MCB Exam 2 Review 11/3/2018

Questions from Emails

Defining type and # of cooperative binding sites: Hill plots The Hill equation accounts for the possibility that not all receptor sites are independent, and states that Fractional occupancy = Lfn/ (Kd + Lfn) n= slope of the Hill plot and also is the avg # of interacting sites For linear transformation, log [B/(Rt - B)] = n(log Lf) - log Kd If slope = 1, then single class of binding sites The Hill equation is an equation used in enzyme characterization, which should not be confused with the Hill differential equation that is also sometimes referred to as simply the Hill equation.In biochemistry, the binding of a ligand to a macromolecule is often enhanced if there are already other ligands present on the same macromolecule (this is known as Cooperative binding). The Hill coefficient, named for Archibald Vivian Hill, provides a way to quantify this effect.It describes the fraction of the enzyme saturated by ligand as a function of the ligand concentration; it is used in determining the degree of cooperativity of the enzyme. It was originally formulated by Archibald Hill in 1910 to describe the sigmoidal O2 binding curve of hemoglobin.[1]A coefficient of 1 indicates completely independent binding, regardless of how many additional ligands are already bound. Numbers greater than one indicate positive cooperativity, while numbers less than one indicate negative cooperativity. The Hill coefficient was originally devised to explain the cooperative binding of oxygen to Hemoglobin (a system which has a Hill coefficient of 2.8-3).Hill equation:θ - fraction of ligand binding sites filled[L] - ligand concentrationKd - dissociation constant derived from the law of mass action (equilibrium constant for dissociation)KA - ligand concentration producing half occupation (ligand concentration occupying half of the binding sites)n - Hill coefficient, describing cooperativity (and many more, depending on the system, in the case of which the Hill equation is used)Taking the logarithm on both sides of the equation leads to an alternative formulation of the Hill equation: When appropriate, the value of the Hill constant describes the cooperativity of ligand binding in the following way:▪ n > 1 - Positively cooperative reaction: Once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules increases.▪ n < 1 - Negatively cooperative reaction: Once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules decreases.▪ n = 1 - Noncooperative reaction: The affinity of the enzyme for a ligand molecule is not dependent on whether or not other ligand molecules are already bound.The Hill equation (as a relationship between the concentration of a compound adsorbing to binding sites and the fractional occupancy of the binding sites) is equivalent to the Langmuir equation. log [B/(Rt - B)] If slope > 1, then positive cooperativity Slope= n If slope < 1, then negative cooperativity log Lf

Cooperativity indicated by non-linear Scatchard plots Positive cooperativity: binding of ligand to first subunit increases Affinity of subsequent binding events. Example: hemoglobin binding O2 (Bound Lig) (Free) (Bound Lig) Negative cooperativity: binding of ligand to first subunit decreases affinity of subsequent binding events.

EGF activates the MAPK pathway in multiple steps, with multiple mechanisms EGF Extracellular GF EGFR RTK EGFR~P Phospho-RTK Grb2 Adapter SOS Ras-GEF Ras Small GTPase Raf Mechanism Ser kinase Mek Proximity Tyr/thr kinase Ser kinase ERKs Allostery C-Jun Covalent modification Transcription factor

The best understood MAPK cascade MAPK = Mitogen-activated protein kinase . Phos’n of T-loop Ser residues Raf-1 A-raf B-raf . P Phos’n of T-loop Thr and Tyr MEK1 MEK2 . P MAPKKK Phos’n of Ser/Thr MAPKK ERK1 ERK2 C-Jun MAPK Altered gene expression

EGFR Activation of PI3K combines Proximity & Allostery . . PIP2 PIP3 P SH2 p85 P p110 P P Activated by EGFR/p85 SH2 p110 Recruitment from cytoplasm to PM, via SH2 domains p85 Can also be activated by Rac or Ras! SH2 How do we know proximity is not enough? 1. p85 mutants that activate without binding to RTKs 2. Tethering to membrane does not activate

1973: (Rowley) Studies showed that the Ph1 chromosome is the result of a reciprocal translocation between chromosomes 9 and 22 (t 9;22)(q34.1; q11.21)

The BCR-ABL fusion protein activates several signaling pathways Deininger et al., Blood 2000

BCR-ABL can recapitulate the CML phenotype Leukocytosis Splenomegaly Extramedullary hematopoiesis Death

Mechanism of imatinib (Gleevec) BCR-ABL is a tyrosine kinase Imatinib is a kinase inhibitor kinase kinase imatinib ATP substrate substrate SH2 SH2 Phosphotyrosine residues bind SH2 domains No phosphorylation, no interaction

Cytokine Receptors – JAK/STAT Pathway There are three key parts of JAK-STAT signalling: Janus kinases (JAKs), Signal Transducer and Activator of Transcription proteins (STATs), and receptors (which bind the chemical signals Baker et al., Oncogene (2007) 26, 6724–6737

Jak/STAT activation mechanism The binding of various ligands causes the receptors to dimerize, which brings the receptor-associated JAKs into close proximity. The JAKs then phosphorylate each other on tyrosine residues located in regions called activation loops, which increases the activity of their kinase domains The activated JAKs then phosphorylate tyrosine residues on the receptor, creating binding sites for proteins possessing SH2 domains. STATs then bind to the phosphorylated tyrosines on the receptor using their SH2 domains, and then they are tyrosine-phosphorylated by JAKs, causing the STATs to dissociate from the receptor. These activated STATs form hetero- or homodimers, where the SH2 domain of each STAT binds the phosphorylated tyrosine of the opposite STAT The dimer then translocates to the cell nucleus to induce transcription of target genes Jatiani, et al., Genes and Cancer, 2011

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Trypsin – a protease that cleaves after basic residues (R or K). Protein of Interest: Slide courtesy of Andrew Link

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Products from Trypsin digest. Average length of tryptic peptides = 10 aa residues Slide courtesy of Andrew Link

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides The mass difference between the peaks corresponds directly to the amino acid sequence. B-ions contain the N-terminus Slide courtesy of Andrew Link

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Y-ions contain the C-terminus Slide courtesy of Andrew Link

Exam Review

Blumer Cell-surface receptors Scatchard plots How is calcium regulated? What are second and third messenger systems? How are G proteins regulated; what are features of G protein-coupled receptors How is EGFR activation of Ras controlled and executed? How are receptor tyrosine kinases activated? What are the effectors and inputs?

Bose Understand characteristics of nuclear hormone receptors, and how their functions were identified in vivo How is the JAK/STAT pathway regulated? What are the players and what is the downstream result? What are the characteristics of the mTORC complexes? Understand protein kinase regulation How are samples prepared and analyzed for mass spec? Are mass spec experiments quantifiable? How? What are other proteomics techniques; how do they work?

Oh How was BCR-ABL studied in the context of CML? What techniques were used to further our understanding of this disease? What are MPNs and what do we know about them? How do we treat the causative mutations in MPNs?