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A Few Good Domains
(http://www.cellsignal.com/reference/domain/index.jsp)
Starring (in alphabetical order)
14-3-3
C1
EF-hand
PDZ
PH
PTB
RGS
RING
SH2
SH3
SNARE
TPR
TRAF
TUBBY
VHS
WD40
WW
14-3-3
14-3-3 proteins are 30 kDa polypeptides with nine closely related members in mammals.
They are also found in plants and fungi. They are involved in regulating various
pathways including signaling apoptosis and passage through the cell cycle. 14-3-3
proteins form homo and heterodimeric cup-like structures that bind to discrete
phosphoserine-containing motifs. In some instances, 14-3-3 proteins appear to export
their binding partners from the nucleus to the cytoplasm in a phosphorylation and Crm1dependent manner.
Examples of Domain Proteins
Binding Examples
14-3-3 Binding Partners
Functions
cdc25 tyrosine phosphatase Cell cycle regulation
Bad Bcl-xL binding partner Regulation of apoptosis
c-Raf Ser/Thr Kinase
Regulation of kinase activity; Signal transduction
PKC Ser/Thr Kinase
Signal transduction
MEKK1,2,3 Ser/Thr Kinase Signal transduction
C1
C1 domains are approximately 50 amino acids long, enriched in cysteines, and are
involved in the recruitment of proteins to the membrane. Typically, C1 domains 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 hydrophobic
surface in the domain. This enables the region to be buried into the lipid bilayer
stabilizing membrane insertion. The middle portion of the domain contains a number of
basic residues that can interact with lipid head groups in the membrane, while the lower
half of the C1 domain contains two zinc-binding sites that are important to maintain the
fold of the domain.
Examples of Domain Proteins
Binding Examples
C1 Domain Proteins
Binding Partners
PKC Isoforms (classical and novel)
Diacylglycerol or phorbol esters
Diacylglycerol Kinase
Diacylglycerol or phorbol esters
c-Raf Ser/Thr Kinase
Diacylglycerol or phorbol esters
n-Chimaerin Rac GTPase Activating Protein Diacylglycerol or phorbol esters
EF-hand
The EF-hand motif contains approximately 40 residues and is involved in binding
intracellular calcium. EF-hand domains are often found in single or multiple pairs, giving
rise to various structural/functional variations in proteins containing EF-hand motifs.
Proteins containing EF-hands can be grouped into two functional categories—regulatory
or structural. Binding of calcium to regulatory EF-hand domain—containing proteins
induces a conformational change, which is transmitted to their target proteins, often
catalyzing enzymatic reactions. In contrast, binding of calcium to structural EF-hand
domain—containing proteins does not induce a significant conformational change.
Structural EF-hand domains seem to play a role in buffering intracellular calcium levels.
Examples of Domain Proteins
Binding Examples
EF-hand Domain Proteins Binding Partners
Functions
2+
Calmodulin
Ca
Regulatory proteins
2+
S-100
Ca
Regulatory proteins
2+
Recoverin
Ca
Regulatory proteins
2+
Calbindin
Ca
Structural proteins
2+
Parvalbumin
Ca
Structural proteins
PDZ
PDZ domains bind to the C-terminal 4–5 residues of their target proteins, frequently
transmembrane receptors or ion channels. These interactions can be of high affinity (nM
Kd). The consensus binding sequence contains a hydrophobic residue, commonly Val or
Ile, at the very C-terminus. Residues at the —2 and —3 positions are important in
determining specificity. PDZ domains can also heterodimerize with PDZ domains of
different proteins, potentially regulating intracellular signaling. In addition to engaging in
protein-protein interactions, several PDZ domains including those of syntenin, CASK,
Tiam1 and FAP are capable of binding to the phosphoinositide PIP2. PIP2-PDZ domain
binding is thought to control the association of PDZ domain-containing proteins with the
plasma membrane.
Examples of Domain Proteins
Binding Examples
PDZ Domain Proteins
Post-synaptic Density
Protein 95 (PSD-95)
Post-synaptic Density
Protein 95 (PSD-95)
Post-synaptic Density
Protein 95 (PSD-95)
Binding Partners
NMDA receptor B via PDZ1 and
PDZ2 of PSD-95
Kvl1.4 Shaker-type K+ channel via
PDZ1 and PDZ2 of PSD-95
Neural Nitric Oxide Synthase (nNOS)
via PDZ2
Domain Binding
Sites
– IESDV-COOH
– VETDVCOOH
PDZ/PDZ
interaction
PH
Pleckstrin-homology (PH) domains are found in a wide variety of signaling proteins that
associate with membranes. Some PH domains bind with high affinity (low µM or nM Kd)
to specific phosphoinositides such as phosphatidylinositol- 4,5-bisphosphate, PI-3, 4-P2
or PI-3,4,5-P3. Binding to phosphoinositides may allow PH proteins to respond to lipid
messengers for example by relocation to membranes. The C-termini of some PH domains
have also been reported to bind the β/γ subunits of heterotrimeric G-proteins.
Examples of Domain Proteins
Binding Examples
PH Domain Proteins Specific Phosphoinositide Ligands
Phospholipase C-δ; mSos1 PI-(4,5)-P2
Btk Tyr Kinase; Grp1
PI-(3,4,5)-P3
Akt/PKB Ser/Thr Kinase PI-(3,4)-P2
PTB
Phosphotyrosine binding (PTB) domains are 100–150 residue modules that commonly
bind Asn-Pro-X-Tyr motifs. The PTB domains of the docking proteins Shc and IRS-1
require ligand phosphorylation on the tyrosine residue (NPXpY) for binding. More Nterminal sequences are also required for high affinity binding and conferring specificity.
The peptide binds as a β-strand to an anti-parallel β-sheet, while the NPXpY motif makes
a turn, positioning the pY for recognition by basic residues. The PTB domains of proteins
such as X11, Dab, Fe65 and Numb apparently recognize NPXY or related peptide motifs,
but are not dependent on ligand phosphorylation. In addition, the Numb PTB domain can
bind an unrelated peptide that forms a helical turn.
Examples of Domain Proteins
Binding Examples
PTB Domain
Proteins
Shc docking protein
Binding Partners & Peptide Ligands
TrkA Nerve Growth Factor Receptor: Ile-Ile-Glu-Asn-Pro-GlnpTyr
PIRS-1 docking
Insulin receptor: Leu-Tyr-Ala-Ser-Ser-Asn-Pro-Glu-pTyr
protein
X11 neuronal protein β-amyloid precursor protein: Tyr-Glu-Asn-Pro-Thr-Tyr
RGS
The RGS (Regulator of G protein Signaling) domain has been found in over 20 proteins
in humans and is typically about 120 amino acids in length. RGS domains act
allosterically by stabilizing the transition intermediate of the GTP binding pocket of the α
subunit of heterotrimeric G proteins. This results in the acceleration of the intrinsic
GTPase activity of that α subunit. The discovery of the RGS domain therefore answered
the longstanding question of why the intrinsic rate of hydrolysis of many heterotrimeric
G proteins was often slower than the apparent cycling time for a signaling process
requiring that G 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 as they control the time of transmission of each of
these agonists.
Examples of Domain Proteins
Binding Examples
RGS Domain proteins Binding Partners
RGS-4
Gαi, Gαq
p115 RhoGEF
Gα12, Gα13
RGS-2
Gαq
GAIP
Gαi, Gαq
RING
The RING finger is a specialized type of Zn finger consisting of 40–60 residues that
binds two atoms of zinc, and is involved in mediating protein—protein interactions. The
presence of a RING finger domain is a characteristic of RING-class E3 ubiquitin protein
ligases capable of transfering ubiquitin from an E2 enzyme to a substrate protein. The
RING domain mediates the interaction with the appropriate E2 enzyme. Unlike HECT
E3s that form a thioester with ubiquitin, RING fingers likely mediate ubiquitination by
facilitating the direct transfer of ubiquitin from E2s to lysine residues on the target
substrate. RING finger proteins include the Hrt1/Roc1/Rbx1 proteins found in both the
SCF and VCB-like E3 complexes, the APC1 component of the Anaphase Promoting
Complex, Cbl family proteins, MDM2 and many other proteins with demonstrated E3
activity, E2 binding or involvement in ubiquitination. In addition to the involvement of
RING finger domains in ubiquitin transfer, this domain has also been associated with
certain transcription factors such as TIF1β, the PML-family, NFX1 and XPRF.
Examples of Domain Proteins
Binding Examples
RING Domain Proteins
Cbl
RAD5
RAD6
HHARI
Binding Partners
UbcH7
UBC13-MMS2 complex
RAD18
UbcH7
SH2
Src-homology 2 (SH2) domains are modules of ~100 amino acids that bind to specific
phospho (pY)-containing peptide motifs. Conventional SH2 domains have a conserved
pocket that recognizes pY, and a more variable pocket that binds 3-6 residues C-terminal
to the pY and confers specificity. The SAP SH2 domain recognizes Y as well as pY in
the context of residues N and C terminal, suggesting an alternate 3-pronged model may
apply in some cases. Phosphopeptides of optimal sequence bind to SH2 domains with
dissociation constants of ~50-500 nM.
Examples of Domain Proteins
Binding Examples
SH2 Domain Proteins
Binding
Partners
Phosphopeptide Ligand
SH2 Specificity
Residues
Regulation Specificity
Src Tyrosine Kinase
Focal Adhesion
Kinase
Phospho-lipase C-γ Cterminal SH2
Grb2 adaptor
-Ala-Glu-Ile
Tyr βD5
PDGF β receptor pTyr
-Ile-Ile-Pro-LeuPro-Asp
Cys βD5
Shc docking
protein
-Val-Asn-Val
Trp EF1
pTyr
pTyr
SH3
Src-homology 3 (SH3) domains bind to Pro-rich peptides that form a left-handed polyPro type II helix, with the minimal consensus Pro-X-X-Pro. Each Pro is usually preceded
by an aliphatic residue. Each in the aliphatic-Pro pair binds to a hydrophobic pocket on
the SH3 domain. The ligand can, in principle, bind in either orientation. An additional
non-Pro residue, frequently Arg, can form part of the binding core and contacts the SH3
domain. Such peptides usually bind to the SH3 domain with a Kd in the µM range. The
binding affinity and specificity can be markedly increased by tertiary interactions
involving loops on the SH3 domain.
Examples of Domain Proteins
Binding Examples
SH3 Domain
Proteins
Src Tyrosine
Kinase
Crk Adaptor
Protein
Binding Partners
SH3 Domain Binding Sites
RPLPVAP Class l N-terminal to C-terminal
binding site
C3G guanidine nucleotide PPPALPPKKR Class ll C-terminal to Nexchanger
terminal binding site
p85 subunit of Pl3 kinase
SNARE
While the mechanism by which a vesicle fuses with its proper membrane target is poorly
understood, it appears to involve a highly conserved set of proteins called SNAREs
(Soluble NSF Attachment protein [SNAP] Receptors). SNARE proteins are believed to
mediate most, if not all, cellular membrane fusion events. Most SNAREs are Cterminally anchored integral membrane proteins capable of entering into a coiled-coil
interaction with other SNARE proteins. All SNARE proteins share a homologous domain
of approximately 60 amino acids referred to as the SNARE domain. The SNARE domain
acts as a protein—protein interaction module in the assembly of a SNARE protein
complex. While monomeric SNARE motifs are largely unstructured, they assemble into a
protease resistant core complex. Interestingly, different SNARE family members are
distributed on distinct membranes throughout the cell, suggesting they may play a role in
targeting during vesicular transport. However, the formation of SNARE core complexes
appears to be rather promiscuous with little specificity.
Examples of Domain Proteins
Binding Examples
SNARE complexes
SNARE domain proteins in complexes
Syntaxin-1A (Sx), Synaptobrevin-II (Sb),
Rat Synaptic Fusion SNARE Complex
SNAP-25B
Yeast Exocytic post-Golgi SNARE
Snc2, Sso1, Sec9
Complex
Rat Endosomal SNARE Complex
Syntaxin-7, Vti 1b, Syntaxin 8
TPR
The tetratricopeptide repeat (TPR) motif was originally identified in yeast as a proteinprotein interaction module in cell cycle proteins. It has since been found in organisms
ranging from bacteria to humans. The TPR motif is a degenerate sequence 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. TPR motifcontaining proteins act as scaffolds for the assembly of different multiprotein complexes
including the anaphase promoting, the peroxisomal import receptor and the NADPH
oxidase complexes.
Examples of Domain Proteins
Binding Examples
TPR Domain Proteins
Binding Partners
Peptide Ligands
PEX5
PTS-1 target signal
S-K-L-COOH
Hsp70 - C-term heptapeptide E-E-V-D-COOH
Hop
Hsp90 - C-term pentapeptide E-E-V-D-COOH
phox
p67
GTP-Rac
surface contacts
TRAF
The approximately 150 amino acid TRAF domain is found in Tumor Necrosis Factor
(TNF) receptor-associated factors. TRAF proteins appear to be a relatively recent
evolutionary development as there is just one C. elegans TRAF protein and only two
Drosophila, and six mammalian TRAF proteins. All mammalian TRAFs localize to the
cytoplasm except TRAF4 which is found in the nucleus. TRAF proteins are recruited to
the membrane through interactions of their TRAF domains with activated TNF receptors,
IL-1/Toll receptors or through intermediate proteins such as the TRADDs. TRAFs
primarily act in cell survival upon interacting with TNF receptors by activating the NFkB
and AP-1 transcription factors. The six mammalian TRAF proteins have distinct
functions. For example, TRAF3 regulates T-cell dependent antigen responses, TRAF4 is
required for formation of the trachea and 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 and transformation.
Examples of Domain Proteins
Binding Examples
TRAF Domain Proteins Binding Partners
TRAF 1,2,3,5
CD40
TRAF 1,2
TRADD
TRAF6
IRAK
TRAF 2
TNFR1
TRAF 6
IL-1
TUBBY
The Tubby domain was first identified in the tubby protein implicated in mature-onset
obesity. Spanning approximately 260 amino acids, the Tubby domain has a remarkable
dual binding function as it is capable of interacting with both DNA and
phosphotidylinositol. The Tubby domain of the tubby and TULP proteins binds with high
specificity to biphosphorylated phosphoinositides that are phosphorylated at the 4position on the 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 of trimeric G-proteins that activate phospholipase C (PLC). PLC hydrolysis
of PI(4,5)P2 releases the Tubby domain from the membrane, from whence it tranlocates
into the nucleus. Once in the nucleus, the Tubby domain binds DNA allowing the tubby
protein amino-terminal transcription factor-like activation domain to promote
transcription.
Examples of Domain Proteins
Binding Examples
TUBBY Domain Proteins Binding Partners
Tubby
PI(4,5)P2; PI(3,4)P2
VHS
The approximately 150 amino acid VHS (Vps27p, Hrs and STAM) domain has been
identified in over 40 different eukaryotic proteins. VHS domains can be 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 tyrosine kinases
(RTKs). VHS domains are found in proteins such as STAM, EAST and Hrs that have
been linked to RTK-mediated endocytosis. The VHS domain of GGA proteins binds to
an acidic 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-phosphate receptor to the lysosome, where the receptor functions to mediate lysosomal
enzyme sorting.
Examples of Domain Proteins
Binding Examples
VHS Domain Proteins Binding Partners
Hrs
Hrs FYVE domain
WD40
WD40 repeats are found in a number of eukaryotic proteins that cover a wide variety of
functions including adaptor/regulatory modules in signal transduction, pre-mRNA
processing, cytoskeleton assembly and cell cycle control. The only common functional
theme of WD40 domains is to serve as a stable propeller-like platform to which proteins
can bind either stably or reversibly. Unlike the non-WD40 propeller family of proteins,
there are no cases of WD40 proteins with catalytic activity. The WD40 domains of βTRCP and Cdc4 have been implicated in recognizing phosphorylated serine and
threonine containing peptides, demonstrating that in some cases WD40 repeat forming βpropeller structures can serve in phospho-peptide recognition.
Examples of Domain Proteins
Binding Examples
WD40 Domain Proteins
G-Protein β-Chain
Prp4 Splicing Factor
Tup1 Transcriptional
Repressor
Binding Partners
G-protein α,γ-chain
Prp3 Splicing Factor
α2 Transcriptional
repressor
Cdc4
phosphorylated Sic1
Functions
Signal transduction
Splicing
Transcription
Ubiquitination and Cell Cycle
control
WW
WW domains are small 38 to 40 amino acid residue modules that have been implicated in
binding to Pro-rich sequences. WW domains and SH3 domains can potentially bind
overlapping sites. In addition, the Pin1 WW domain functions as a phospho-serine or
phosphothreonine binding module, suggesting that certain WW domains have evolved an
alternate mode of action. WW domains bind peptide ligands with dissociation constants
in the µM range.
Examples of Domain Proteins
Binding Examples
WW Domain
Proteins
Yes-Associated
Protein (YAP)
Nedd4 E3 Ubiquitin
Ligase
FBP – 11
Binding Partners
Yes, Src-like Tyrosine Kinase
βENaC amiloride-E3 ubiquitin ligasesensitive epithelial Na+ channel
Formin
WW Domain
Binding Sites
PPPPY
PPPNY
PPLP