site [22] (Figure 1A). Categorized by binding modes, kinase inhibitors can be grouped into two classes: irreversible and reversible. The former tend to covalently bind with a reactive nucleophilic cysteine residue proximal to the ATP-binding site, resulting in the blockage of the ATP site exclusively in an allosteric pocket adjacent to ATP without making any interaction with the ATP-binding pocket. Type IV inhibitors bind to an allosteric site remote from the ATP-binding pocket [25]. Some kinase inhibitors, such as bisubstrate and bivalent inhibitors (type V) [26], exhibit more than one of these binding modes. Small-molecule kinase inhibitors are useful reagents to investigate and elucidate kinase functions in various Type I (B) Ala 162 Ser 160 Asp 223 Ser 94 Ser 92 Glu 166 (A) N-lobe C-lobe Hinge Type II Type III Type IV TRENDS in Pharmacological Sciences Type II kinase pocket is inactive conformation of the kinase with the flipped aspartate residue facing outward of the binding pocket.
  48. 48. DISCUSSION & CONCLUSIONS Cryptic Pocket 48 The opening of this pocket is facilitated by a flap-like rearrangement of residues 495–498, associated with the“gating”of the conserved residue W505, which switches to an alternative hydrophobic environment supported by residues in or nearby to the mobile N-lobal region. Closer examination of the normal modes reveals that the phosphorylated states exhibit concerted motions along the entire activation loop, whereas these motions are much more localized to the region following the two tyrosines Y492 and Y493 in the non-phosphorylated state.
  49. 49. DISCUSSION & CONCLUSIONS Mechanisms of activation 49 Phosphorylation was observed to lead to a global increase in dynamics, irrespective of the precise phosphorylation state. From simulations allow to identify changes that are specific to Y493 phosphorylation and therefore let trace the activation cascade: Y493 phosphorylation (green triangle) causes the salt bridge R369-E386 connecting the N-lobe with the αC helix (red) to weaken. Concurrently, the activation loop (black) becomes more dynamic, thus allowing easier access to the active center. stronger mobilizing effect than single phosphorylation of either residue. However, structural changes were distinctly different for the individual mono-phosphorylated complexes. Muta- tional studies suggest that Y492 is only weakly implicated in biological activation of ZAP–70. However, Y493 phosphorylation is of crucial importance to biological function as the Y493F mutation abolishes catalytic activity. Our simulations allow us to identify changes that are spe- cific to Y493 phosphorylation and therefore let us trace the activation cascade: Y493 phosphor- ylation causes the salt bridge R369-E386 connecting the C-lobe with the αC helix to weaken. In contrast to Y492 phosphorylation, Y493 phosphorylation also promotes rearrangement of the αC helix towards an active conformation already observed for members of the Src kinase fam- ily. Concurrently, flexibility of the activation loop increases significantly, thus allowing for eas- ier access to the catalytic center. A similar increase in activation loop exposure has been observed in the activation of focal adhesion kinase (FAK). However, the activation cascade of FAK is not directly analogous to ZAP–70, as FAK has an additional FERM domain involved in forming an auto-inhibited complex. [21] Our proposed activation cascade for ZAP–70 is sum- marized in Fig 7. Normal mode analysis further confirms these motions as characteristic for the biologically relevant Y493 phosphorylation. Fig 7. Proposed activation cascade of ZAP–70 kinase domain through the phosphorylation of Y493: upon phosphorylation of Y493 (green triangle), the salt bridge K369-E386 weakens and thus allows for movement of the αC helix (red) towards an active conformation. Concurrently, the activation loop (black) becomes more dynamic, thus allowing easier access to the active center. doi:10.1371/journal.pcbi.1004560.g007 Regulation of ZAP-70 Kinase Domain
  50. 50. THANKYOU
  51. 51. The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain COMPUTATIONAL BIOLOGY Presented by: Bundit Boonyarit 5814400587 Dept. Biochemistry, Fac. Science, Kasetsart University Huber R. G., et.al (2015) IMAGE CREDIT: ANSGAR PHILIPPSEN Nov 8, 2016 11.30 am - 12.00 pm

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