Modular proteins Flashcards
define domain
a folded structural unit; the sequence need not be contiguous e.g. MHC II
Define a module
A domain with a contiguous sequence, repeatedly used in diverse proteins e.g. F3
Define a Repeat
A unit that does not fold in isolation; several copies are needed e.g. leu-rich repeat
True or false, a module is a specific type of domain
TRUE
Are domain combinations random?
NO
Few domains combine with many
Most combine only with one or few other domains
E.g. tandem repeats of the same domain are frequent (gene duplication)
Examples of popular domains
IG, F3, F1, EGF, SH2, SH3, Kinase, MHC II
F3 is a module and a domain (because its contiguous)
What are the biological implications of protein domains?
many surfaces using the same scaffold
presentation of binding sites - binding sites can be in many places
Assembly processes can be controlled
Regulation- control the regulation of the function by a range of domains
poly-valency – interaction constants – control or increase
Variable connections
position of the domain influences its function
Regulation via domain rearrangement
can control the activity of some domains by the controlling the domain interface (gives flexibility and control) e.g. SH2 and SH3 domains of Src
Binding constant of bivalent interactions is…
The product of the individual binding constants
Which binding is tighter (mono- vs bi-valent)
Bivalent
Give an example of a protein that has polyvalent interactions
IgA (teravalent)
IgM (decavalent)
What are the two forms of fibronectin (FN)?
Circulating blood (soluble form)
In matrix (activated) (ECM form)
Features of FN
Two S-S linked glycosylated chains (~ 250kDa, ~70nm) (several alternatively spliced forms)
Binds integrins, bacteria and other ECM components
Role in development, growth, wound healing and cancer
gene knockout is lethal
Explain the different things that bind to FN and some of their functions
Fibrin - blood clotting
Heparin - ECM component
S. aureus - Virus
Collagen - ECM component
Gelatin
C1q (complement)
Cell membranes
Bacterial FN binding proteins
Pathogenic bacteria e.g. Streptococci use FN in the ECM of the host to adhere and gain access to host cells
Fibronectin-binding ashesins
These are on the surface of pathogenic bacteria, they bind to FN on host cell (e.g. epithelial cell)
share overall organisation
subtypes - S.aureus: FnbpB, FnbA
S. Dysgalactiae: FnbB
What did sequence alignment allow when it came to FN
showed sequences of around 8 AAs bound to F1
allowed finding of patterns - conserved in multiple places
need at least 4 AAs in the right place to bind to FN
so in a run of 40 AAs, this occured multiple times, meaning up to 11 FNs could bind (multivalency)
Explain the model for FN mediated bacterial uptake
1) Each fibronectin binding repeat (FnBR) binds one fibronectin (Fn) molecule via 4 to 5 N-terminal F1 domains. Multiple Fn’s are bound depending on the number of FnBR in the bacterial receptor (recond layer of polyvalancy)
2) Clustering of RGD sequences on Fn activates integrins leading to
3) signalling, actin rearrangement and potentially uptake
why is clustering of integrins necessary for FN mediated bacterial uptake
clustering integrins of the host – by doing this they can engulf the bacteria
Explain why bacteria want to be engulfed by host cells
Persistent infection occurred unless the bacteria had somewhere to hide, this is why it hijacks the migration capability of the cell to be engulfed
What is cell attachment to the matrix mediated by
8th-10th F3 module of fibronectin:
Primary binding site is Arg-Gly-Asp (RGD) tri-peptide
RGD peptide is located on 10F3
Additional synergy site on 9F3
Explain how the two different binding sites on the F3 scaffolds are an example of poly-valence
Same scaffold (F3) two different binding sites:
RGD loop
Synergy region part of β-strand
Explain functional regulation via domain rotation in FN
engineer 9F3-10F3 domain interface by a disulfide bond
Disulphide bond oxidation leads to a 30O rotation of 9F3 wrt to10F3
i.e. synergy site wrt RGD site
No disulphide bind - ability to have a range of conformation
With bond - range is smaller - stiffened
How do you get specificity into the tyrosine signalling interaction
residue sequence upstream of the tyrosine
e.g. SH2 domain of Src recognises Y*EEI
Grb2 - recognises hydrophobic residues
General features of SH2
Phosphotyrosine recognition domain
~ 100 aa
central 5 stranded β-sheet
two flanking helices
‘Socket’ with two holes to plug in peptide
Describe how the SH2 domain has two distinct sides
The spine of the domain is an anti-parallel sheet formed by strands A, B, C, D, and G –
This central sheet divides the domain into two functionally distinct sides
One side, flanked by helix A, is concerned primarily with binding phosphotyrosine
other side, flanked by helix B and the EF and BG loops, provides residues that interact with side-chains of the peptide that are C-terminal to the phosphotyrosine - a small sheet (strand D0, E, and F) closes off one part of this side
Is SH2’s recognition mode always the same?
NO, you can have variation on classic recognition things e.g. phosphotyrosine
you can have specificity by different means e.g. SLAM peptide recognises unphosphorylated SH2
Give an example of the use of protein domains in signalling
Grb2 has SH2 that recognises phosphotyrosine on membrane bound signalling receptor, and SH3 that recruits SOS for downstream signalling
Explain features of the SH3 domain
Poly proline binding
Domain recognises PxxP motif
around 60 aas
2 beta sheets fold against each other, forming a flat surface that can recognise PXXP motifs
Explain the domain arrangement of the inactive form of Src Kinase
Kinase domain - with N lobe, C lobe and ATP (analogue) wedged in-between
SH3 domain, SH2 domain wedged on back of kinase - this is inactive state - very low activity due to this
Purpose of the kinase is to phosphorylate tyrosine by using the tertiary phosphate from ATP
How is the Src kinase kept in its inactive domain form?
SH3 domain interacts with a poly proline motif pXXp on the linker between SH3, SH2 and the kinase domain
Also in the case of Src - C-terminal tail has a phosphorylated tyrosine that plugs into the SH2 domain
Explain the activation of Src Kinase
Unclamping then switching
Unclamping:
Take polypeptide e.g. from focal adhesion kinase (FAK) - also have a poly proline (pXXp) and phosphotyrosine that interact more strongly with the SH3 and SH2 domain, causing competing of the self interaction out with this external ligand (unlatching)
Unlatching is also helped by dephosphorylating the phosphotyrosine 527
Switching:
Now that SH2 and SH3 domains have unlatched and N and C lobe are free the kinase function can start operating - enhances the catalytic rate, in doing so the ATP will use a tertiary phosphate to phosphorylate the tyrosine on the activation loop (Tyr416)
Once this is phosphorylated and activation loop is fully activated - it is switched on (this is the switching process)
Explain what is meant my pleiotropic and redundant an example of a molecule that demonstrates these properties
Cytokines
pleitropic: act on multiple cells in multiple locations
Redunant: cannot work in isolation
Features of haematopoietic receptors (Class 1)
- small intracellular region that associates with Jak
- can be homodimers (ligands = GH, EPO) or hetero-oligemers (ligands = IL-6, IL11, LIF)
- all have a cytokine homology region (CHR), 2 domains, a disulphide bond + WSXWS sequence motif
- some have F3 modules, some have IG domains
Explain the structure of Haematopoietic receptor ligands
e.g.s hGH, G-CSF, IL-6, LIF
4 helix bundle cytokines
around 19KDa
170-190aas
up-up, down-down topology
Explain the structure of CHR recognition domains
wo domains
ca 100 aa each
7 β-strands
β-sheet sandwich
variant of IG domain
contain a WSXWS motif
A sheet = N terminus
G sheet = C terminus
arranged as GFCC’, ABE
Give three examples of Haematopoietic Receptors (Class I) and their topology (ratio)
Growth hormone (homo dimer 1:2)
GCSF (homo dimer 2:2)
IL6 (hetero mutlimer 2:2:2)
Explain the GH-Gh receptor homodimer
GH comes along and forms a 1:2 homodimer with the receptors (once two tails are together = on state)
How is the stoichiometry determined for homo/heterodimers e.g. GH/GH receptor
know it has a 2:1 ratio by:
-mix components at different molar ratios and
-run each mixture over a gel filtration column
-analyse the size of the traces
for GH:
GH mw~22Kd,
GHreceptor ~20Kda
therefore can look and see right amunf to make 2:1
Explain simply the binding of GH to its receptors
Gh binds in the junction between two receptors, same part of the receptor from both, interacts with a different part (epitope) of the ligand
Explain the different epitopes of the GH ligand
2 epitopes
site 1: Helices D & AB loop, large, concave
site 2: Helices AC, smaller, flat
Buried surface area:
site 1: 1230 A2
site 2: 900 A2
explain epitopes on the CHR
Epitopes are well conserved across the cytokine receptor family
- local structure similar
- primarily hydrophobic
- key residues “hot spots” W104 & W169 - important for interaction energy
Explain the local geometry of site 1 & 2 of GH and how they interact with key epitope ‘hot spots’ on the GH receptors
Receptor W104 - interacts with Helix D (Site 1) GH: K172, T175
Receptor W104 (other receptor) - interacts with helix C (site 2) GH: G120, D116
Which interaction between GH and GHR is weaker?
Site 2
Explain common features of both interaction sites (1&2) of GH
- Both domains contribute to ligand interaction
- domain orientation the same in both sides
- locally both interaction sites are structurally similar
Explain characteristics of interaction site 1 (of GH)
- central hydrophobic residues contribute most of binding energy W104, W169)
- hydrophobic patch surrounded by polar residues
- electrostatic interactions less important
Explain structure/on state of the GCSF receptor
GCSF-GCSF receptor homodimer
2:2 homodimer for on state
IG domain, CHR region, 3 ‘spacer’ F3 domains variant of IG domain
Briefly compare Ligand interactions of GCSF vs hGH
GCSF:
Site 2 retained
new site 3
hGCSF:hGCSFR complex residues site 2
Y173
hGCSF:hGCSFR complex residues site 3
I88, D90, Q91
IL-6 function
a cytokine with a wide variety of biological functions
- essential for final differentiation of B-cells into Ig-secreting cells
- induces myeloma and plasmacytoma growth
- induces nerve cells differentiation
- in hepatocytes it induces acute phase reactants.
Explain the receptor that IL-6 binds to
IL6a/gp130 homodimer (active complex is a hexamer)
2 IL-6s bind
on state: 2 of each (2IL-6, 2IL6a, 2gp130)
IL-6a vs gp130
gp130:
has extra f3 spacer domain (3)
can intercat with a variety of cytokines
Il-6a:
has one less f3 spacer domian (2)
makes it an IL-6 specific cytokine interaction
In the case of the active IL-6 complex, what is a hexamer?
a dimer of trimers
one trimer constist of : IL6-Rα : IL6 : gp130
Stoichiometry: 1:1:1
Homodimer consists of (IL6-Rα/IL6/gp130)
Stoichiometry: (1:1:1)2
Describe the binding sites for the IL-6 complex
Site 1: IL6(A,D) & IL6-Rα(D2,D3)
Site 2: (a): IL6(A,C) & gp130(D2,D3)
(b): IL6-Rα(D3) & gp130(D3)
Site 3: (a): gp130(D1) & IL6(ABloop,D )
(b): gp130(D1) & IL6-Rα
Describe the site 1 interaction for IL-6
Reminicent of the GHR site 1
interaction site between IL6 and the specific receptor IL6-Rα
IL6: interface
helix D and helix A
Helix D: K171,E175,R179
hydrogen bonds to IL6-Rα
IL6-Rα buried
surface area 1230 A2
70% D3, 30% D2
F229: hot spot
Describe the site 2a interaction of IL-6
interaction site between IL6 and the public receptor gp130 (D2,D3)
IL6: interface
helix A & C
flat surface
gp130 buried surface area 1270 A2
F169: hot-spot
Describe the site 3a interaction of IL-6
interaction site between IL6 and the public receptor gp130 (D1)
IL6 donated by trimer 2 (AB loop)
gp130 donated by trimer 1(side of D1)
AB loop and side of D1 - brings two trimers together
key Y157 residue
Describe site 2b and 3b innteractiuons
Receptor-receptor interactions
Viral IL6
Mimics IL6 interactions in a very specific way
vIL6 derives from Kaposi’s Sarcoma-associated herpesvirus
KSHV probably causes Kaposi sarcoma
role in KSHV associated disorders: e.g. AIDS
vIL6 multi functional cytokine that can induce
angiogenesis and heamatopoiesis
direct role in human disease pathogenesis
explain the viral subversion strategy of vIL6
uses two gp130 to from an active complex
Cuts out the alpha chain (IL6a specific receptor)
It has two interaction sites (1&2) that bring together 2 gp130s which is enough to bring about the biological response of formation of new blood cells
Do IL6 and vIL6 share sequence similarity
NO
they lack sequence similarity
only 22% of residues identical
Secondary structure is conserved (A-D helices)
Binding sites in viral IL6 complex
Site 1: empty, not used
Site 2: (a): IL6(A,C) & gp130(D2,D3)
Site 3: (a): gp130(D1) & IL6(ABloop,D )
vIL6 compared tp GCSF
Very simnilar in interaction sites
Viral vIL6 receptor complex:
- tetramer: 2x vIL6, 2x gp130
uses site 2 & site 3,
does not use site 1
hGCSF receptor complex:
- tetramer: 2x hGCSF, 2x hGCSFr
uses site 2 & site 3, but not site 1
Explain engineering novel signals by receptor alteration
(Findeisen 2019)
IL-6 and CNTF improve metabolic homeostasis
but have limited therapeutic use for the treatment of type 2 diabetes
Created cytokine IC7Fc with CNTF-like, but IL-6Rα-dependent, signalling by
replacing gp130-binding site 3 on IL-6 with the LIF-receptor-binding site from CNTF
IC7Fc shows high potential for treating type 2 diabetes
Explain the novel graphted cytokine IC7
(Kallen 1999)
Taken IL-6 and did sequence alignment with CNTF
Grafting LIFR binding site 3 of CNTF onto IL6:
IC7Fc: IC Fused with Fc region from IGg for better immune compatibility
multistep activation of receptors and STAT response
Site 1:
Cytokine concentration modulates EC50 of pSTAT response
Site 2:
Cytokine concentration modulates maximal strength of pSTAT response
Modulation of cytokine receptor affinity at site 1 alters the dose sensitivity (EC50) of STAT activation, whereas modulation of site 2 alters the maximal strength (Emax)
Functional pleiotropy
Single cytokine may activate multiple transcription factors
Cellular pleiotropy
Single cytokine may activate multiple cell types
Tunable parameters via cytokine engineering
3 areas that have been engineered:
Receptor affinity - RW
Receptor geometry - exploiting conserved binding sites to affect how they associate physicaly
Receptor composition - use chimera - RW
How can cellular pleiotropy be controlled
Partial agonist
IL-2 potent pro-inflammatory cytokine via stimulation of CD8+ cells
IL-2 partial agonist is immunosuppressive by expanding preferentially CD4+ & Treg over CD8+ cells
Done via engineering affinity to site 2.
Example of IL2 affinity modulation/engineering
Super IL-2 engineered to increase IL-2Rβ affinity
H9T: reduced affinity for γc CD8+ to TCF+ stem-like state
IL-2 REH further reduced affinity for γc CD4+ FoxP3+ Treg cell
Example receptor geometry modulation
E.g. EPO - homodimer in 2:1 geometry
Make a Diabody - 2 molecules that are placed at a distance - instead of getting full agonist get partial
can also change the angles/orientation of two receptors, depending on angle you can get a higher or lower pSTAT output
Receptor composition modulation
Making an IL-2/IL-4 hybrid
- taking IL-4Ra chain and IL-2Rb chain
- instead of having Il-2 profile (JAK-1, JAK3 common together) STAT 5 STAT6 activation profile, non-natural signals created
How can we engineer pharmacological responses of cytokines
Affinity engineering
Geometry engineering
Composition engineering
- Could end up with a set pf pharmacological agents that are extremely potent
Examples of implications of domain structure for..
binding site presentation
assembly
regulation
Future in directing and controlling biological processes
Building new materials with precisely tuneable properties
Synthetic biology and material sciences
