HEDGEHOG PROTEIN FAMILY Structural Bioinformatics Oriol Senan Alexandros Pittis Amadís Pagès

HEDGEHOG PROTEIN FAMILY

Structural Bioinformatics

Oriol SenanAlexandros PittisAmadís Pagès

CONTENTS

4. CARBOXY-TERMINAL HOG DOMAIN (HhC)

0

2. HEDGEHOG PROTEIN FAMILY

1. INTRODUCTION

5. QUESTIONS

3. AMINO-TERMINAL HEDGE DOMAIN (HhN)

CONTENTS


0


5. QUESTIONS


1. INTRODUCTION

INTRODUCTION1

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

INTRODUCTION1

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

INTRODUCTION1

> 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

THE HEDGEHOG PROTEIN FAMILY

4. CARBOXY-TERMINAL HOG DOMAIN (HhC) (oriol)

2


1. INTRODUCTION

5. QUESTIONS


1. > Overview

1. > Structural conformation

1. > Conservation

1. > Interactions with other proteins

THE HEDGEHOG PROTEIN FAMILY2

> 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


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 catalyzesan 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


Role of the hedgehog protein in the hedgehog signaling pathway




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


Role of the hedgehog protein in the hedgehog signaling pathway




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.


> The Hh gene family is present throughout Eumetazoa although it is absent in some nematodes

One single gene in Drosophila melanogaster

Three paralogous genes in most eumetazoa : Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh)

Due to genome duplication of ray-finned fishes in zebrafish five hh genes are present

>Caenorhabditis elegans has no hh genes but has hh-relates genes via the Hog domain

Dhh

Ihh

Shh

Drosophila single hh

C.elegans hh-like


Sequence alignment of Hedgehog family

Hedge domain

Hog domain


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


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

AMINO-TERMINAL HEDGE DOMAIN (HhN)2

Evolution of hh and hh-related genes and domain architecture

AMINO-TERMINAL HEDGE DOMAIN (HhN)


3


1. > Overview


1. > Conservation

1. > Interactions with other proteins


1. INTRODUCTION

5. QUESTIONS

HhN -> Overview Domain organization and evolution3.1

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 very few

uncharacterized proteins

Hedgehog

proteins

Hedgling

proteins

3.1

Domain organization and evolution


> 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)

HhN -> Overview Domain organization and evolution

Scenario 1

Hedge domain evolved from a secreted amino-terminal domain already associated with the Hog domain. Hedgling is then derived from Hh by a ‘split’ of Hedge from Hog

Scenario 2

Hedge domain evolved in a extracellular protein such as hedgling. Then Hedge is ‘merged’ with a Hog protein giving rise to Hh

HhN -> Structural conformation SCOP Classification3.2

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-peptidasealpha-beta(2)-alpha-beta(2); 2 layers:alpha/beta

Superfamilies:

1. Superfamily: Hedgehog/DD-peptidasezinc-binding motif

Families:

• Muramoyl-pentapeptide carboxypeptidase1. MepA-like• VanX-like• Hedgehog, N-terminal signaling domain• VanY-like

HhN -> Structural conformation Fold3.2


SCOP Classification

Lineage:

1. Root: scop



Superfamilies:


Families:


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

HhN -> Structural conformation Zinc-binding motif3.2


SCOP Classification

Lineage:

1. Root: scop



Superfamilies:


Families:


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



SCOP Classification

Lineage:

1. Root: scop



Superfamilies:


Families:


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



SCOP Classification

Lineage:

1. Root: scop



Superfamilies:


Families:


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

HIS183HIS141

ASP148

GLU177

Zn

H2O

HhN -> Conservation Sequence Alignment3.3

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%

HhN -> Conservation Sequence Alignment3.3


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

HhN -> Conservation Structural Alignment3.3


Structural Alignment

> 1VHH : SHh – Mus musculus> 2IBG:E : Hh – Drosophila Melanogaster> 2WFQ : DHh – Homo Sapiens> 3HO5:H : SHh – Homo Sapiens

HhN -> Conservation Structural Alignment3.3


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

HhN -> Interactions with other proteins Hh -> Igoh/CDO/BOC3.4

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

SCOP Classification of interacting domains

Lineage:

1. root: scop

• Class : all beta proteins

• Fold : Immunoglobulin-like b sandwichSandwich; 7 strands in 2 sheets; greek keysome 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

> Ihog binds Hh in the first FNIII repeat : IhogFn1 domain

> CDO bind SHh (and homologous genes) in the third FNIII> repeat : CDOFn3 domain

Immunoglobulinrepeats

Fibronectin type IIIrepeats

Cell membrane


Interaction of HhN domain with Ihog/CDO proteins

Overview of Ihog/CDO proteins

> Homologous genes> > 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

• Class : all beta proteins

• Fold : Immunoglobulin-like b sandwichSandwich; 7 strands in 2 sheets; greek keysome 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

FNIII repeats of the Ihog proteinin Drosophila Melanogaster(IhogFn1-2)

Third FNIII repeat of CDO proteinin Homo Sapiens(CDOFn3)



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




> IhogFn1-2 froms a symetric dimer complex in the presence of heparin> Each HhN molecule contacts only one single IhogFn1-2 domain


Core of three hydrophobic residues surrounded by predominantly polar interactions

V103

V553

L551






Hydrogen bonds surrounding the hydrophobic core, one through a water molecule

R238

D558

N559

E561






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



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 and Glu91

Asp96

Glu127

Asp130 and Asp132

> Calcium ions are coordinated by three aspartate and three glutamate residues> Each ion is coordinated by eight oxygen atoms






His134

Glu90

Val198, Met919 and Ile920

> 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






> Calcium coordinating residues absolutely > conserved






> 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

CARBOXY-TERMINAL HOG DOMAIN (HhC)

3. AMINO-TERMINAL HEDGE DOMAIN

4

4. CARBOXY-TERMINAL HOG DOMAIN

1. > Overview


1. > Structural homologs

1. > Other homologs


1. INTRODUCTION

5. QUESTIONS

HhC -> Overview Domain evolution and history4.1

HhC components and evolution


> 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

>

HhC -> Overview Function4.1

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)

>

HhC -> Structural conformation SCOP Classification4.2


SCOP Classification

Lineage:

1. Root: scop

2. Class: Alll beta proteins

3. Fold: Hedgehog/intein (Hint) DomainComplex fold made of five beta-hairpin units and a b-ribbon arc

Superfamilies:

1. Superfamily: Hedgehog/intein (Hint) domain

Families:

• Hedgehog, C-terminal (Hog) signaling domain


Structural conformation (homologues)

SCOP Classification

Lineage:

1. Root: scop

2. Class: Alll beta proteins

3. Fold: Hedgehog/intein (Hint) DomainComplex fold made of five beta-hairpin units and a b-ribbon arc

Superfamilies:

1. Superfamily: Hedgehog/intein (Hint) DomainDuplicated (Contaiins interwined structural repeats

Families:

• Intein (protein splicing domain)


> Several aminoacids crucial for the function

Structural conformation (conserved aminoacids)

4.2

> Cys 258 (in protein Hehgehog Drosophila


Does the first nucleophilic atack, crucial for autocleavage

HhC -> Structural conformation Aminoacid conservation

4.2

> His 329 (in protein Hehgehog Drosophila


Essencial for autocleavage


4.2

> Thr 326 (in protein Hehgehog Drosophila


Involved in autocleavage



4.2

> Thr 303 (in protein Hehgehog Drosophila


Involved in cholesterol transfer

HhC -> Structural homologs Inteins4.3

> 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.

Hedgehog Hint is homolog to Inteins


> Example: Protein Hedgehog HhC of drosophila


> Example: Mtu recA intein splicing domain


> The proteins are structurally very similar


> Orange Protein Hedgehog

> Cyan Intein domain


> 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

HhC -> Other HhC homologs Other homologs4.4

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.

QUESTIONS


5


1. INTRODUCTION

5. QUESTIONS


HEDGEHOG PROTEIN FAMILY Structural Bioinformatics Oriol Senan Alexandros Pittis Amadís Pagès

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