Domains Signaling

7/31/2019 Domains Signaling

1/58

1

Signaling Pathways

and

Interaction Domains

in

Cell Biology

A supplement to Path 230, Molecular Biology of the Cell

A general survival supplement


2/58

2

Introduction 3Receptors coupled to various signaling system 5Jak/STAT combinations used by various cytokines 6Pathways 7

Receptor tyrosine kinase family 7Platelet-derived growth factor (PDGF) receptor signaling 8Ras-Raf signaling 9

Mitogen-activated protein kinases (MAP kinases) signaling 10AKT-PI3-kinase signaling 11Jak/STAT5 signalingErythropoietin receptor 12Jak/STAT1/2 signalingInterferon alpha receptor 13Wnt signaling 14NF-kB signaling 15Hedgehog-Gli signaling 16SMAD signaling: TGFbeta, Activin, Bone morphogenetic proteins 17Notch signaling 18G1S checkpoint--cell cycle regulation 19G2M damage checkpoint--cell cycle regulation 20Apoptosis inhibition 21Apoptosis activation: Death receptor signaling 22Apoptosis control by mitochondria 23

Toll Receptor signaling 24G protein coupled signaling 25Fc receptor signaling 26T cell antigen receptor signaling 27B cell antigen receptor signaling 28

Domains14-3-3 Domain 29 DH Domain 38 PTB Domain 46ADF Domain 29 EF-hand Domain 38 PX Domain 46ANK Domain 30 EH Domain 39 RGS Domain 47ARM Domain 30 ENTH Domain 39 RING Domain 47BH1-4 Domain 31 EVH1 Domain 39 SAM Domain 48BIR Domain 31 F-box Domain 40 SH2 Domain 48BRCT Domain 32 FERM Domain 40 SH3 Domain 49Bromo Domain 32 FHA Domain 41 SNARE Domain 50BTB/POZ Domain 33 FYVE Domain 41 SOCS Domain 50C1 Domain 34 GEL Domain 41 START Domain 51C2 Domain 34 GYF Domain 42 TIR Domain 51CARD Domain 35 HECT Domain 42 TPR Domain 52CC Domain 35 LIM Domain 42 TRAF Domain 53Chromo Domain 36 LRR Domain 43 TUBBY Domain 53CH Domain 36 MH-2 Domain 44 UBA Domain 54CSD Domain 37 PB1 Domain 44 VHS Domain 54Death Domain 37 PDZ Domain 45 WD40 Domain 54DED Domain 38 PH Domain 45 WW Domain 55

MotifsITAM and ITIM 56

Transcription Factor DomainsBasic-Helix-loop-Helix Domain (bHLH proteins) 58Leucine Zipper motif 59


3/58

3

Introduction

Cellular behavior is controlled in a dynamic fashion by receptors for external and intrinsic signals, which activate intracellularsignaling pathways that regulate virtually every aspect of cellular function. Signaling events of this sort are critical fordevelopmental processes during embryogenesis, and also for responses of cells in the adult organism to changes in theirenvironment. For example, the signaling molecules that are important in axon guidance, and formation of the mammalianbrain, also play an important role in synaptic functions associated with post-natal learning and memory. Investigating howthese signaling pathways are assembled is relevant not only for understanding how normal cells work, but also forappreciating the molecular basis for disease, since many human disorders result from breakdowns in signal transduction.

Two common mechanisms through which signaling systems are regulated involve the phosphorylation of proteins by kinases,on the one hand, and the ability of proteins to associate with one another, and with other macromolecules, on the other. Thesedistinct regulatory devices are in fact inseparably linked, since the principal means by which protein phosphorylation exerts aneffect on cellular behavior is by creating binding sites for a set of protein interaction domains whose ability to bind their targetsis phosphorylation-dependent.

Protein-protein interactions recruit cytoplasmic polypeptides to activated receptors, direct their assembly into largercomplexes, target them to defined subcellular locations, and determine the specificity with which enzymes interact with theirsubstrates. Typically, protein interaction domains are independently folding modules of ~35-150 amino acids, that can beexpressed in isolation from their host proteins while retaining their intrinsic ability to bind their physiological partners. Their N-and C-termini are often closely juxtaposed in space, while their binding surface lies on the opposite face of the domain. Thisarrangement allows the domain to be inserted into a host polypeptide, while projecting its ligand-binding site to engageanother protein. Phospho-dependent protein interaction domains typically recognize specific peptide motifs on their bindingpartners, in a fashion that depends on the phosphorylation of a tyrosine or serine/threonine residue in the recognition

sequence. The mechanisms by which protein kinases and phospho-dependent interaction domains work hand-in-glove toactivate biochemical pathways is illustrated by receptor tyrosine kinases (RTK) signaling.

RTKs are often activated by growth factor-mediated dimerization (or by oncogenic mutations which induce constitutiveoligomerization), which result in the cross-phosphorylation of one receptor chain by its neighbor. This autophosphorylation hastwo consequences. Phosphorylation within the activation segment of the kinase domain results in a conformational changethat stimulates catalytic activity, while phosphorylation of tyrosine residues within the juxtamembrane region, C-terminal tail orkinase insert created binding sites for the SH2 domains or PTB domains of cytoplasmic targets. The human genome iscurrently estimated to encode 114 SH2 domains, which are found in 104 distinct proteins. SH2 domains bind their ligands asan extended strand; they all recognize phosphotyrosine, through a conserved, basic binding pocket, and also bind at leastthree residues immediately C-terminal to the phosphotyrosine, in a fashion that differs from one SH2 domain to another andprovides an element of specificity in signal transduction. Thus the sequence contexts of a receptors autophosphorylation sitesdetermine the identities of the SH2-containing proteins that bind the activated RTK, and the spectrum of signaling pathwaysactivated in the cell. Consistent with the view that the SH2 domain serves as a portable module to couple phosphotyrosinesignals to intracellular targets, SH2-containing proteins can have a wide range of biological functions, including the regulationof Ras-like GTPases, phospholipid metabolism, gene expression, cytoskeletal organization, and protein phosphorylation. Insome cases, SH2 are not covalently linked to catalytic domains, but rather are found in adaptor proteins, composedexclusively of interaction domains, such as SH2 and SH3 domains. Such adaptors can nucleate the formation of multi-proteinsignaling complexes that regulate particular aspects of cellular function.

Phosphotyrosine-containing motifs are also recognized by PTB domains, found in docking proteins such as IRS-1, a principalsubstrate of the insulin-receptor. PTB domains typically bind NPXY sequences, which form b-turns; PTB domains of proteinsinvolved in tyrosine kinase signaling (i.e. IRS, Shc, FRS2 and Dok family members) require phosphorylation of the NPXYtyrosine for stable binding. Such docking proteins themselves possess multiple tyrosine phosphorylation sites, which engageSH2-containing proteins, exemplifying how a succession of phospho-dependent protein-protein interactions can be used toconstruct signaling pathways and networks. Although SH2 and PTB domains both recognize phosphotyrosinecontainingsequences, they are structurally unrelated, and engage their phospho-peptide ligands in different ways (albeit that basicarginine and lysine residues are a common feature of their phosphotyrosine-binding pockets). Similarly, there is a growingnumber of quite different interaction domains that share an ability to selectively bind phosphoserine/threonine-containing

motifs.

The majority of protein kinases in eukaryotes phosphorylate serine/threonine residues, and regulate facets of cellular functionranging from the cell cycle to gene expression and metabolism. 14- 3-3 proteins, FHA domains, MH2 domains, WD40 repeatdomains and WW domains all have the ability to bind specific phosphoserine/ threonine-containing peptide motifs. FHAdomains, for example, are found in protein kinases involved in DNA damage repair, and recognize phosphothreonine, withselectivity being provided by recognition of the +3 residue. The interactions mediated by the FHA domains of the Rad53protein kinase in yeast are required for the cellular response to DNA damage. MH2 domains are structurally similar to FHAdomains, and are found in SMAD proteins, that serve as targets for activated TGF] receptor serine/threonine kinases (RSK).Autophosphorylation of the serine-rich juxtamembrane region of the type I TGF] receptor apparently creates a binding site forthe SMAD MH2 domain, which recognizes pSer-X-pSer motifs; the receptor-bound SMAD is subsequently phosphorylatedwithin a C-terminal motif, leading to the formation of a phospho-dependent SMAD complex that leaves the receptor and moves


4/58

4

to the nucleus to regulate gene expression. Although the details are quite different, there are significant parallels in therecognition of specific phosphorylated motifs of activated RTKs and RSKs by interaction domains on their targets.

Protein ubiquitylation, and resulting proteolysis or receptor internalization, is frequently asociated with phosphorylation of thetarget protein. Tyrosine phosphorylated receptors are recognized by the variant SH2 domain of the E3 protein ubiquitin ligasec-Cbl, which recruits an E2 ubiquitin ligase and induces receptor ubiquitylation. Serine/threonine phosphorylation of proteinsdestined for degradation can created binding sites for the WD40 repeat or leucine rich repeat domains of F-box proteins, thetargeting subunits of SCF E3 ubiquitin ligase complexes. In these cases, phosphodependent protein-protein interactions serveto induce another modification, ubiquitylation, which itself can create binding sites for ubiqutin interaction motifs (UIM). Taken

together, these observations highlight the importance of phosphorylation as a device to control the assembly of proteincomplexes, and thereby to regulate the dynamic behavior of the cell.

Tony Pawson


5/58

5

Receptors coupled to various signaling systems

Signaling system Cytokine/Receptor

JAK/STAT IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15(see supplement Interferon alpha, Interferon beta, Interferon gamma

sheet for specific OSM, CNTF, LIF, CT-1, Leptincategories, associated Growth Hormone, Erythropoietin, TPO, PROJak kinases, and Platelet-derived growth factor (PDGF)target STAT proteins) Epidermal growth factor (EGF)

Macrophage colony stimulating factor (M-CSF, aka CSF-1)Granulocyte-Macrophage colony stimulating factors (GM-CSF)Granulocyte colony stimulating factor (G-CSF)Insulin, Basic FGF, Hepatocyte growth factor

Smads TGF beta, ActivinBone morphogenetic proteins

IRAK/TRAF6/NFkB IL-1Toll-like receptor (TLR) ligands (bacterial products)

Ras/Raf Tyrosine protein kinasesGrowth factorsCytokinesCXC chemokines

MAP kinases Tyrosine protein kinasesGrowth factorsTrophic FactorsOsmotic ShockGamma radiationFAS ligandInflammatory cytokines

UV radiation

PI3-Kinase/AKT Tyrosine protein kinasesGrowth factorsCytokinesCXC chemokines (e.g. CXCR4)

Apoptosis/Caspase TNF alpha, FAS, Trail (Activate)Growth Factors/Cytokines (Inhibit)

Frizzeled/B-catenin Wnt proteins

NF-kB UV, TLRs (e.g. LPS Receptor, TLR4)

TNF receptor, IL1CXC chemokines

Smoothened/Patched/Gli Shh, Hh


6/58

6

Jak/STAT signal trasduction

Receptor Family Receptor Jak kinase STATgp130 IL-6, IL-11, OSM, CNTF, G-CSF, LIF, CT-1 Jak1, Jak2, Tyk2 STAT1, STAT3, STAT5

IL-12 Jak2, Tyk2 STAT4Leptin Jak2 STAT3, STAT5

gp140 IL-3, GM-CSF, IL-5 Jak2 STAT5

Receptor tyrosine kinases EGF, TGFalpha, PDGF, CSF-1 STAT1, STAT3, STAT5Insulin STAT3, STAT5BFGF STAT1, STAT3HGF STAT3

IL-2 IL2, IL7, IL9, IL15 Jak1, Jak3 STAT1, STAT3, STAT5IL4 Jak1, Jak3 STAT6IL13 Jak1, Jak2, Tyk2 STAT6

Growth Hormone GH Jak2 STAT1, STAT3, STAT5TPO Jak2 STAT3, STAT5PRO, EPO Jak2 STAT5

Interferon IFN alpha, IFN beta Jak1, Tyk2 STAT1, STAT2, STAT3,STAT5

IFN gamma Jak1, Jak2 STAT1IL-10 Jak1, Tyk2 STAT1, STAT3

STAT1 homodimer binds GAS elementSTAT1:STAT2 heterodimer binds ISRE elementSTAT5 homodimer binds PIE element


7/58

7

Receptor tyrosine kinase Family


8/58

8

Platelet-derived growth factor (PDGF) receptor signaling


9/58

Ras-Raf signaling


10/58

10

Mitogen-activated protein kinases (MAP kinases) signaltransduction


11/58

11

AKT-PI3 kinase signaling


12/58

12

Jak/STAT5 Signaling Erythropoietin Receptor


13/58

13

Jak/STAT 1-2 signaling Interferon alpha receptor


14/58

14

Wnt signaling


15/58

15

NF-kB signaling


16/58

16

Hedgehog-Gli signaling


17/58

17

SMAD signaling: TGF-beta, activin, bone morphogenetic proteins


18/58

18

Notch signaling


19/58

1

G1S CheckpointCell cycle regulation


20/58

20

G2M damage checkpointCell cycle regulation


21/58

21

Apoptosis Inhibition


22/58

22

Apoptosis activation: Death Receptor Signaling


23/58

23

Apoptosis Control by Mitochondria


24/58

24

Toll Receptor Signaling


25/58

25

G Protein Coupled Signaling


26/58

26

Fc Receptor Signaling


27/58

27

T cell antigen receptor signaling


28/58

28

B cell antigen receptor signaling


29/58

2

14-3-3 DomainDomain binding and function: 14-3-3 proteins are 30 kDa polypeptides with nine closelyrelated members in mammals. They are also found in plants and fungi. They are involved inregulating various pathways including signaling apoptosis and passage through the cell cycle.14-3-3 proteins form homoand heterodimeric cup-like structures that bind to discretephosphoserine-containing motifs. In some instances, 14-3-3 proteins appear to export theirbinding partners from the nucleus to the cytoplasm in a phosphorylationand Crm1-dependentmanner.

Structure Reference: Rittinger, K. et al. (1999) Mol. Cell4, 153.

14-3-3 dimer bound to TrkA phospho-peptide

14-3-3 binds cdc25 tyrosine phosphatase, c-Raf Ser/Thr Kinase, PKC Ser/Thr Kinase,MEKK1,2,3 Ser/Thr Kinase

ADF DomainDomain binding and function: The Actin-Depolymerizing Factor (ADF) homology domain or

ADF domain is a 130170 amino acid domain, first identified in the ADF family of proteins, that

is associated with proteins involved in F-actin severing. The domain functions as an actin-binding module present in an extensive family of proteins, including ADF/Cofilin, the Twinfilinsand Drebrin/Adp1. This evolutionarily primitive domain pre-dates the divergence of fungi andanimals and is found in all eukaryotic organisms. The ADF-containing proteins ADF, Cofilin,Depactin and Actophorin bind to monomeric and filamentous actin and that act to sever Actinfilaments. This creates more plus (barbed) and minus (pointed) ends allowing faster Actinturnover and results in the observation that these proteins both rapidly depolymerizefilamentous Actin in vitro, as well as increase the rate of F-actin polymerization. Certain ADF-


30/58

30

containing proteins appear to have developed more specialized functions as Drebrin/Adp1class proteins bind only filamentous actin, while Twinfilins bind only monomeric Actin.

Structure Reference: Fedorov, A.A. et al. (1997) Nat. Struct. Biol. 4(5), 366369.

ANK DomainDomain binding and function: Breedan and Nasmyth first reported a 33 amino acid repeatcommon among a small number of proteins. Subsequently, a cytoskeletal protein named

Ankyrin was identified that was composed almost entirely of these short repeats. To date, ANKrepeats have been identified in over 1700 different proteins from viruses, prokaryotes andeukaryotes. ANK repeats have been implicated in mediating proteinprotein interactionsalthough no common theme among the known ANK domain protein targets has beenidentified.

Structure Reference: Foord, R. et al. (1999) Nat. Struct. Biol. 6(2), 157165.

ANK repeat domain proteins Ank repeats in Swi6

ARM DomainDomain binding and function: The approximately 40 amino acid Armadillo (ARM) repeat wasfirst identified in the Drosophila segment polarity gene product Armadillo (the homologue ofmammalian beta-catenin). It has since been identified in over 240 different proteins of diversecellular function from yeast to man. The ARM domain is implicated in mediating protein-proteininteractions, but no common features among the target proteins recognized by the ARMrepeats have been identified. The ARM repeat has a common phylogenetic origin with theHEAT repeat. Both ARM and HEAT repeats contain a set of seven highly conservedhydrophobic residues and both mediate protein-protein interactions. Although structurallysimilar, the ARM repeat consists of three helices (H1, H2, and H3) whereas HEAT repeatsconsist of two helices (A and B). However, the strongly bent helix A of HEAT repeats

corresponds to helices 1 and 2 of ARM repeats.

Structure Reference: Conti, E. et al. (1998) Cell94(2), 193204.


31/58

31

BH1-4 DomainDomain binding and function: Bcl-2 Homology (BH14) domains are found in proteins thatinhibit apoptosis including Bcl-2, Bcl-xL and Bcl-xW. Bcl-2 family members form homodimersand heterodimers between pro- and antiapoptotic family members. Homodimerization of Bcl-2involves a head-totail interaction. The N-terminal region, where the BH4 domain resides,interacts with the more distal region of Bcl-2 where BH1, BH2 and BH3 are located. The BH3domain is required for dimerization and apoptosis induction. Conversely, Bcl-2/Baxheterodimerization involves a tail-to-tail interaction that requires the BH1, BH2 and BH3 regionof Bcl-2 and a central region in Bax where the BH3 domain is located.

Structure Reference: Sattler, M. et al. (1997) Science 275(5302), 983986.

BIR DomainDomain binding and function: The Baculovirus IAP Repeat (BIR) domain is anapproximately 70 amino acid zinc-binding domain, first identified by sequence homologyamong proteins belonging to the Inhibitors of Apoptosis (IAP) family. Present in one to three

tandem copies per protein, the BIR domain has been identified in over 80 different proteins ineukaryotic organisms. Most of what is known about BIR domains come from their role in IAPproteins. IAPs bind to and inhibit caspases, a class of cysteine proteases involved inpropagating apoptotic signals within the cell. The BIR domain has been shown to be necessaryfor the interaction of IAP proteins with diverse proapoptotic factors, including invertebratedeath inducers such as Reaper, Grim, HID and Doom from Drosophila and vertebrate andinvertebrate members of the caspase family of proteases. BIR domains appear to have twopotential modes of action. In the case of the inhibition of caspase-9 by XIAP, the third BIRdomain of XIAP acts as a peptide binding motif that interacts with an ATPF/AVPY motif at theN-terminus of the linker peptide on the p12 small subunit of caspase-9, which becomesexposed after proteolytic activation of procaspase-9. This interaction is in turn regulated by the

Smac/Diablo protein which competes for XIAP BIR3 binding by presenting a high affinity BIR3interacting peptide, and thereby sequestering XIAP away from caspase-9. In contrast, thesecond BIR domain of XIAP appears to exert its anti-apoptotic effect simply by acting as aregulatory element for caspase binding while the N-terminal linker interacts with, and blocks,the substrate groove of caspase-3 and -7. This peptide lies across the capase active site in anorientation reverse to that of substrate binding. In this context, deletion of the BIR domainabrogates antiapoptotic function, possibly because in the absence of the BIR domain theadjacent peptide fails to adopt a caspase inhibitory conformation. Finally, the BIR domainappears to be capable of mediating homophilic interactions.

Structure Reference: Wu, G. et al. (2000) Nature 408, (6815), 10081012.


32/58

32

Bir domain proteins 3rd BIR domain of XIAP, showing zinc atomand binding of Smac N-terminal residues (red).

BRCT DomainDomain binding and function: The BRCT domain (BRCA1 C-terminus) is a conservedproteinprotein interaction region of approximately 95 amino acids found predominantly inproteins involved in cell cycle checkpoint functions responsive to DNA damage. It was firstidentified in the breast cancer suppressor protein BRCA1 but is also found in DNA repairproteins such as DNA ligase III and XRCC1, which form strong heterodimers through theirBRCT domains. The C-terminal BRCT domain of BRCA1 has been reported to bind to thecentral domain of p53, allowing BRCA1 to act as a coactivator of p53.

Structure Reference: Zhang, X. et al. (1998) EMBO J. 17(21), 64046411.

BROMO DomainDomain binding and function: Approximately 110 amino acids in length, the Bromo domainis found in many chromatin-associated proteins such as histone acetylases and the ATPasecomponent of certain nucleosome-remodeling complexes. Bromo domains have beenidentified in over 100 proteins from yeast to man. The Bromo domains of PCAF and Gcn5phave been shown to interact specifically with peptides containing acetylated lysine residues.Recognition of acetyl-lysine is similar to that of acetyl-CoA by histone acetyltransferases,though the bromodomain is the only domain known to interact with acetylated lysinecontaining peptides.Structure Reference: Owen, D.J. et al. (2000) EMBO J. 19(22), 61416149.

Bromo domain proteins Bromodomain of Gcn5pbinding acetylatede lysine


33/58

33

BTB/POZ DomainDomain binding and function: Domain binding and function: The BTB domain is a protein-protein interaction module consisting of approximately 120 amino acids that is found in over600 different proteins in organisms ranging from yeast to humans. The domain was firstidentified as a conserved sequence element in the developmentally regulated Drosophilaproteins Broad-complex, Tramtrack and Bric-abrac. The BTB domain, also known as the POZ(poxvirus and zinc finger) domain, is often found at the N-termini of several zinc fingertranscription factors as well as Shaw-type potassium channels. Experimental studies havestrongly implicated the BTB domain in the regulation of gene expression through the localcontrol of chromatin conformation. In several cases, the BTB domain has been shown tomediate protein oligomerization which subsequently prevents high affinity DNA binding. Bothhomotypic and heterotypic protein-protein interactions have been observed because the BTBdomain can form dimers as well as mediating interactions with non-BTB domaincontainingproteins.

Structure Reference: Ahmad, K.F. et al. (1998) Proc. Natl. Acad. Sci. USA 95(21),1212312128.

Poz domain proteins Poz domain from human PLZF


34/58

34

C1 DomainDomain binding and function: C1 domains are approximately 50 aminoacids long, enrichedin cysteines, and are involved in the recruitment of proteins to the membrane. Typically, C1domains bind phorbol esters or diacylglycerol, which are necessary for membrane localization.With phorbol ester bound, the upper surface of the C1 domain forms a contiguous hydrophobicsurface in the domain. This enables the region to be buried into the lipid bilayer stabilizingmembrane insertion. The middle portion of the domain contains a number of basic residuesthat can interact with lipid headgroups in the membrane, while the lower half of the C1 domaincontains two zinc-binding sites that are important to maintain the fold of the domain.

Structure Reference: Zhang, G. et al. (1995) Cell81(6), 917924.

C1 domain proteins C1 domain of PKCdelta bound to phorbol ester

C2 DomainDomain binding and function: The C2 domain, a region containing approximately 130residues, is involved in binding phospholipids in a calcium-dependent manner or calcium-

independent manner. C2 domains are found in over 100 different proteins with functionsranging from signal transduction to vesicular trafficking. Calcium binding to the C2 domain ofsynaptotagmin induces little conformational change in the C2 domain but rather induces achange in electrostatic potential, thereby enhancing phospholipid binding. This suggests thatthe C2 domain functions as an electrostatic switch. In addition to electrostatic interactions, sidechains in the calcium binding loops influence the binding of different C2 domains to eitherneutral or negatively charged phospholipids.

Structure Reference: Sutton, R.B. et al. (1995) Cell 80(6), 929938.

C2 domain proteins C2 domain


35/58

35

CARD DomainDomain binding and function: Caspase Recruitment Domains (CARDs) are modules of90100 amino acids involved in apoptosis signaling pathways. CARDs mediate the associationof adaptor proteins and procaspases through heterodimerization of their respective CARDs,recruiting procaspases to upstream signaling complexes and allowing autoactivation.Dimerization of CARDs is believed to be mediated primarily by electrostatic interactionsbetween complementary charged surfaces with a binding specificity achieved by particularcharge patterns between CARD binding partners.

Structure Reference:Vaughn, D.E. et al. (1999) J. Mol. Biol. 293(3), 439447.

CARD domain proteins CARD domain of Apaf-1

CC DomainDomain binding and function: Coiled-coils (CC) function as oligomerization domains for awide variety of proteins including structural proteins, motor proteins and transcription factors.The coiled-coil structure is conserved from viruses to plants and mammals and it has beenpredicted that approximately 5% of proteins encoded in sequenced genomes contain coiled-

coils. Coiled-coils typically consists of two or more alpha-helices that wrap around each otherwith a superhelical twist. Sequences with a propensity to assume coiled-coil structures arecharacterized by the heptad repeat pattern (abcdefg)n, where a and d are hydrophobic, and eand g are charged or polar. Coiled-coils may interact with each other to form homotypicoligomers, or with other coiled-coil domains to form heterotypic oligomers.

Structure Reference: Nooren, I.M. et al. (1999) Nat. Struct. Biol. 6(8), 755759.

CC domain proteins Coiled coils in the tetramericMnt repressor protein


36/58

36

Chromo DomainDomain binding and function: The Chromatin Organization Modifier (Chromo) domain isdefined as a 3070 amino acid residue protein module found in a number of proteins involvedin the assembly of protein complexes on chromatin. This domain was first described inDrosophila modifiers of variegation, which are proteins that modify the structure of chromatin tothe condensed morphology of heterochromatin, a cytologically visible condition where geneexpression is repressed. Examples of chromo-domain-containing proteins include the HP1molecule involved in repression of gene expression in heterochromatin, the Polycomb (Pc)transcriptional repressors of homeotic genes in which the chromodomain is essential forchromatin targeting, and human retinoblastoma binding protein (RBP-1). Some chromodomain (CD) proteins contain an N-terminal chromo domain and a C-terminal Shadow ChromoDomain (CSD).

Structure Reference: Ball, L.J. et al. (1997) EMBO J. 16(9), 24732481.

Chromo domain proteins Chromo domain from mouse modifier protein 1

CH DomainDomain binding and function: The calponin homology (CH) domain is a protein module ofapproximately 110 amino acids present in cytoskeletal and signal transduction proteins. TwoCH domains in tandem form an F-actin binding region at the N-termini of spectrin-like proteinssuch as dystrophin and alpha-actinin. Such tandem CH domains bind F-actin with 5 - 50 Maffinity and cross-link actin filaments into bundles and networks. CH domains can besubdivided into at least three types. Type 1 and 2 are found together in tandem in cytoskeletalproteins such as dystrophin, spectrin and filamin. Type 3 CH domains are found in proteinsthat regulate muscle contraction, such as calponin, as well as in signaling proteins such asVav, ARHGEF6 and IQGAP. Type 3 CH domains may not interact directly with actin, but rather

act as regulatory domains or protein-protein interaction scaffolds to modulate the activity ofproteins in which they are present.

Structure Reference: Banuelos, S. et al. (1998) Structure 6(11), 1419-1431.


37/58

37

CSD DomainDomain binding and function: The Shadow Chromodomain (CSD) is a 4070 amino aciddomain that occurs only in the context of proteins containing a chromodomain (CD). While theShadow chromodomain resembles the chromodomain structurally, it appears to function in adistinct manner with respect to proteinprotein interactions. CSD lacks the hydrophobic sashbelieved to mediate CD interactions. Shadow chromodomains form stable dimers, anddimerization generates an interaction pit that may allow docking with partner proteinscontaining an extended hydrophobic pentapeptide motif.

Structure Reference: Cowieson, N.P. et al. (2000) Curr. Biol. 10(9), 517525.

CSD domain proteins CSD domain from Swi6

Death DomainDomain binding and function: Death domains (DD) are 80100 residues long motifsinvolved in apoptotic signal transduction. They are found both in cytoplasmic proteins and intransmembrane proteins including members of the tumor necrosis factor receptor superfamily.

Death domains serve as recruiting modules through their ability to heterodimerize with thedeath domains of distinct proteins, including adaptor proteins such as FADD. Due to thesignificant polarization of charged residues on the surface of the death domain, dimerization isbelieved to arise primarily through electrostatic interactions. Binding has been shown to bespecific and is thought to arise through complementary charge patterns on dimerizationpartners.

Structure Reference: Jeong, E.-J. et al. (1999) J. Biol. Chem. 274(23), 16337-16342.

Death domain proteins Death domain from FADD


38/58

38

DED DomainDomain binding and function: The Death Effector Domain (DED) is a protein interactiondomain found in inactive procaspases (cysteine proteases) and proteins that regulate caspaseactivation in the apoptosis cascade. Similar to CARDs, DEDs recruit procaspases intocomplexes with members of the TNF-receptor superfamily. This recruitment is mediated by ahomotypic interaction between the procaspase DED and a second DED in an adaptormolecule that is directly associated with activated TNF receptors. Complex formation allowstransprocessing of procaspase to the active form. This in turn activates downstream caspasesand initiates apoptosis.

Structure Reference: Eberstadt, M. et al. (1998) Nature (6679)392, 941945.

DH DomainDomain binding and function: The Dbl homology (DH) or RhoGEF domain consists of an ~150 amino acid region that induces Rho family GTPases to displace GDP. This effectivelyactivates the Rho GTPase by allowing binding to GTP, which is in excess over GDP in the cell.

The DH domain is invariably proceeded by a pleckstrin homology (PH) domain. While notabsolutely required for catalysis of nucleotide exchange, the PH domain appears to greatlyincrease catalytic efficiency in many cases. Rho proteins control actin dynamics, geneexpression, membrane trafficking, growth factor signaling, and cellular transformation. Proteinsencoding DH domains (RhoGEFs) also play a role in these events as they function as theprimary activators of Rho GTPases. In fact, many RhoGEFs were identified based on theirtransforming activity, which was abrogated upon disruption of their DH domain.

Structure Reference: Worthylake, T. et al. (2000) Nature 408, (6813), 682-688.

DH domain proteins DH domain of mouse TIAM-1

EF-hand DomainDomain binding and function: The EF-hand motif contains approximately 40 residues and isinvolved in binding intracellular calcium. EF-hand domains are often found in single or multiplepairs, giving rise to various structural/functional variations in proteins containing EF-handmotifs. Proteins containing EF-hands can be grouped into two functional categoriesregulatoryor structural. Binding of calcium to regulatory EF-hand domaincontaining proteins induces aconformational change, which is transmitted to their target proteins, often catalyzing enzymaticreactions. In contrast, binding of calcium to structural EF-hand domaincontaining proteins does


39/58

3

not induce a significant conformational change. Structural EF-hand domains seem to play arole in buffering intracellular calcium levels.

Structure Reference: Taylor, D.A. et al. (1991) J. Biol. Chem. 266(32), 2137580.

EH DomainDomain binding and function: The EH domain is a module of ~100 amino acids originallyidentified in the tyrosine kinase substrate Eps15, and thus termed the Eps15-Homology (EH)domain. There is considerable evidence to suggest that EH domain proteins are primarilyinvolved in regulating endocytosis and vesicle transport. Typically, EH domains recognizepeptides with core NPF motifs. Most EH proteins have multiple copies of the EH domain, andmay bind cooperatively to proteins with several NPF motifs. EH domain proteins frequentlyhave other repeated motifs (i.e., DPF, PXXP, coiled-coil) and modules (i.e., SH3 domains),suggesting that they may serve a scaffolding function in endocytosis. Indeed, the DPF motifsof Eps15 interact with the N-terminal appendage region of the clathrin adaptor AP-2component, alpha-Adaptin. Eps15, and other EH domain proteins such as Intersectin, can bindproteins implicated in endocytosis, such as the GTPase Dynamin and the lipid phosphataseSynaptojanin. Genetic data in yeast have directly demonstrated the importance of an EHdomain protein, Pan1, in endocytosis.

Structure Reference: De Beer, T. et al. (1998) Science 281(5381), 13571360.

ENTH DomainDomain binding and function: First identified in the endocytotic protein epsin 1, the epsinNH2-terminal homology (ENTH) domain is a membrane binding motif of approximately 150amino acids. Proteins containing this domain have been shown to bind to phospholipids

including PtdIns(4,5)P2 and PtdIns(1,4,5)P3. Consistent with these findings, the primaryfunction suggested for the ENTH domain containing proteins is to act as clathrin adaptors inendocytosis, with binding of the ENTH domain to the phospholipid bilayer allowing recruitmentof clathrin components and clathrin accessory factors to the cell membrane. In addition, twoENTH containing proteins (HIP1, HIP1R), shown to localize to clathrin coated pits, also containa putative actin binding motif (ILWEQ) providing evidence for the elusive link between the actincytoskeleton and endocytosis.

Structure Reference: Hyman, J. et.al. (2000) J. Cell Biol. 149(3), 537-46.

EVH1 DomainDomain binding and function: Domain binding and function: The EVH1 domain is a proteinmodule of ~110 amino acids found in a number of scaffolding proteins that mediate theassembly of multiprotein complexes involved in control of the actin cytoskeleton. This domainwas originally identified at the N-terminus of the Drosophila protein Enabled (Ena), itsmammalian counterpart (Mena) and the closely related protein Vasp (hence the termEna/Vasp Homology domain 1). EVH1 domains are also found in an additional member of theMena family, Evl, in the WASP docking protein that is affected by mutations that cause theimmunodeficiency Wiskott-Aldrich syndrome, and in the Homer family of synaptic proteins thatinteract with group 1 metabotropic glutamate receptors. EVH1 domains recognize related


40/58

40

proline- rich motifs, such as E/DFPPPPXD/E in the case of Mena. These motifs are commonlyfound in components of the cytoskeleton, such as Vinculin and Zyxin, as well as in the ActAprotein of the pathogenic bacterium Listeria monocytogenes, which regulates bacterial motilityby controling actin polymerization in the infected cell.

Structure Reference: Federov, A.A. et al. (1999) Nat. Struct. Biol. 6(7), 661665.

F-box DomainDomain binding and function: The F-box domain is a 4248 amino acid conserved domainfound at the N-terminus of F-box proteins. F-box proteins act as adaptor components of themodular E3 ubiquitin ligase SCF complex that functions in phosphorylation-mediatedubiquitination. The F-box domain mediates interaction with SKP1, which links F-box proteins toa core ubiquitin-ligase complex composed of Rbx1, cdc53/Cul1 and the E2 conjugatingenzyme cdc34. The C-terminal region of F-box proteins are also composed of various modulardomains that interact with target substrates, often in a phosphorylation-dependent manner.

Structure Reference: Schulman, B.A. et al. (2000) Nature 408(6810), 381386.

FERM DomainDomain binding and function: Previously known as the B4.1 (band 4.1) homology and ERMdomain, the FERM domain is named for the four proteins in which this domain was originallydescribed: F for Band 4.1, E for Ezrin, R for Radixin, M for Moesin. The FERM domain isapproximately 150 amino acids in length and is found in a number of cytoskeletal-associatedproteins that are found at the interface between the plasma membrane and the cytoskeleton.The FERM domain is responsible for PIP2 regulated membrane binding of ERM(Ezrin/Radixin/Moesin) proteins that play a role in formation of membraneassociated

cytoskeleton by linking actin filaments to adhesion proteins. The structure of the Radixin FERMdomain bound to IP3 has been solved, and surprisingly, phosphoinositide binding is notmediated by the PH-fold subdomain of FERM, but occurs at a cleft between two subdomainson a relatively flat face of the module. The FERM domain is also postulated to bind to adhesionproteins, in a PIP2 -regulated fashion, providing a link between cyctoskeletal signals andmembrane dynamics.

Structure Reference: Hamada, K. et al. (2000) EMBO J. 19(17), 44494462.

FERM domain proteins FERM domain with PIP2 bound


41/58

41

FHA DomainDomain binding and function: The FHA domain, or Forkhead-Associated domain, wasoriginally identified as a conserved region of forkhead transcription factors. It is 65100 aminoacids long, contains several highly conserved key residues, and is found primarily in eukaryoticnuclear proteins. FHA domaincontaining proteins are also found in certain prokaryotes, suchas mycoplasma bacteria. The FHA domain mediates phosphopeptide interactions with proteinsphosphorylated by serine/threonine kinases. The first FHA domain of Rad53 binds to a pTXXDmotif with a Kd = 1.6 M, while other FHA domains also bind to pTXXX peptides.

Structure Reference: Durocher, D. et al. (2000) Mol. Cell6 (5), 11691182.

FHA domain proteins FHA domain bound to P-Thr peptide

FYVE DomainDomain binding and function: The FYVE (Fab-1, YOTB, Vac1 and EEA1) domain is a small,cysteine-rich Zn2+ binding domain of approximately 60 amino acids. To date, FYVE domains

have been identified in over 200 different proteins from yeast to man. The FYVE domain hasbeen shown to specifically bind PI(3)P. This observation has implicated FYVEdomaincontaining proteins in a signaling role downstream of PI3 kinase. Furthermore, FYVE-containing proteins have been implicated in the regulation of the vacuolar/lysosomalmembrane trafficking pathway and in regulation of signaling by TGFbeta-receptors.

Structure Reference: Misra, S. and Hurley, J.H. (1999) Cell97(5), 657666.

GEL DomainDomain binding and function: Also know as the gelsolin/severin/villin homology domain, thegelsolin homology domain (GEL) is a 120 - 150 amino acid domain found in a variety ofproteins involved in cytoskeletal regulation, particularly in proteins that function in actinsevering. The GEL domain has both calcium binding and actin binding activity, such that actinbinding is calcium regulated. The gelsolin protein, composed of six GEL domains, binds to thebarbed ends of actin filaments preventing monomer exchange and acting as an end-blockingor capping protein for the actin filament. In addition, gelsolin can promote actin nucleation tocreate new filaments and sever existing filaments.

Structure Reference: Robinson, R.C. et al. (1999) Science 286(5446), 1939-1942.


42/58

42

GYF DomainDomain binding and function: The glycine-tyrosine-phenylalanine, or GYF domain was firstreported in the CD2 binding protein CDBP2 as a domain capable of binding to a proline-richpeptide sequence in the CD2 tail region. Despite functioning as a proline-rich peptide bindingdomain, the GYF fold is structurally unrelated to the SH3 or WW domains. The GYF domain ofCDBP2 binds to a PPPPGHR repeat in the CD2 tail via a relatively smooth, concave surfacethat forms a continuous hydrophobic patch containing many of the GYF domainconservedresidues.

Structure Reference: Freund, C. et al. (1999) Nat. Struct. Biol. 6, 656660.

HECT DomainDomain binding and function: The HECT domain, short for Homologous to the E6-APCarboxyl Terminus, is an approximately 40 kDa (350 amino acid) catalytic domain found at thecarboxy-terminus of Hect-class E3 ubiquitin protein ligases. This domain functions to bindspecific E2s, accepts ubiquitin from the E2 to form a ubiquitin-thioester intermediate with the

HECT active cysteine, and then transfers ubiquitin to either the epsilon-amino groups of lysineside chains of the substrate or to the growing end of multiubiquitin chains. The formation of athioester intermediate with Ub is unique to Hect E3s and has not been observed with otherclasses of E3s.

Structure Reference: Structure reference: Huang, L. et al. (1999) Science 286 (5443),13211326.

Hect domain proteins Hect domain of E6AP ubiquitin-protein ligase

LIM DomainDomain binding and function: The LIM domain was first identified in three developmentallyregulated transcription factors Lin-1, Isl-1 and Mec-3. It consists of approximately 60 aminoacids and has been identified in over 300 proteins from organisms ranging from yeast tohumans. The LIM domain is a zincbinding, cysteine-rich motif consisting of two tandemlyrepeated zinc fingers. Unlike GATA-type zinc fingers, LIM domains do not seem to bind DNAbut instead appear to mediate proteinprotein interactions. Functionally, LIMdomaincontaining proteins have been implicated in a variety of biological processes includingcell lineage specification, cytoskeletal organization and organ development. Some LIMdomaincontaining proteins appear to function solely as adapters to bring together other


43/58

43

components into a complex (i.e., LMO and CRP) while other LIM domain containing proteinsclearly have other functions conferred by additional functional domains such as the DNAbinding homeodomain or a catalytic kinase domain. In addition, certain LIM domains havebeen observed to form dimers with other LIM domains. An overall consensus LIM domainbinding site has not been defined. Factors that confer the specificity of LIM domain interactionsremain to be determined.

Structure Reference: Konrat, R. et al. (1997) J. Biol. Chem. 272(18), 1200112007.

LIM domain proteins LIM domain from CRP2

LRR DomainDomain binding and function: Domain binding and function: Leucine-Rich Repeats (LRR)are 2228 amino acid motifs that are found in a number of proteins with diverse functions andcellular locations. These repeats are usually involved in proteinprotein interactions, and inseries they form nonglobular, crescent-shaped structures. The crescent shape adopted byseries of leucine-rich repeats creates a solventexposed elongated concave surface of parallel

beta-strands that acts as a scaffold for proteinprotein interactions. The particular function ofeach LRR crescent is specified by different residues arranged in an appropriate orientation onthe surface of the structurally conserved three-dimensional fold. For example, RNAse inhibitorand U2A' LRR scaffolds appear to interact with their targets via the concave inner surface ofthe crescent, while in the case ofS. pombe Rna1p, the Ran binding site appears to be locatedon the side face of the crescent within the loop regions connecting the beta-strands and alpha-helices.Structure Reference: Hillig, R.C. et al. (1999) Mol. Cell3(6), 781791.

LRR-containing proteins Leucine rich repeats in Rna1p


44/58

44

MH2 DomainDomain binding and function: The MH2 domain of R-Smads allows the interaction with theSmad binding domain (SBD) of SARA. SARA recruits R-Smads to the type I TGFbetareceptor, and this is believed to be stabilized by a charge-mediated interaction between theMH2 domain and the cytoplasmic domain of the type I TGFbeta-R. The MH2 of the co-Smad,Smad4, appears to be responsible for homooligomerization of Smad4 trimers into disk-likestructures and heterooligomerization between Smad4 trimers and Smad2 trimer disks.Mutations in the Smad4 trimer interface disrupt homo-oligomerization and result in inactivationof Smad4 tumor suppression function observed in pancreatic carcinomas and other cancers.

Structure Reference: Wu, G. et al. (2000) Science 287(5450), 9297.

MH2 domain proteins SMAD2 MH2 domain

PB1 DomainDomain binding and function: Phox and Bem1 (PB1) domains contain approximately 80

amino acids and are found in a number of cytoplasmic signaling proteins. The PB1 domain isinvolved in the heterodimerization with a paired PB1 domain, although not all PB1 domains willassociate with one another. A highly conserved internal sequence known as OPR, PC or AIDmotifs is necessary for PB1 domain function. Regions outside the OPR, PC and AID helpconfer specificity for binding.

Structure Reference: Terasawa, H. et al., (2002) EMBO 20(15), 3947-3956.

PB1 proteins PB1 domain from yeast Bem1


45/58


46/58

46

PTB DomainDomain binding and function: Phosphotyrosine binding (PTB) domains are 100150 residuemodules that commonly bind Asn-Pro-X-Tyr motifs. The PTB domains of the docking proteinsShc and IRS-1 require ligand phosphorylation on the tyrosine residue (NPXpY) for binding.More N-terminal sequences are also required for high affinity binding and conferring specificity.The peptide binds as a beta-strand to an anti-parallel beta-sheet, while the NPXpY motifmakes a turn, positioning the pY for recognition by basic residues. The PTB domains ofproteins such as X11, Dab, Fe65 and Numb apparently recognize NPXY or related peptidemotifs, but are not dependent on ligand phosphorylation. In addition, the Numb PTB domaincan bind an unrelated peptide that forms a helical turn.

Structure Reference: Zhou, M.M. et al. (1995) Nature 378(6557), 584592.

PTB domain proteins Shc PTB domain bound to TrkA phosphopeptide

PX DomainDomain binding and function:The Phox homology (PX) domain is the most recentlyidentified member of the family of phospholipid-binding domains. Consisting of ~120 amino

acids, the PX domain is found in more than 100 proteins, including the p40phox and p47phoxcomponents of the NADPH oxidase complex, sorting nexins, phospholipases D1 and 2 and thekinases PI3K and CISK. Biochemical and cell biology studies have established that PXdomains function predominantly as D3-phosphorylated phosphoinositide [PI(3)P] bindingmodules, targeting the PX domain-containing proteins to the membranes.

Structure Reference: Bravo, J. et al. (2001), Mol. Cell8 (4), 829-839.

PX domain proteins PX domain of p40phox bound to PI(3)P


47/58

47

RGS DomainDomain binding and function: The RGS (Regulator of G protein Signaling) domain has beenfound in over 20 proteins in humans and is typically about 120 amino acids in length. RGSdomains act allosterically by stabilizing the transition intermediate of the GTP binding pocket ofthe alpha subunit of heterotrimeric G proteins. This results in the acceleration of the intrinsicGTPase activity of that alpha subunit. The discovery of the RGS domain therefore answeredthe longstanding question of why the intrinsic rate of hydrolysis of many heterotrimeric Gproteins was often slower than the apparent cycling time for a signaling process requiring thatG protein. Heterotrimeric G proteins transmit signaling from seven transmembrane receptors,which, in turn, are activated by many important agonists such as hormones, neurotransmitters,light and odorants. Proteins that encode RGS domains also modulate such signaling events asthey control the time of transmission of each of these agonists.

Structure Reference: Tesmer, J.J. et al. (1997) Cell89(2), 251-261.

RGS domain proteins RGS domain of RGS-4

RING DomainDomain binding and function: Domain binding and function: The RING finger is aspecialized type of Znfinger consisting of 4060 residues that binds two atoms of zinc, and isinvolved in mediating proteinprotein interactions. The presence of a RING finger domain is acharacteristic of RING-class E3 ubiquitin protein ligases capable of transfering ubiquitin froman E2 enzyme to a substrate protein. The RING domain mediates the interaction with theappropriate E2 enzyme. Unlike HECT E3s that form a thioester with ubiquitin, RING fingerslikely mediate ubiquitination by facilitating the direct transfer of ubiquitin from E2s to lysineresidues on the target substrate. RING finger proteins include the Hrt1/Roc1/Rbx1 proteinsfound in both the SCF and VCB-like E3 complexes, the APC1 component of the AnaphasePromoting Complex, Cbl family proteins, MDM2 and many other proteins with demonstratedE3 activity, E2 binding or involvement in ubiquitination. In addition to the involvement of RINGfinger domains in ubiquitin transfer, this domain has also been associated with certaintranscription factors such as TIF1beta, the PML-family, NFX1 and XPRF.

Structure Reference: Structure reference: Zheng, N. et al. (2000) Cell102(4), 533539.


48/58

48

Ring domain proteins Ring domain in c-Cbl

SAM DomainDomain binding and function: The approximately 70 amino acid SAM (Sterile Alpha Motif)domain has been identified in over 400 different proteins with diverse cellular function, fromyeast to man. SAM domains have been implicated in mediating proteinprotein interaction viathe formation of homo- and heterotypic oligomers. The residues at the interface of the EphA4and EphB2 SAM domain homodimers have been mapped, but the factors that determinespecificity remain to be determined.

Structure Reference: Stapleton, D. et al. (1999) Nat. Struct. Biol. 6(1), 4449.

SAM domain proteins SAM domain of the EphA4 receptor

SH2 DomainDomain binding and function: Src-homology 2 (SH2) domains are modules of ~100 aminoacids that bind to specific phospho (pY)-containing peptide motifs. Conventional SH2 domainshave a conserved pocket that recognizes pY, and a more variable pocket that binds 3-6residues C-terminal to the pY and confers specificity. The SAP SH2 domain recognizes Y aswell as pY in the context of residues N and C terminal, suggesting an alternate 3-prongedmodel may apply in some cases. Phosphopeptides of optimal sequence bind to SH2 domainswith dissociation constants of ~50-500 nM.


49/58

4

Structure Reference: Waksman, G. et al. (1993) Cell72(5): 779-90.

SH2 domain proteins Src SH2 domain bound toPhosphotyrosine peptide

SH3 DomainDomain binding and function:Src-homology 3 (SH3) domains bind to Pro-rich peptides thatform a left-handed poly-Pro type II helix, with the minimal consensus Pro-X-X-Pro. Each Pro isusually preceded by an aliphatic residue. Each in the aliphatic-Pro pair binds to a hydrophobicpocket on the SH3 domain. The ligand can, in principle, bind in either orientation. An additionalnon-Pro residue, frequently Arg, can form part of the binding core and contacts the SH3domain. Such peptides usually bind to the SH3 domain with a Kd in the M range. The bindingaffinity and specificity can be markedly increased by tertiary interactions involving loops on theSH3 domain.

Structure Reference: Lim, W.A. et al. (1994) Nature 372(6504), 375379.

SH3 domain proteins SEM5 SH3 domain bound to proline peptide


50/58

50

SNARE DomainDomain binding and function: While the mechanism by which a vesicle fuses with its propermembrane target is poorly understood, it appears to involve a highly conserved set of proteinscalled SNAREs (Soluble NSF Attachment protein [SNAP] Receptors). SNARE proteins arebelieved to mediate most, if not all, cellular membrane fusion events. Most SNAREs are C-terminally anchored integral membrane proteins capable of entering into a coiled-coilinteraction with other SNARE proteins. All SNARE proteins share a homologous domain ofapproximately 60 amino acids referred to as the SNARE domain. The SNARE domain acts asa proteinprotein interaction module in the assembly of a SNARE protein complex. Whilemonomeric SNARE motifs are largely unstructured, they assemble into a protease resistantcore complex. Interestingly, different SNARE family members are distributed on distinctmembranes throughout the cell, suggesting they may play a role in targeting during vesiculartransport. However, the formation of SNARE core complexes appears to be ratherpromiscuous with little specificity.

Structure Reference: Sutton, R.B. et al. (1998) Nature 395(6700), 347353.

Tsnare domain proteins Syntaxin-1A, Synaptobrevin-II, SNAP 25B

SOCS DomainDomain binding and function: The SOCS box is an approximately 40 amino acid region ofhomology that is invariably located at the C-terminus of the proteins in which it is present.Initially identified in the SOCS, or supressors of cytokine signaling family of proteins, the SOCSbox appears to be involved in targeting proteins for ubiquitination. The SOCS box contains asub-domain known as the BC-box that is also found in the VHLa domain. The BC box in bothSOCS box and VHLa domain facilitates binding to the Elongin BC complex. Elongin BC, in

turn, interacts with the von-Hippel Lindau (VHL) tumor suppressor protein to form the core ofthe larger VCB E3 ubiquitin protein ligase complex. Thus, the SOCS box/VHLa domain mayserve a function analogous to the structurally related F-box protein linking substrates to E3complexes to allow ubiquitination. Five classes of N-terminal regions are found associated withthe C-terminal SOCS box. These include SH2 domains (SOCS proteins), WD40 repeats (WSBproteins), a SPRY domain (SSB proteins), Ankyrin repeats (ASB proteins), and a GTPasedomain (RAR family).

Structure Reference: Stebbins, C.E. et al. (1999) Science 284(5413), 455-461.


51/58

51

SOCS domain proteins VHLalpha domain

START DomainDomain binding and function: Both the CSD and BA START domain families share acommon topology consisting of a seven stranded core beta sheet centered between a pair ofalpha helices at the N-terminus and an alpha helix at the C-terminus. The C-terminal alphahelix is packed tightly against the sheet, consistent with a Helix-Grip type fold. A strikingfeature of the START domains is the long hydrophobic tunnel expanding throughout the lengthof the domain. By adopting a structure similar to an incomplete beta barrel, the interior face ofthe beta sheet forms the bottom and sides of this tunnel. The tunnel is in turn, roofed by the C-terminal helix.

Structure Reference: Tsujishita, Y. and Hurley, J.H. (2000) Nat. Struct. Bio 7(5), 408-444.

START domain proteins START domain of MLN64

TIR DomainDomain binding and function: The Toll/Il-1 Receptor (TIR) domain was first characterizeddue to homology between the intracellular regions of the mammalian IL-1 receptor (IL-1R) andthe Drosophila protein Toll. Subsequently, six Toll-like receptors (TLRs) have been identified in


52/58

52

Drosophila and more than twenty TLRs and IL-1Rs have been recognized in humans. Severaladaptor proteins containing TIR domains have also been described. The domain consists ofthree 'boxes' of conserved residues set in a core sequence ranging from 135 to 160 aminoacids. Intervening residues may vary, as sequence conservation between domains is only 20-30%. Two interfaces are responsible for mediating TIR domain interactions, which includereceptor/adaptor oligomerization and association between receptors and adaptors. TLR andIL-1R signaling pathways are key mediators of the innate immune response to bacteria andfungi in both Drosophila and mammals. TIR domain interactions between receptors andadaptors play a key role in activating conserved cellular signal transduction pathways inresponse to bacterial LPS, microbial and viral pathogens, cytokines and growth factors.Homotypic and heterotypic interactions are thought to mediate receptor signaling. Activationinvolves liberation of NF-kappaB resulting in lymphocyte activation, immunoglobulin isotypeswitching and expression of cytokines and their receptors.

Structure Reference: Xu, Y. et al. (2000) Nature 408(6808), 111-115.

TIR domain proteins TIR domain of TLR-2

TPR DomainDomain binding and function: The tetratricopeptide repeat (TPR) motif was originallyidentified in yeast as a protein-protein interaction module in cell cycle proteins. It has sincebeen found in organisms ranging from bacteria to humans. The TPR motif is a degeneratesequence of ~34 amino acids loosely based around the consensus residues -W-LG-Y-A-F-A-P-. The sequence occurs in tandem arrays and is present in over 800 different proteins. TPRmotif-containing proteins act as scaffolds for the assembly of different multiprotein complexesincluding the anaphase promoting, the peroxisomal import receptor and the NADPH oxidasecomplexes.

Structure Reference: Scheufler, C. et al. (2000) Cell101(2), 199-210.

TPR domain proteins TPR domain bound to Hsp70 C-terminal peptide


53/58

53

TRAF DomainDomain binding and function:The approximately 150 amino acid TRAF domain is found inTumor Necrosis Factor (TNF) receptor-associated factors. TRAF proteins appear to be arelatively recent evolutionary development as there is just one C. elegans TRAF protein andonly two Drosophila, and six mammalian TRAF proteins. All mammalian TRAFs localize to thecytoplasm except TRAF4 which is found in the nucleus. TRAF proteins are recruited to themembrane through interactions of their TRAF domains with activated TNF receptors, IL-1/Tollreceptors or through intermediate proteins such as the TRADDs. TRAFs primarily act in cellsurvival upon interacting with TNF receptors by activating the NFkB and AP-1 transcriptionfactors. The six mammalian TRAF proteins have distinct functions. For example, TRAF3regulates T-cell dependent antigen responses, TRAF4 is required for formation of the tracheaand TRAF6 modulates IL-1, CD40, and LPS signaling. TRAFs are also important in Epstein-Barr Virus replication by binding to LMP1 and subsequently potentiating growth andtransformation.

Structure Reference: Park, Y.C. et al. (2000) Cell101 (7), 777-787.

TRAF domain proteins TRAF domain of human TRAF2

TUBBY DomainDomain binding and function: The Tubby domain was first identified in the tubby proteinimplicated in mature-onset obesity. Spanning approximately 260 amino acids, the Tubbydomain has a remarkable dual binding function as it is capable of interacting with both DNAand phosphotidylinositol. The Tubby domain of the tubby and TULP proteins binds with highspecificity to biphosphorylated phosphoinositides that are phosphorylated at the 4-position onthe inositol ring, such as PI(4,5)P2. This allows the Tubby domain to function downstream of

receptors such as the 5HT2C serotonin receptor. 5HT2C activation leads to stimulation oftrimeric G-proteins that activate phospholipase C (PLC). PLC hydrolysis of PI(4,5)P2 releasesthe Tubby domain from the membrane, from whence it tranlocates into the nucleus. Once inthe nucleus, the Tubby domain binds DNA allowing the tubby protein amino-terminaltranscription factor-like activation domain to promote transcription.

Structure Reference: Santagata, S. et al. (2001), Science 292(5524), 2041-2050.


54/58

54

UBA DomainDomain binding and function: The ubiquitin-associated (UBA) domain is an approximately40 amino acid motif that was first recognized in proteins associated with ubiquitination but isalso found in proteins involved in DNA nucleotide excision-repair and other proteins. UBAdomains have been shown to bind mono-, di-, tri-, and tetra-ubiquitin in vitro but appear to bindto polyubiquitin with a higher affinity and it is thought that polyubiquitinated proteins representthe true in vivo binding substrates. As well, some UBA domains appear to homo andheterodimerize and to bind other substrates. Functionally, the UBA domain has been proposedto limit ubiquitin chain elongation and to target ubiquitinated proteins to the 26S proteasome fordegradation.Structure Reference: Mueller, T.D. and Feigon, J. (2002) J. Mol. Biol. 319 (5), 1243-1255.

VHS DomainDomain binding and function: The approximately 150 amino acid VHS (Vps27p, Hrs andSTAM) domain has been identified in over 40 different eukaryotic proteins. VHS domains canbe found in the context of other modular domains such as the SH3 domain and the FYVE

domain in EAST and Hrs proteins, respectively. This domain is also found at the amino-terminus of several proteins that have been implicated in signaling from receptor tyrosinekinases (RTKs). VHS domains are found in proteins such as STAM, EAST and Hrs that havebeen linked to RTK-mediated endocytosis. The VHS domain of GGA proteins binds to anacidic di-leucine motif in the cytoplasmic domain of sorting receptors including the mannose 6-phosphate receptor. GGA proteins are required for the targeting of mannose 6-phosphatereceptor to the lysosome, where the receptor functions to mediate lysosomal enzyme sorting.Structure Reference: Misra, S. et al. (2000) Biochemistry39(37), 1128211290.

WD40 Domain

Domain binding and function: WD40 repeats are found in a number of eukaryotic proteinsthat cover a wide variety of functions including adaptor/regulatory modules in signaltransduction, pre-mRNA processing, cytoskeleton assembly and cell cycle control. The onlycommon functional theme of WD40 domains is to serve as a stable propeller-like platform towhich proteins can bind either stably or reversibly. Unlike the non-WD40 propeller family ofproteins, there are no cases of WD40 proteins with catalytic activity. The WD40 domains ofbeta-TRCP and Cdc4 have been implicated in recognizing phosphorylated serine andthreonine containing peptides, demonstrating that in some cases WD40 repeat forming beta-propeller structures can serve in phospho-peptide recognition.

Structure Reference: Wall, M.A. et al. (1995) Cell83(6), 10471058.

WD40 repeat proteins WD40 repeats in G beta


55/58

55

WW DomainDomain binding and function: WW domains are small 38 to 40 amino acid residue modulesthat have been implicated in binding to Pro-rich sequences. WW domains and SH3 domainscan potentially bind overlapping sites. In addition, the Pin1 WW domain functions as aphospho-serine or phosphothreonine binding module, suggesting that certain WW domainshave evolved an alternate mode of action. WW domains bind peptide ligands with dissociationconstants in the M range.

Structure Reference: Ranganathan, R. et al. (1997) Cell89(6), 875886.

WW domain proteins The Pin1 WW domain


56/58

56

MOTIFS

ITAM and ITIM

I. ITAM (immunoreceptor tyrosine-based activation motif)

The acronym ITAM was first proposed in 1994 [8] to designate the di-tyrosine-based YxxLactivation motifs that had been first described by Reth as a module responsible for the cell-triggering properties of receptors belonging to the family of MIRR. The tyrosine-basedactivation motif exists in one or more copies in each of the receptor-associated signal-transducing molecules and it contains two repeats of the consensus sequence YXXL/I spacedby six to eight amino acids (EX2YX2L/IX6-8YX2L/I, in the single-letter code for amino acids,with X signifying any amino acid). Receptor clustering results in a rapid and transientphosphorylation of tyrosine residues within their ITAMs, thereby creating binding sites forseveral SH2-domain containing cellular proteins including protein tyrosine kinases (PTK) andadaptor molecules coupling to downstream processes [9].

Evidence that the two YxxL are functionally distinct, emerged from mutations of either the Y orL residues in the N-terminal YxxL segment of the membrane-proximal ITAM of CD3 whichabolished all signal transduction functions of their ITAM, while mutations at the Y or L in the C-terminal YxxL abrogated signals for IL-2 production but did not prevent Y phosphorylation ofthe N-terminal tyrosine of the ITAM and of other ITAM mediated functions [10].

It is noteworthy that the various ITAMs of the TCR/CD3 complex can interact with distinctcytosolic effector molecules, indicating that differential ITAM phosphorylation during T-cellactivation could be a mechanism to generate the signaling diversity by the TCR complex [11].

A critical event in signaling in immune cells is the interaction of Syk or ZAP-70 protein tyrosine

kinases with the multisubunit receptors that contain ITAM. Thus studies of the binding of signaltransducing molecules to the ITAMs of the TCR- chain showed [12] that ZAP-70 boundspecifically to bisphosphorylated but not to the mono- or unphosphorylated peptides. Incontrast, Shc, PI3-K, Grb-2, and GAP bound with different affinities to the bis- ormonophosphorylated peptides, while Fyn did not bind specifically to any of the tested peptides.The different preferences of signaling molecules for distinct ITAMs, and the preferential bindingof some of them to monophosphorylated peptides, suggest that each ITAM may bind a uniqueset of such molecules. This may mean that ITAM-bearing receptors can couple to varioussignaling pathways under different conditions of receptor triggering ( Table 1).

II. ITIM (immunoreceptor tyrosine-based inhibition motif)

Studying the mechanism of FcRIIb-mediated inhibition of B-cell activation (see later) a highlyconserved 13 amino acid region was described within the intracytoplasmic tail of the murineFcRIIb containing a single YxxL motif. The tyrosine and leucine within this motif is essential forthe FcRIIb mediated inhibitory function [13, 14 and 15]. It turned out recently that in addition tothe FcRIIb a family of ITIM-bearing negative co-receptors exists [16 and 17]. The characteristicfeature of the ITIM is that the tyrosine is followed by a leucine or valine at position Y+3 and it isgenerally preceded by a hydrophobic residue (I, V, L,S) at the -2 position. Inhibitory activity of


57/58

57

ITIM-bearing receptors is only seen upon co-crosslinking with an ITAM-bearing receptor [18],and the inhibitory mechanism depends on the phosphorylation of the tyrosine in the ITIM by anITAM-containing PTK [14, 19 and 20]. From the functional point of view it is important to notethat in contrast to the ITAMs which in phosphorylated form bind SH2 domain-bearing proteintyrosine kinases, the phosphorylation of the tyrosine residue in ITIM leads to the recruitment ofSH2-containing phosphatases such as protein tyrosine phosphatases SHP-1 and SHP-2 andthe polyinositol 5'-phosphate phosphatase SHIP ( Table 2).


58/58

TRANSCRIPTION FACTOR DOMAINS

Basic-Helix-Loop-Helix motif, (bHLH motif)

Domain binding and function: The bHLH motif is a dimerization motif found in transcriptionfactors controling proliferation (c-Myc), as well as differentiation (e.g. E2a and MyoD). Some

bHLH motifs permit homodimerization (e.g. E2a), while others do not (e.g. MyoD).Heterodimerization with common partners, such as E2a, permit tissue-specific partners, suchas MyoD, to form a complex that targets specific DNA sequences within the promoters of aspecific subset of cellular genes, in this case those controling myogenesis.

Documents

Domains Signaling