Transcript Substrate targeting mechanisms

Protein Kinase Structure and Function
Introduction
 500 protein kinases encoded in the human genome
 1.5 % of our genetic diversity
Protein kinases regulate at some level almost all cellular processes.
Cellular proliferation
Cell cycle
Replication
Translation …..
The kinase domain is a catalytic entity with one basic function
- transfers the gamma phosphate moiety from ATP to a hydroxyl moiety of a
serine/threonine or tyrosine residues
Ser
HO
ThrTyr
HO
HO
half life = weeks
In aqueous solution
Protein phosphorylation modification is stable in solution
Phospho-moiety is hydrolyzed in the cell by protein phosphatases
Role of protein phosphorylation: Phosphorylation induces a conformational
change or generates a protein interaction ligand.
i. Conformational change: Phosphorylation of a residue causes a gross conformational change in
target protein that affects function.
-e.g. protein kinases themselves (very tight ON/OFF switch)
Insulin receptor kinase
ii. Promote complex formation by the generation of protein interaction ligands:
-phosphorylation generates a product that is recognized (or no longer recognized) by a protein
interaction module. This can serve to define networks of interacting proteins.
-SH2 domain (src homology 2) Src SH2 binds pYEEI ligands
-PTB domain (phosphotyrosine binding domain) Shc and IRS-1 NPXpY ligands
-14,3,3 domain (phosphoserine binding domains) XXXpSerXXX ligands
-FHA domain (phosphothreonine binding domains) XXXpThrXXX ligands
-WD40 repeat domain, cdc4 b-trcp – diphophopeptide motif pThrPXXpThr
SH2 Domain: The prototypical phospho recognition module
The prototype ‘interaction module’ discovered and characterized by Dr. Tony Pawson
-100 amino acid module consists of a central b-sheet of (4 to 6 b-strands) and two a-helices
-binding site lies across the sheet structure flanked by the two helices
-recognizes phospho-tyrosine containing motifs (reads out sequence Yxxx after phospho-tyrosine)
- SH2 domains bind the phosphotyrosine using a conserved mechanism
- Single Arg mutation disables all phospho-peptide binding –surgical for dissecting biological function
The phosphotyrosine binding site is most highly conserved part of SH2 domains
Invariant arginine and deep pocket are most critical binding features
Y 416
Y 527
The eukaryotic protein kinase domain
12 highly conserved
motifs
Structural elements of the kinase domain
N-lobe –b sheet + one helix aC
C-lobe –mainly a-helical
Cleft region between lobes
-nucleotide binding
-location of phospho acceptor site
-catalytic residues
-activation segment (blue)
C-lobe = phospho-acceptor binding site
N-lobe
35kDa 300a.a. core
-12 highly conserved motifs termed sub domains
-12 near invariant residues
PKA = prototype structure (Susan Taylor Lab)
Hundreds of structures now available in PDB
Sub-domain
1 glycine rich loop
function
flexible flap that tethers sugar and
non- hydrolyzable phosphate
groups
2 invariant Lys
3 invariant Glu
glu-lys salt bridge orients a & b
phosphate
5 hinge region
coordinates adenine ring of ATP
6 invariant Asp
(HRDxxxxN)
catalytic base in catalytic loop
removes proton from target
hydroxyl site (Asn orients Asp)
7 invariant Asp
(DFG)
binds mg2+ that in turn binds and
orients b & g phosphates of ATP
7.5 Activation
segment
regions spanning DFG and APE
motif usually contains site of
phophoregulation
8 Helix aEF
(APE)
Determines Ser/Thr vs Tyrosine
specificity and flanking sequences
(P+1 loop)
D5
C-lobe
Catalytic mechanism is well understood
Catalytic mechanism is conserved for all kinases
All eukaryotic protein kinases transfer phosphate the same way and
look very much alike in their active states
How do the 500 kinases differ to allow each to regulate specific
aspects of biology?
Origin of diversity lies with:
1) Catalytic switching mechanisms
-the ability to switch on and off in response to specific upstream
signals
2) Substrate targeting mechanisms
- The ability to select a restricted set of substrates in the cell any
one time
-15,000+ proteins in the genome
- each with many phosphorylatable sites (ser/thr, tyr)
Features of the kinase that facilitate diversification of function
i. the protein kinase catalytic domain is structurally pliant / flexible
bilobal nature – connection by flexible hinge
N-terminal sheet – easily deformed
aC helix –position easily modulated
g-loop – gly is inherently flexible
activation segment
ii. strict conformational requirements for catalysis – perturb structure of any
conserved element and kinase is inactivated
iii. extensive surface provides opportunities for the evolution of secondary peptide
binding sites to complement the peptide binding site at the catalytic site
-although core catalytic structure is conserved exposed surfaces are highly
variable
iv. modular – kinase domain is commonly linked to non-catalytic interaction
domains (eg. SH2 PTB, …)
These factors provide many opportunities for diversification of substrate
recognition and regulation and indeed diverse mechanisms of regulation
have been uncovered and many more remain to be solved.
We will survey paradigms uncovered by x-ray crystallographic methods.
Substrate targeting mechanisms
Active site directed specificity
i. Ser/Thr versus Tyrosine
ii. Proline directed
iii. Phospho-priming
P+1 loop of activation segment defines Ser/Thr versus Tyr preference
- serves as a platform for main chain of substrate
Ser
Thr
Tyr
Held close
Held far
What about dual specificity kinases like PKR?
Tyrosine kinases
Tyr
Serine/Threonine kinases
Ser/Thr versus Tyrosine kinases
(easy to predict)
Unique highly constrained
back bone conformation
necessitates specialized
infrastructure
MAP kinases
cyclin dependent
Protein kinases
PROLINE (S/T-P) DIRECTED
protein kinases
(easy to predict)
SRPK
GSK3a,b
Phospho-priming dependent
protein kinases
variable mechanisms make prediction difficult
Higher order Substrate Targeting Mechanisms
(augmenting specificity of the active site)
Secondary Peptide
Docking Sites
Highly specific
to subfamilies
Docking site motif
Acceptor site motif
N
hypothetical
substrate
C
OH
Very versatile and pervasive but hard to predict substrate-kinase relationship
due to variation in placement and degenerate nature of the motifs
Substrates may not look anything like each other except for presence of two short motifs
Each kinase can / has evolved tens to hundreds of substrates
MAP kinases
Cyclin dependent
protein kinases
Mechanisms are conserved across closely
related subfamilies
Higher order Substrate Targeting Mechanisms – cont.
Best characterized example = PKR
(an eIF2a kinase)
eIF2a
Substrate targeting based on Domain - Domain recognition
-Highly specialized
-Relatively monogamous
Substrate recognition by the eIF2a protein Kinases
heme
deficiency
viral
invasion
Cellular
Stress
amino
acid
starvation
misfolded
proteins
distinct
regulatory
domains
Diverse Sensing
HRI
P Ser51
PKR
eIF2a
GCN2
Inhibition of
Protein
Synthesis
PERK
similar
catalytic
domains
Common Target
Molecular basis for specificity revealed by x-ray crystallography
eIF2a
PKR
Specificity arise from non-cononical
aG helix – very diagnostic
Second example of domain based substrate targeting
Type I TGF-b receptor kinases (7 members)
/ SMAD substrates (5 members)
Globular MH2 domain
- TGFB family kinases phoshorylate
Smad family of proteins and almost
nothing else
Acceptor site
C-terminal to
MH2 domain
- Presence of MH2 domain in Smad proteins
very diagnostic
- Recognition is phosphorylation dependent
Structure of the complex has not yet been determined
TGF-b receptor family
eIF2a kinase family
PKR
GCN2
PERK
HRI
Domain-Domain dependent substrate recognition
Substrate targeting mechanisms
Use of interaction domains
Y 416
Y 527
Splicing modules together is easier than evolving
entirely new interaction surfaces
Very versatile
Same issues as kinases using peptide docking sites
SH3
Docking motif 1
N
pxxp
Acceptor site motif
pYEEI
Docking motif 2
xYxxx
Hypothetical
substrate
C