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 proliferationCell 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

Protein phosphorylation modification is stable in solutionPhospho-moiety is hydrolyzed in the cell by protein phosphatases

Ser Thr Tyr

HO HO HO

half life = weeksIn aqueous solution

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 -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 527

Y 416

The eukaryotic protein kinase domain

Structural elements of the kinase domain N-lobe – sheet + one helix C C-lobe –mainly -helical Cleft region between lobes

-nucleotide binding-location of phospho acceptor site-catalytic residues-activation segment (blue)

C-lobe = phospho-acceptor binding site

12 highly conservedmotifs

Sub-domain function1 glycine rich loop flexible flap that tethers sugar and

non- hydrolyzable phosphate groups

2 invariant Lys glu-lys salt bridge orients 3 invariant Glu phosphate

5 hinge region coordinates adenine ring of ATP

6 invariant Asp catalytic base in catalytic loop (HRDxxxxN) removes proton from target

hydroxyl site (Asn orients Asp)

7 invariant Asp binds mg2+ that in turn binds and (DFG)orients phosphates of ATP

7.5 Activation regions spanning DFG and APE segmentmotif usually contains site of phophoregulation

8 Helix EF Determines Ser/Thr vs Tyrosine

(APE) specificity and flanking sequences

(P+1 loop)

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

N-lobe

C-lobe

D5

EF

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 / flexiblebilobal nature – connection by flexible hingeN-terminal sheet – easily deformedC helix –position easily modulatedg-loop – gly is inherently flexibleactivation 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?

Ser/Thr versus Tyrosine kinases (easy to predict)

Tyrosine kinases

Tyr

Serine/Threonine kinases

cyclin dependentProtein kinases

MAP kinases

PROLINE (S/T-P) DIRECTED protein kinases (easy to predict)

Unique highly constrainedback bone conformationnecessitates specialized infrastructure

Phospho-priming dependent protein kinasesvariable mechanisms make prediction difficult

GSK3

SRPK

Secondary Peptide Docking Sites

Highly specific to subfamilies

Very versatile and pervasive but hard to predict substrate-kinase relationship due to variation in placement and degenerate nature of the motifsSubstrates may not look anything like each other except for presence of two short motifs Each kinase can / has evolved tens to hundreds of substrates

Acceptor site motifDocking site motif

hypothetical substrate

N C

Higher order Substrate Targeting Mechanisms (augmenting specificity of the active site)

OH

MAP kinases

Cyclin dependentprotein kinases

Mechanisms are conserved across closely related subfamilies

Substrate targeting based on Domain - Domain recognition -Highly specialized

-Relatively monogamous

eIF2

Best characterized example = PKR (an eIF2 kinase)

Higher order Substrate Targeting Mechanisms – cont.

Substrate recognition by the eIF2 protein Kinases

Cellular Stress

heme deficiency

viralinvasion

aminoacid starvation

misfolded proteins

Inhibition ofProtein Synthesis

HRI

PKR

GCN2

PERK

distinct regulatorydomains

similar catalyticdomains

Common TargetDiverse Sensing

P

eIF2

Ser51

eIF2

PKR Specificity arise from non-cononical G helix – very diagnostic

Molecular basis for specificity revealed by x-ray crystallography

- TGFB family kinases phoshorylate Smad family of proteins and almost nothing else

- Presence of MH2 domain in Smad proteinsvery diagnostic

- Recognition is phosphorylation dependent

Second example of domain based substrate targeting

Structure of the complex has not yet been determined

Globular MH2 domain

Acceptor siteC-terminal to MH2 domain

Type I TGF- receptor kinases (7 members) / SMAD substrates (5 members)

Domain-Domain dependent substrate recognition

eIF2a kinase familyPKRGCN2PERKHRI

TGF- receptor family

Substrate targeting mechanisms

Y 527

Y 416 Use of interaction domainsSplicing modules together is easier than evolving entirely new interaction surfacesVery versatileSame issues as kinases using peptide docking sites

SH3

SH2

pxxp xYxxx

Acceptor site motifDocking motif 1

N CpYEEI

Docking motif 2

Hypothetical substrate