Protein and molecular recognition Flashcards
SH2 domains
- 100 amino acids
- two antiparallel beta strands surrounded by 2 helices
- phosphotyrosine binds as an extended strand
- there are conserved Arg/Lys residues that contribute to interact with the negative tyrosine
- variable residues contribute to specificity
- two binding pockets:
1. for the phosphotyrosine (conferring selectivity for the activate RTK/EGFR receptor)
2. for the downstream binding peptide (conferring sequence specificity to that protein)
Grb2
- one SH2 and two SH3 domains
- function– recruit Sos
Gbr2-Sos interaction
- the SH3 domain of Grb2 consists of 5 anti-parallel beta strands a a group of aromatic residues forming a hydrophobic patch
- the SH3 binds a left handed poly-proline helix (consensus=PxxP)
- the specificities of individual SH3 domains are different due to two charged variable loops
- as the binding surface of Grb2 is hydrophobic, the interactions between it and the hydrophobic Sos are strong and permanent !!
- 9 proline residues bind to a long groove in Grb2
Sos-Ras interaction
- binding is through a large interface– mainly hydrogen bonds and electrophilic interactions
- binding causes a change in the Ras nucleotide binding site (this releases GDP)
conformational changes in Ras
Ras has different conformations depending on which nucleotide is present;
the difference is because of conformational changes in two regions (“switch” 1 and 2) which form hydrogen bond with the “gamma” (third) phosphate of GTP
what does the GTPase activity of Ras require
- GAP
- inserts Arg residue in the active site to stimulate hydrolysis
Binding between Ras and Raf
- an intermolecular beta sheet (extending the anti-parallel beta sheet in both)
- Raf has NO direct contact with the GTP; Raf only contacts one of the switches which change conformation
Raf
- Ser/Thr kinase
- N terminal inhibitory domain loops back and blocks the catalytic domain normally
- Ras binds to inhibitory domain and displaces it, allowing activation
Fos and Jun
- DNA binding proteins causing transcription of growth factor responsive genes
- leucine zippers
bonds:
- hydrophobic interactions between the helices
- non-specific ionic interactions with phosphate backbone
- specific H bonds with base pairs (in the major groove)–>this gives the specificity of promoter interaction
binding specificity of antibodies
- determined by CDR sequence
- CDR3 is the most variable
- variability is achieved through recombination
FcgR (g=>gamma)
- it is a receptor on cell that binds the CH2 and CH3 constant domains of the antibody IgG
- leads to phagocytosis or cytotoxicity– mediated by the cell that expresses the receptor
FcRn
-is a receptor expressed in neonates that protects IgG from degradation
complement cascade and constant domains
- the constant domains of Ig are recognised by the first component of complement (C1q)
- conformational change and proteolysis leads to activation of the complement cascade and activation of the membrane attack complex
- the membrane attack complex forms pores in the cell and leads to lysis and inflammation response
what is the concept behind immunity to flu
- haemagglutinin binds sialic acid on target
- immunity comes from antibodies that block the site that binds to the sialic acid
human antibody fragments? their role?
- FAb–>(contain VL, CL, VH, and CH1)
- Fv–>(contains VL and VH)
- scFv–>(also contains VL and VH but with a peptide linker)
-used in drugs to bind to pathogens or toxins and simply block their function
humira (adalimumab)
- therapeutic antibody generated from phage display
- prevents TNF binding to its receptor
- used for autoimmune diseases (RA, crohns, psoriasis)
mechanism of action of humira
- reduced TNF-alpha activity
- reduces the expression of TNF inducible genes
- decreases inflammation
- decreased angiogenesis and invasion of basement membrane
Herceptin (Trastuzumab)
- is a drug used in chemotherapy
- it binds to ErbB2 near membrane and doesn’t disrupt dimerization
- thought to cause receptor-mediated internalisation
- also mediates antibody dependant cytotoxicity
which cancers is EGFR involved in?
-Colorectal, head and neck etc.
Cetuximab
chimaeric antibody to ErbB2
disrupt dimerization
Pertuzumab
humanised antibody to ErbB2
disrupt dimerization
Panitumumab
Transgenic mice dervied antibody to Erb2
disrupt dimerization
ErbB2
- it is a member of EGF family
- it doesn’t have a ligand binding domain to bind to GFs
- but, it can bind tightly to other EGFRs to form a heterodimer
- this stabilizes the ligand binding and enhances the signalling pathway
- overexpression of ErbB2 has been proposed in various cancers (incl. breast)
what’s antibody-dependant cytotoxicity?
-a cell mediated immune defense whereby an effector cell lyses a target cell whose membrane-surface antigens have been bound by specific antibodies
Erlotinib (Tarceva)
-EGF receptor kinase inhibitor
-competes with ATP for binding the active site
(ATP forms H bonds with some residues and binds weakly; tarceva has many hydrophobic interactions–binds 100x more tightly)
Vemurafinib
- it’s a Raf inhibitor used in treatment of melanoma once the V600E mutation has been confirmed by sequencing;
- it is generated by fragment-based drug theory
around half malignant melanomas have a Raf (V600E) inhibitor– Val in the activation loop is replaced by a Glu; this leads to activated Raf without a signal
