1. MCB Exam 2 Review
11/3/2018

2. Questions from s

3. 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

4. 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.

5. 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

6. 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

7. 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

8. 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)

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

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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

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

18. Exam Review

19. 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?

20. 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?

21. 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?