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BIOL3013 - Molecular recognition > Modular proteins > Flashcards

Modular proteins Flashcards

1
Q

define domain

A

a folded structural unit; the sequence need not be contiguous e.g. MHC II

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2
Q

Define a module

A

A domain with a contiguous sequence, repeatedly used in diverse proteins e.g. F3

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3
Q

Define a Repeat

A

A unit that does not fold in isolation; several copies are needed e.g. leu-rich repeat

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4
Q

True or false, a module is a specific type of domain

A

TRUE

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5
Q

Are domain combinations random?

A

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)

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6
Q

Examples of popular domains

A

IG, F3, F1, EGF, SH2, SH3, Kinase, MHC II

F3 is a module and a domain (because its contiguous)

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7
Q

What are the biological implications of protein domains?

A

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

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8
Q

Variable connections

A

position of the domain influences its function

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9
Q

Regulation via domain rearrangement

A

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

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10
Q

Binding constant of bivalent interactions is…

A

The product of the individual binding constants

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11
Q

Which binding is tighter (mono- vs bi-valent)

A

Bivalent

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12
Q

Give an example of a protein that has polyvalent interactions

A

IgA (teravalent)
IgM (decavalent)

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13
Q

What are the two forms of fibronectin (FN)?

A

Circulating blood (soluble form)
In matrix (activated) (ECM form)

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14
Q

Features of FN

A

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

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15
Q

Explain the different things that bind to FN and some of their functions

A

Fibrin - blood clotting
Heparin - ECM component
S. aureus - Virus
Collagen - ECM component
Gelatin
C1q (complement)
Cell membranes

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16
Q

Bacterial FN binding proteins

A

Pathogenic bacteria e.g. Streptococci use FN in the ECM of the host to adhere and gain access to host cells

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17
Q

Fibronectin-binding ashesins

A

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

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18
Q

What did sequence alignment allow when it came to FN

A

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)

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19
Q

Explain the model for FN mediated bacterial uptake

A

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

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20
Q

why is clustering of integrins necessary for FN mediated bacterial uptake

A

clustering integrins of the host – by doing this they can engulf the bacteria

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21
Q

Explain why bacteria want to be engulfed by host cells

A

Persistent infection occurred unless the bacteria had somewhere to hide, this is why it hijacks the migration capability of the cell to be engulfed

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22
Q

What is cell attachment to the matrix mediated by

A

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

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23
Q

Explain how the two different binding sites on the F3 scaffolds are an example of poly-valence

A

Same scaffold (F3) two different binding sites:

RGD loop

Synergy region part of β-strand

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24
Q

Explain functional regulation via domain rotation in FN

A

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

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25
Q

How do you get specificity into the tyrosine signalling interaction

A

residue sequence upstream of the tyrosine

e.g. SH2 domain of Src recognises Y*EEI

     Grb2 - recognises hydrophobic residues
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26
Q

General features of SH2

A

Phosphotyrosine recognition domain

~ 100 aa
central 5 stranded β-sheet
two flanking helices

‘Socket’ with two holes to plug in peptide

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27
Q

Describe how the SH2 domain has two distinct sides

A

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

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28
Q

Is SH2’s recognition mode always the same?

A

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

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29
Q

Give an example of the use of protein domains in signalling

A

Grb2 has SH2 that recognises phosphotyrosine on membrane bound signalling receptor, and SH3 that recruits SOS for downstream signalling

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30
Q

Explain features of the SH3 domain

A

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

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31
Q

Explain the domain arrangement of the inactive form of Src Kinase

A

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

32
Q

How is the Src kinase kept in its inactive domain form?

A

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

33
Q

Explain the activation of Src Kinase

A

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)

34
Q

Explain what is meant my pleiotropic and redundant an example of a molecule that demonstrates these properties

A

Cytokines
pleitropic: act on multiple cells in multiple locations
Redunant: cannot work in isolation

35
Q

Features of haematopoietic receptors (Class 1)

A
  • 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
36
Q

Explain the structure of Haematopoietic receptor ligands

A

e.g.s hGH, G-CSF, IL-6, LIF
4 helix bundle cytokines
around 19KDa
170-190aas
up-up, down-down topology

37
Q

Explain the structure of CHR recognition domains

A

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

38
Q

Give three examples of Haematopoietic Receptors (Class I) and their topology (ratio)

A

Growth hormone (homo dimer 1:2)
GCSF (homo dimer 2:2)
IL6 (hetero mutlimer 2:2:2)

39
Q

Explain the GH-Gh receptor homodimer

A

GH comes along and forms a 1:2 homodimer with the receptors (once two tails are together = on state)

40
Q

How is the stoichiometry determined for homo/heterodimers e.g. GH/GH receptor

A

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

41
Q

Explain simply the binding of GH to its receptors

A

Gh binds in the junction between two receptors, same part of the receptor from both, interacts with a different part (epitope) of the ligand

42
Q

Explain the different epitopes of the GH ligand

A

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

43
Q

explain epitopes on the CHR

A

Epitopes are well conserved across the cytokine receptor family
- local structure similar
- primarily hydrophobic
- key residues “hot spots” W104 & W169 - important for interaction energy

44
Q

Explain the local geometry of site 1 & 2 of GH and how they interact with key epitope ‘hot spots’ on the GH receptors

A

Receptor W104 - interacts with Helix D (Site 1) GH: K172, T175

Receptor W104 (other receptor) - interacts with helix C (site 2) GH: G120, D116

45
Q

Which interaction between GH and GHR is weaker?

A

Site 2

46
Q

Explain common features of both interaction sites (1&2) of GH

A
  • Both domains contribute to ligand interaction
  • domain orientation the same in both sides
  • locally both interaction sites are structurally similar
47
Q

Explain characteristics of interaction site 1 (of GH)

A
  • central hydrophobic residues contribute most of binding energy W104, W169)
  • hydrophobic patch surrounded by polar residues
  • electrostatic interactions less important
48
Q

Explain structure/on state of the GCSF receptor

A

GCSF-GCSF receptor homodimer
2:2 homodimer for on state
IG domain, CHR region, 3 ‘spacer’ F3 domains variant of IG domain

49
Q

Briefly compare Ligand interactions of GCSF vs hGH

A

GCSF:
Site 2 retained
new site 3

50
Q

hGCSF:hGCSFR complex residues site 2

A

Y173

51
Q

hGCSF:hGCSFR complex residues site 3

A

I88, D90, Q91

52
Q

IL-6 function

A

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.
53
Q

Explain the receptor that IL-6 binds to

A

IL6a/gp130 homodimer (active complex is a hexamer)
2 IL-6s bind
on state: 2 of each (2IL-6, 2IL6a, 2gp130)

54
Q

IL-6a vs gp130

A

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

55
Q

In the case of the active IL-6 complex, what is a hexamer?

A

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

56
Q

Describe the binding sites for the IL-6 complex

A

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

57
Q

Describe the site 1 interaction for IL-6

A

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

58
Q

Describe the site 2a interaction of IL-6

A

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

59
Q

Describe the site 3a interaction of IL-6

A

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

60
Q

Describe site 2b and 3b innteractiuons

A

Receptor-receptor interactions

61
Q

Viral IL6

A

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

62
Q

explain the viral subversion strategy of vIL6

A

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

63
Q

Do IL6 and vIL6 share sequence similarity

A

NO
they lack sequence similarity
only 22% of residues identical
Secondary structure is conserved (A-D helices)

64
Q

Binding sites in viral IL6 complex

A

Site 1: empty, not used
Site 2: (a): IL6(A,C) & gp130(D2,D3)
Site 3: (a): gp130(D1) & IL6(ABloop,D )

65
Q

vIL6 compared tp GCSF

A

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

66
Q

Explain engineering novel signals by receptor alteration

A

(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

67
Q

Explain the novel graphted cytokine IC7

A

(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

68
Q

multistep activation of receptors and STAT response

A

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)

69
Q

Functional pleiotropy

A

Single cytokine may activate multiple transcription factors

70
Q

Cellular pleiotropy

A

Single cytokine may activate multiple cell types

71
Q

Tunable parameters via cytokine engineering

A

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

72
Q

How can cellular pleiotropy be controlled

A

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.

73
Q

Example of IL2 affinity modulation/engineering

A

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

74
Q

Example receptor geometry modulation

A

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

75
Q

Receptor composition modulation

A

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

76
Q

How can we engineer pharmacological responses of cytokines

A

Affinity engineering
Geometry engineering
Composition engineering
- Could end up with a set pf pharmacological agents that are extremely potent

77
Q

Examples of implications of domain structure for..

A

binding site presentation
assembly
regulation
Future in directing and controlling biological processes
Building new materials with precisely tuneable properties
Synthetic biology and material sciences

BIOL3013 - Molecular recognition (6 decks)

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