Signal Transduction Phosphorylation Protein kinases. Misfolding diseases. Protein Engineering Lysozyme variants

Transcription

1 Signal Transduction Phosphorylation Protein kinases Misfolding diseases Protein Engineering Lysozyme variants

2 Cells and Signals

3 Regulation The cell must be able to respond to stimuli Cellular activities need tight regulation Transcriptional level Inhibitors Allosteric effectors Proteolytic activation Reversible covalent modification - phosphorylation

4 Human Growth Hormone Binding site Extra cellular domain of receptor The hormone is a 4 helix bundle.

5 Human Growth Hormone The hormone also binds to the prolactin receptor, but in a 1:1 complex Two receptors can bind to the hormone, which thus induces dimerization of the receptor.

6 Growth Hormone Interactions with Different Receptors Both sequence differences and different orientations of receptor domains are important for ligand binding

7 Signal Transduction and Regulation Phosphorylation G-proteins Tyrosine kinases Growth hormone Conformational change - interactions - complexes

8 The Phosphorylation Reaction The γ-phosphoryl group of ATP is transferred to Ser/Thr or Tyr residues by different kinases Not on extracellular proteins The net outcome of the cycle is hydrolysis of ATP to ADP and P i The cycle is unidirectional

9 Thermodynamics, kinetics ATP hydrolysis: -12 kcal/mol half used to make reaction irreversible, the other half is stored in the phosphorylated protein 1.4 kcal/mol corresponds to a 10-fold change in K eq phosphorylation can change a conformational equilibrium by 10 4 Wide kinetic range: <seconds to >hours Tunable, depending on activity of kinases and phosphatases Amplification: A single activated kinase can phosphorylate hundreds of targets in a short time

10 Phosphoryl Interactions ATP Two negative charges are added May induce conformational change Three hydrogen bonds can be formed Quite specific. Substrate binding and catalytic activity may be influenced

11 Protein Kinase Specificity Dedicated protein kinases: Single protein target (or small set of closely related targets) Multifunctional protein kinases: Different targets, coordination Related sequences are recognized

12 Protein Kinase A specificity (cyclic AMP-dependent protein kinase) Consensus motif for PKA: Arg-Arg-X-Thr/Ser-Z X is small, Z is large hydrophobic 1atp.pdb

13 PKA activation Cyclic AMP works by activating PKA

14 G proteins

15 G proteins Amplifiers Bind guanine nucleotides Switched off by GTP hydrolysis, on by GTP rebinding Heterotrimers with α,β, and γ subunits

16 G α activation Conformation in three switch regions depends on GTP/GDP being bound Active, GTP bound Inactive, GDP bound GTPase domain Helical domain

17 G α Conformational Switch

18 G α Conformational Switch

19 G α Conformational Switch

20 G β and G γ domains

21 G β and G γ domains

22 Light Adaptation in the Retina Phosphorylation of Ser73 of phosducin in dark adapted rods reduces binding to G βγ Phosducin binds to G βγ and moves it to the cytosol in light adapted rods.

23 Enzyme-linked receptors Receptor Tyrosine kinases Tyrosine kinase-associated receptors Receptor Tyrosine phosphatases Transmembrane Ser/Thr kinases Transmembrane guanyl cyclases Modular with similar kinase domains in the Tyr and Ser/Thr kinases

24 Tyrosine kinases Receptor Tyrosine kinases Tyrosine kinases associated with receptors

25 Receptor Tyrosine kinases Ligand binding induces oligomerization Kinase activity is turned on Cross-phosphorylation Phosphotyrosines serve as docking points for intracellular proteins Formation of ensemble of immobilized proteins at the membrane, and we have an active multisignaling complex

26 Src Tyrosine Kinase

27 Src Tyrosine Kinase SH2

28 Src Tyrosine Kinase SH2 SH2 domain phosphotyrosine binding pocket SH2 domain w/ bound phosphotyrosine peptide (1sha.pdb)

29 Src Tyrosine Kinase SH3

30 Src Tyrosine Kinase SH3

31 Transmission of information between SH2 and SH3 How does the (de)phosphorylation of Tyr527, which interacts with SH2 domain, get known by the SH3 domain? Young, M. A., Gonfloni, S., Superti-Furga, G., Roux, B., and Kuriyan, J. (2001). Dynamic Coupling between the SH2 and SH3 Domains of c-src and Hck Underlies Their Inactivation by C-Terminal Tyrosine Phosphorylation. Cell 105,

32 Dynamic SH2 SH3 coupling SH3 Perform computer simulations of motions in SH2-SH3 domains Monitor which aa move together (connected w/ red lines in figure) Test effect of Tyr527 dephosphorylation coupling patterns change, with less C-tail- SH2-linker-SH3 coupling in the dephosporylated, active form SH2

33 SH2 SH3 coupling, importance of the linker A rigid SH2-SH3 linker is required for the switch to work. Introducing 3 Gly mutations in linker reduces coupling, resembling the effect of tail dephosphorylation. Shown to give constitutively active kinase!

34 Regulation of catalytic activity Conformational change

35 Protein Structure and Disease Hemoglobin: 2α+2β subunits Sickle-cell hemoglobin polymerizes - the effect of one single point mutation: Glu6Val in the β chain

36 Sickle-cell anemia, a molecular disease [Hb] is extremely high in red blood cells - 340mg/ml! Glu6 Val6 introduces hydrophobic patch on the surface In deoxygenated Hb, as after oxygen delivery in capillaries, this patch can interact with a pocket on another Hb and fibers are formed, changing the shape of the red blood cells Lethal to homozygotes, but gives increased malaria resistance to heterozygotes

37 Proteins precipitate! Prion Diseases Protein is the infectious agent Amyloidosis - normally soluble globular proteins form stable insoluble fibrils which are deposited in the extracellular space, e g in the brain or eye. BSE, CJD, Alzheimer, type II diabetes Transthyretin The fibrils have β structures (carries thyroxine hormone) Single point mutation (Val Met) causes fibril formation!

38 Fibril formation (model!) Strands C & D unfold, giving a conformation which can form fibrils

39 Protein Engineering Change specificity or affinity - specific interactions in the active site are modified Optimize stability - e g temperature, ph Native state is entropically unfavored Reduce the number of conformations of unfolded state and the native state should be stabilized How? Carefully chosen disulfide bridges Remove Gly, add Pro

40 T4 Lysozyme wt has two Cys but no disulfide Cys-Cys has quite rigid conformation select pairs of aa in wt where it would be possible to introduce Cys-Cys

41 Introduce negative residues at the N-terminal end of α helices gives ca 2K increased T m T4 Lysozyme

42 More T4 lysozyme PERM1 PERMEXT Either C- or N- terminal helix could be in native position (in crystal structure it is the N- terminal one) Change position of N- and C-terminus: less stable than wt, folds faster Add extra helix: as stable as wt, folds slower due to competition from wrong helix for the correct position Sagermann, M., Baase, W. A., Mooers, B. H. M., Gay, L., and Matthews, B. W. (2004). Relocation or Duplication of the Helix A Sequence of T4 Lysozyme Causes Only Modest Changes in Structure but Can Increase or Decrease the Rate of Folding. Biochemistry 43,

43 Ligand Triggered Conformational Switch in Engineered Lysozyme Duplicated helix With or without stabilizing ligand Yousef, M. S., Baase, W. A., and Matthews, B. W. (2004). Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme. Proc Natl Acad Sci USA 101,

44 Mutating the Arg gives a form that can change conformation in the presence of guanidinium Fluctuations per residue Accessible surface area per residue