HEDGEHOG PROTEIN FAMILY
Structural Bioinformatics
Oriol Senan
Alexandros Pittis
Amadís Pagès
0 CONTENTS
1. INTRODUCTION
2. HEDGEHOG PROTEIN FAMILY
3. AMINO-TERMINAL HEDGE DOMAIN (HhN)
4. CARBOXY-TERMINAL HOG DOMAIN (HhC)
5. QUESTIONS
0 CONTENTS
1. INTRODUCTION
2. HEDGEHOG PROTEIN FAMILY
3. AMINO-TERMINAL HEDGE DOMAIN (HhN)
4. CARBOXY-TERMINAL HOG DOMAIN (HhC)
5. QUESTIONS
1 INTRODUCTION
Hedgehog signaling pathway
>Responsible for controled cell growth and
differentiation in embryogenesis in metazoa
regulating a wide range of patterning events in a
local and long-range scale
Left-right asymmetry
Anterior-Posterior patterning
Neural tube patterning
Limb patterning
>Homeostatic role in postembryonic tissues
(maintenance of stem cell populations)
Continuous Hh pathway activity plays a
pathological role in the growth of cancers
1 INTRODUCTION
Overview of the Hh signaling
pathway
> The signal transduction pathway regulate
the production of either a transcriptional
repressor (CiR) or a transcriptional activator
(CiA), active and inactive forms of Ci, a
zinc-finger transcription factor
> Patched (Ptc) is a 12-transmembrane
receptor which acts as an inhibitor for
Smoothened (Smo) another
transmembrane protein which acts as a
signal transducer
1 INTRODUCTION
> Downstream of Smo is a multi-protein
complex known as the Hedgehog signaling
complex (HSC)
> Ptc represses Smo, preventing the
activation of Hedgehog signalling via
proteolytic cleavage of Ci. Cleavage results in
a repressor form of Ci, which enters the
nucleus and inhibits Hedgehog target gene
expression.
> The binding of Hedgehog protein to Ptc
inactivates it, prevents the inhibition of Smo
and induces signal transduction leading to the
full length form of Ci (CiA)
> The release of the active CiA stimulates the
transcription of several target genes
2 THE HEDGEHOG PROTEIN FAMILY
1. INTRODUCTION
2. HEDGEHOG PROTEIN FAMILY
3. AMINO-TERMINAL HEDGE DOMAIN (HhN)
1. > Overview
1. > Structural conformation
1. > Conservation
1. > Interactions with other proteins
4. CARBOXY-TERMINAL HOG DOMAIN (HhC) (oriol)
5. QUESTIONS
2 THE HEDGEHOG PROTEIN FAMILY
> The Hedgehog family consists of secreted signal proteins which comprise different domains
and several motifs
Signal peptide for protein export (SS)
Amino-terminal 'Hedge' secreted signaling domain (HhN)
Carboxy-terminal 'Hog' autocatalytic domain (HnC), containing the 'Hint' module and the
Sterol Recognition site (SRR)
Signaling domain Autocatalytic domain
2 THE HEDGEHOG PROTEIN FAMILY
Role of hedgehog protein in the hedgehog signaling pathway
> To become an active ligand requires:
> > Autoprocessing reaction
> > Palmitoylation of the most amino-terminal cysteine
> Once released into the extracellular environment, interacts with different proteins in multimeric form
> Targets Patched (Ptc) protein
(1) the signal sequence is cleaved
Autoprocessing reaction
(2) the C-terminal domain of the Hh polypeptide catalyzes
an intramolecular cholesteroyl transfer reaction, resulting in
(3) the formation of a C-terminally cholesterol-modified
Nterminal Hh signaling domain (HhN). This causes association
of HhN with membranes, which facilitates the final
modification,
Palmitoylation
(4) the addition of a palmitic acid moiety to the N terminus by
an acyltransferase, resulting in the formation of dually
modified Hh signaling domain
> Skinny hedgehog (Ski or Ras) in Dros. Mel.
> Hedgehog AcetylTransferase (HHAT) in mammals
2 THE HEDGEHOG PROTEIN FAMILY
Role of the hedgehog protein in the hedgehog signaling pathway
> To become an active ligand requires:
> > Autoprocessing reaction
> > Palmitoylation of the most amino-terminal cysteine
> Once released into the extracellular environment, interacts with different proteins in multimeric form
> Targets Patched (Ptc) protein
Growth Arrest Specific 1 (GAS1)
> Attenuates signaling, reduces effective range
Hedgehog-Interacting Protein (Hip)
> Membrane-bound glycoprotein
> Binds to SHH, DHH and IHH
> Attenuates signaling, reduces effective range
Glypical Dally-Like (Dlp)
> Acts as an accessory receptor
> Helps in HhN transport
Interference Hedgehog (Ihog) - Dros. Mel.
Boc / Cdo - Mammals
> Required for normal HhN signaling
> Facilitate binding to responding cells
> Increase HhN association with Ptc
2 THE HEDGEHOG PROTEIN FAMILY
Role of the hedgehog protein in the hedgehog signaling pathway
> To become an active ligand requires:
> > Autoprocessing reaction
> > Palmitoylation of the most amino-terminal cysteine
> Once released into the extracellular environment, interacts with different proteins in multimeric form
> Targets Patched (Ptc) protein
The key function of the HhN as an extracellular signal is to
inhibit the activity of the receptor Patched (Ptc). In the
absence of HhN binding, Patched represses a signaling
pathway that acts trough Smoothened.
The downstream signaling pathway is ultimately leading Gli
(mammals) or Ci (dros. mel.) transcription factors to
activate target genes.
2 THE HEDGEHOG PROTEIN FAMILY
Dhh
> The Hh gene family is present
throughout Eumetazoa although it
is absent in some nematodes
One single gene in
Drosophila melanogaster Ihh
Three paralogous genes in
most eumetazoa :
Sonic Hedgehog (Shh),
Indian Hedgehog (Ihh) and
Shh
Desert Hedgehog (Dhh)
Due to genome duplication
of ray-finned fishes in
zebrafish five hh genes are
present
>Caenorhabditis elegans has no hh Drosophila single
genes but has hh-relates genes via hh
the Hog domain
C.elegans
hh-like
2 THE HEDGEHOG PROTEIN FAMILY
Sequence alignment of Hedgehog family
Hedge domain
Hog domain
2 THE HEDGEHOG PROTEIN FAMILY
Hedgehog amino-terminal signalling
domain 'Hedge'
> The N-terminal domain of Hedgehog proteins
> It has been found in sponges and cnidaria in a
large extracellular membrane protein called
Hedling
Contains many additional domains apart
from Hedge but lacks Hog domain
2 THE HEDGEHOG PROTEIN FAMILY
Hedgehog carboxy-terminal
autoproteolytic domain 'Hog'
>The domain characterizes a group of
cysteine peptidases
>High similarity of Hint module to self
splicing inteins
>Several classes of Hint containing
proteins, with various types of processing
activities
2 AMINO-TERMINAL HEDGE DOMAIN (HhN)
Evolution of hh and hh-related genes and domain architecture
3 AMINO-TERMINAL HEDGE DOMAIN (HhN)
1. INTRODUCTION
2. HEDGEHOG PROTEIN FAMILY
3. AMINO-TERMINAL HEDGE DOMAIN (HhN)
1. > Overview
1. > Structural conformation
1. > Conservation
1. > Interactions with other proteins
4. CARBOXY-TERMINAL HOG DOMAIN (HhC)
5. QUESTIONS
3.1 HhN -> Overview Domain organization and evolution
Domain organization and evolution
> Present in Hedhegoh proteins
> Present in Hedgling proteins
> Large extracellular protein that contains a hedge domain at its amino terminus plus many additional
> domains such as VWA domains and numerous cadherin repeats, but lacks a Hog domain.
> Found in sponges and Cnidaria
> Not present in Warthog, Groundhog and Quahog
> Proteins which contain a hog domain at its carboxyl terminus but have an amino terminus distinct from HhN.
> Found in some nematodes (Caenorhabditis elegans)
Mainly hedgehog
fragments and Hedgehog
very few proteins
uncharacterized
proteins
Hedgling
proteins
3.1 HhN -> Overview Domain organization and evolution
Domain organization and evolution
> Present in Hedhegoh proteins
> Present in Hedgling proteins
> Large extracellular protein that contains a hedge domain at its amino terminus plus many additional
> domains such as VWA domains and numerous cadherin repeats, but lacks a Hog domain.
> Found in sponges and Cnidaria
> Not present in Warthog, Groundhog and Quahog
> Proteins which contain a hog domain at its carboxyl terminus but have an amino terminus distinct from HhN.
> Found in some nematodes (Caenorhabditis elegans)
Scenario 1 Scenario 2
Hedge domain Hedge domain
evolved from a evolved in a
secreted amino- extracellular protein
terminal domain such as hedgling.
already associated Then Hedge is
with the Hog domain. ‘merged’ with a Hog
Hedgling is then protein giving rise to
derived from Hh by a Hh
‘split’ of Hedge from
Hog
3.2 HhN -> Structural conformation SCOP Classification
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
Families:
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
3.2 HhN -> Structural conformation Fold
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
Families:
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
Fold
> Core α + β sandwich of two α-helices and six-
> stranded mixed β -sheet decorated by extensive
> loop regions
> Small, two-stranded antiparallel β -sheet
3.2 HhN -> Structural conformation Zinc-binding motif
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
Families:
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
Zinc-binding motif
> Zinc coordination site
> > Histidine 141 in loop
> > Aspartic acid 148 in β-sheet 4
> > Histidine 183 in β -sheet 6
> > Water molecule
> > Acid glutamic 177 in β –sheet 5
3.2 HhN -> Structural conformation Zinc-binding motif
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
Families:
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
Zinc-binding motif
> Zinc coordination site
> > Histidine 141 in loop
> > Aspartic acid 148 in β-sheet 4
> > Histidine 183 in β -sheet 6
> > Water molecule
> > Acid glutamic 177 in β -sheet 5
3.2 HhN -> Structural conformation Zinc-binding motif
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
Families:
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
Zinc-binding motif
> Zinc coordination site
> > Histidine 141 in loop
> > Aspartic acid 148 in β-sheet 4
> > Histidine 183 in β -sheet 6
> > Water molecule
> > Acid glutamic 177 in β -sheet 5
3.2 HhN -> Structural conformation Zinc-binding motif
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
Families:
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
Zinc-binding motif
> Zinc coordination site
> > Histidine 141 in loop
> > Aspartic acid 148 in β-sheet 4
> > Histidine 183 in β -sheet 6
> > Water molecule
> > Acid glutamic 177 in β -sheet 5
3.2 HhN -> Structural conformation Zinc-binding motif
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
Families:
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
Zinc-binding motif
> Zinc coordination site
> > Histidine 141 in loop
> > Aspartic acid 148 in β-sheet 4
> > Histidine 183 in β -sheet 6
> > Water molecule
> > Acid glutamic 177 in β -sheet 5
3.2 HhN -> Structural conformation Zinc-binding motif
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
Families:
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
Zinc-binding motif
> Zinc coordination site
> > Histidine 141 in loop
> > Aspartic acid 148 in β-sheet 4
> > Histidine 183 in β -sheet 6
> > Water molecule
> > Acid glutamic 177 in β -sheet 5
3.2 HhN -> Structural conformation Zinc-binding motif
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alpha and beta proteins (a+b)
HIS141
Mainly antiparallel beta sheets (segregated alpha and beta regions) HIS183
3. Fold: Hedgehog/DD-peptidase
alpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta
Zn
Superfamilies:
1. Superfamily: Hedgehog/DD-peptidase
zinc-binding motif
ASP148 H2O
Families:
GLU177
• Muramoyl-pentapeptide carboxypeptidase
1. MepA-like
• VanX-like
• Hedgehog, N-terminal signaling domain
• VanY-like
Zinc-binding motif
> Zinc coordination site
> > Histidine 141 in loop
> > Aspartic acid 148 in β-sheet 4
> > Histidine 183 in β -sheet 6
> > Water molecule
> > Acid glutamic 177 in β -sheet 5
3.3 HhN -> Conservation Sequence Alignment
Conservation of the HhN domain in the Hedgehog family of proteins
Sequence Alignment
Domain is highly conserved, especially
in the alpha-beta(2)-alpha-beta(2)
region
Overall percentages:
> Identity percentage: 49%
> Similarity percentage: 23%
3.3 HhN -> Conservation Sequence Alignment
Conservation of the HhN domain in the Hedgehog family of proteins
Sequence Alignment
Conservation of residues near the zinc
binding motif is really high, with zinc-
coordinating histidines and aspartic acid
absolutely conserved among vertebrates
In Drosophila Melanogaster And
Drosophila Hydei there’s only one of
those coordinating residues: HIS141
> Hh is not expected to bind zinc
> Hh achieves activity in a different
> fashion than SHh, IHh and DHh
3.3 HhN -> Conservation Structural Alignment
Conservation of the HhN domain in the Hedgehog family of proteins
Structural Alignment
> 1VHH : SHh – Mus musculus
> 2IBG:E : Hh – Drosophila Melanogaster
> 2WFQ : DHh – Homo Sapiens
> 3HO5:H : SHh – Homo Sapiens
3.3 HhN -> Conservation Structural Alignment
Conservation of the HhN domain in the Hedgehog family of proteins
Structural Alignment
> 1VHH : SHh – Mus musculus
> 2IBG:E : Hh – Drosophila Melanogaster
sequence identity: 62.66 %
pdb1vhh 157 residues -> 4_pdb2ibg 142 residues
matching Ca: 140 ( 89.17% / 98.59% )
rms deviation: 0.584701 min. length: 6
39 49 59 69 79 89
KLTPLAYKQFIPNVAEKTLGASGRYEGKITRNSERFKELTPNYNPDIIFKDEENTGADRL
************************************************ ***
************************************************ ***
YPLVLKQTIPNLSEYTNSASGPLEGVIRRDSPKFKDLVPNYNRDILFR-------DRL
100 110 120 130 140
99 109 119 129 139 149
MTQRCKDKLNALAISVMNQWPGVKLRVTEGWDEDGHHS-EESLHYEGRAVDITTSDRDRS
************************************** * *******************
************************************** * *******************
MSKRCKEKLNVLAYSVMNEWPGIRLLVTESWDEDYHHGQE-SLHYEGRAVTIATSDRDQS
157 167 177 187 197 207
159 169
KYGMLARLAVEAGFDWVYYESKAHIHCSVKAENSVAAK
********************************
********************************
KYGMLARLAVEAGFDWVSYVSRRHIYCSVKSD
217 227 237 247
Non cristallized residues
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO/BOC proteins
Overview of Ihog/CDO/BOC proteins
> Homologous genes
> > Ihog : Drosophila Melanogaster
> > CDO/BOC: Mammals
> Cell-surface proteins
> > Multiple immunoglobulin repeats
> > Multiple fibronectin type 3 (FNIII) repeats Immunoglobulin
repeats
SCOP Classification of interacting domains
Lineage:
1. root: scop
1. Class : all beta proteins
Fibronectin type III
1. Fold : Immunoglobulin-like b sandwich repeats
Sandwich; 7 strands in 2 sheets; greek key
some members of the fold have additional strands
4. Superfamily : Fibronectin type III
5. Family : Fibronectin type III
Protein domains: Cell membrane
2. Fibronectin, different Fn3 modules
30. Brother of CDO precursor > Ihog binds Hh in the first FNIII repeat : IhogFn1 domain
42. Hedgehog receptor iHog
> CDO bind SHh (and homologous genes) in the third FNIII
> repeat : CDOFn3 domain
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Overview of Ihog/CDO proteins
FNIII repeats of the Ihog protein
in Drosophila Melanogaster
> Homologous genes (IhogFn1-2)
> > Ihog : Drosophila Melanogaster
> > CDO (and also BOC) : Mammals
> Cell-surface proteins
> > Multiple immunoglobulin repeats
> > Multiple fibronectin type 3 (FNIII) repeats
SCOP Classification of interacting domains
Lineage:
1. root: scop
1. Class : all beta proteins
1. Fold : Immunoglobulin-like b sandwich
Sandwich; 7 strands in 2 sheets; greek key
some members of the fold have additional strands
4. Superfamily : Fibronectin type III
5. Family : Fibronectin type III
Protein domains:
2. Fibronectin, different Fn3 modules
30. Brother of CDO precursor
42. Hedgehog receptor iHog Third FNIII repeat of CDO protein
in Homo Sapiens
(CDOFn3)
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Iteraction of Hedgehog and Ihog in Drosophila Melanogaster
> IhogFn1-2 forms a symetric dimer complex in the presence of heparin
> Each HhN molecule contacts only one single IhogFn1-2 domain
> HhN presents an heparin binding site
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Iteraction of Hedgehog and Ihog in Drosophila Melanogaster
> IhogFn1-2 froms a symetric dimer complex in the presence of heparin
> Each HhN molecule contacts only one single IhogFn1-2 domain
> HhN presents an heparin binding site
V103
V553
L551
Core of three hydrophobic residues surrounded by predominantly polar interactions
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Iteraction of Hedgehog and Ihog in Drosophila Melanogaster
> IhogFn1-2 froms a symetric dimer complex in the presence of heparin
> Each HhN molecule contacts only one single IhogFn1-2 domain
> HhN presents an heparin binding site
R238
D558
N559
E561
Hydrogen bonds surrounding the hydrophobic core, one through a water molecule
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Iteraction of Hedgehog and Ihog in Drosophila Melanogaster
> IhogFn1-2 froms a symetric dimer complex in the presence of heparin
> Each HhN molecule contacts only one single IhogFn1-2 domain
> HhN presents an heparin binding site
Key residues in the heparin binding site are highly
conserved among invertebrate homologs, but not
among vertebrate homologs
Vertebrate homologs do not bind via heparin binding
site
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Iteraction of Sonic Hedgehog and CDO in Homo Sapiens
> SHhN presents a calcium binding site. SHhN binds to CDOFn3 in the presence of calcium.
> ShhN-CDOFn3 interaction is completely different than Hh-IgohFn1-2 interaction !!
Asp96
Asp130 and Asp132
Glu90 and Glu91
Glu127
> Calcium ions are coordinated by three aspartate and three glutamate residues
> Each ion is coordinated by eight oxygen atoms
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Iteraction of Sonic Hedgehog and CDO in Homo Sapiens
> SHhN presents a calcium binding site. SHhN binds to CDOFn3 in the presence of calcium.
> ShhN-CDOFn3 interaction is completely different than Hh-IgohFn1-2 interaction !!
Glu90
Val198, Met919 and
Ile920
His134
> His134 and Glu90 in ShhN make Van der Waals contacts with Val 198, Met919 and Ile920 in CDOFn3
> Mostly acidic residues in CDOFn3 interact with mostly basic residues in SHhN
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Iteraction of Sonic Hedgehog and CDO in Homo Sapiens
> SHhN presents a calcium binding site. SHhN binds to CDOFn3 in the presence of calcium.
> ShhN-CDOFn3 interaction is completely different than Hh-IgohFn1-2 interaction !!
> Calcium coordinating residues absolutely
> conserved
3.4 HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC
Interaction of HhN domain with Ihog/CDO proteins
Iteraction of Sonic Hedgehog and CDO in Homo Sapiens
> SHhN presents a calcium binding site. SHhN binds to CDOFn3 in the presence of calcium.
> ShhN-CDOFn3 interaction is completely different than Hh-IgohFn1-2 interaction !!
> Key residues in the HhN-CDOFn3 interface are
> highly conserved
> Two non-conserved substitutions:
> > Leucine 124 in Drosophila Hydei (expected)
> > Leucine 134 in Danio Rerio (unexpected)
Conserved substitutions in green
4 CARBOXY-TERMINAL HOG DOMAIN (HhC)
1. INTRODUCTION
2. HEDGEHOG PROTEIN FAMILY
3. AMINO-TERMINAL HEDGE DOMAIN
4. CARBOXY-TERMINAL HOG DOMAIN
1. > Overview
1. > Structural conformation
1. > Structural homologs
1. > Other homologs
5. QUESTIONS
4.1 HhC -> Overview Domain evolution and history
HhC components and evolution
> Present in Hedhegoh proteins
> Present in other proteins families, as a domain homolog.
HhC contains the Hint region. Hint superclass.
> Distribution in three kingdoms, Bil-A,Bil-B,Bil-C, inteins, hog and Vint.
> It seems hog domain has an early origin in eukaryotic evolution. Absence in higher plants.
> HhC also contains a SRR region (Sterol recognition region). It binds to cholesterol.
This region is also found in other proteins families,but the the nature is unknown. ARR
>
4.1 HhC -> Overview Function
HhC function
> Hint function
Mediates de cleavage of the hedge and hog. A
aaconserved cysteine mediates a nucleophilic atack on
aathe carbonyl of the preceding residue.
aaThis includes the formation of a thioester instead of
aathe peptide bond. The thioester is atacked by a
aacholesterol molecule, and results a aminoterminal
aacholesterol modificated residue.
> Also contains a SRR region (Sterol recognition
aaregion). It binds to cholesterol.
This region is also found in other proteins families,but
aathe the nature is unknown. Is called ARR(aduct
recognition region)
>
4.2 HhC -> Structural conformation SCOP Classification
Structural conformation
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alll beta proteins
3. Fold: Hedgehog/intein (Hint) Domain
Complex fold made of five beta-hairpin units and a b-ribbon arc
Superfamilies:
1. Superfamily: Hedgehog/intein (Hint) domain
Families:
1. Hedgehog, C-terminal (Hog) signaling domain
4.2 HhC -> Structural conformation SCOP Classification
Structural conformation (homologues)
SCOP Classification
Lineage:
1. Root: scop
2. Class: Alll beta proteins
3. Fold: Hedgehog/intein (Hint) Domain
Complex fold made of five beta-hairpin units and a b-ribbon arc
Superfamilies:
1. Superfamily: Hedgehog/intein (Hint) Domain
Duplicated (Contaiins interwined structural repeats
Families:
1. Intein (protein splicing domain)
4.2 HhC -> Structural conformation SCOP Classification
Structural conformation (conserved aminoacids)
> Several aminoacids crucial for the function
4.2 HhC -> Structural conformation SCOP Classification
Structural conformation (conserved aminoacids)
> Several aminoacids crucial for the function
4.2 HhC -> Structural conformation Aminoacid conservation
Structural conformation (conserved aminoacids)
> Cys 258 (in protein Hehgehog
Drosophila
Does the first nucleophilic atack, crucial for
autocleavage
4.2 HhC -> Structural conformation Aminoacid conservation
Structural conformation (conserved aminoacids)
> His 329 (in protein Hehgehog
Drosophila
Essencial for autocleavage
4.2 HhC -> Structural conformation Aminoacid conservation
Structural conformation (conserved aminoacids)
> Thr 326 (in protein Hehgehog
Drosophila
Involved in autocleavage
4.2 HhC -> Structural conformation Aminoacid conservation
Structural conformation (conserved aminoacids)
> Thr 303 (in protein Hehgehog
Drosophila
Involved in cholesterol transfer
4.3 HhC -> Structural homologs Inteins
Hedgehog Hint is homolog to Inteins
> Inteins are proteins that split themselves and rejoin to form functional proteins
> There is not a big conservation in sequence, mostly these aminoacids that are involved in the
active center, and some hidrophobic cores.
> However, there is a high structural similarity bewteen families.
4.3 HhC -> Structural homologs Inteins
Hedgehog Hint is homolog to Inteins
> Example: Protein Hedgehog HhC of drosophila > Example: Mtu recA intein splicing domain
4.3 HhC -> Structural homologs Inteins
Hedgehog Hint is homolog to Inteins
> The proteins are structurally very similar
> Orange Protein Hedgehog
> Cyan Intein domain
4.3 HhC -> Structural homologs Inteins
Hedgehog Hint is homolog to Inteins
> The proteins are structurally very similar
> They do the a similar function → Homology in function is translated in homology in structure
> They are evolutonary related.
> Plausible gene duplication.
> Early in eucaryotic evolution
> High gene diferentiation
4.4 HhC -> Other HhC homologs Other homologs
Hedgehog Hint has other homologs
> It has been found in some proteins that contain self autoprocessing C-terminal domains
> But they don't contain HhN part of a Hh protein.
> For instance, in Caenorhabditis elegans 10 genes were found with C-terminal domains.
> Four proteins families related to Hh (refered as Hh related genes):
> Quahog (qua), Warthog (wrt) Groundhog (grd) and Ground-like (grl)
> There is no experimental structural data yet. Solution: In silico model.
> Building of a in silico model is very difficult. There is very few data of structures for the Hh family in the Hh-C
part (only one PDB) and there is very few homology in sequence with other related proteins like inteins. Is not
possible to build a acurated model.
5 QUESTIONS
1. INTRODUCTION
2. HEDGEHOG PROTEIN FAMILY
3. AMINO-TERMINAL HEDGE DOMAIN (HhN)
4. CARBOXY-TERMINAL HOG DOMAIN (HhC)
5. QUESTIONS