SH2 domain

Nuria BonifaciFrancesc Estanyol

Structural BioInformaticsBIOINFO, 2007

INDEX

• Introduction

• General traits of SH2 domain

• Proteins with SH2 domain

• Study of conservation– Sequence – Structure – Phylogenetic

Why are they called SH2?

• 1911 Peyton Rous discovers Sarcoma Rous Virus

• Tyrosine Kinase v-Src

• 3 domains:– Src homolog 1 (Kinase)– Src homolog 2 (SH2)– Src homolog 3 (SH3)

gag pol env V-src

INDEX

• Introduction

• General traits of SH2 domain

• Proteins with SH2 domain

• Study of conservation– Sequence – Structure – Phylogenetic

General traits of SH2 domain

3 beta strands

2 alpha helix

Protein-protein binding on PTyr

SH2 domain

Classification

• Class α/β

• Fold: SH2-like

• Superfamily: SH2

• Family: SH2

INDEX

• Introduction

• General traits of SH2 domain

• Proteins with SH2 domain

• Study of conservation– Sequence – Structure – Phylogenetic

Proteins with SH2 domain

Find the protein function:

-Kinases: Brk, Chk, Csk, Fyn, Hck, p55 (Blk), p56(lck), Pl3Kinase, Proto. oncogenTK, Src, Syk, Zap-70

- Phosphatases: Shp2, Syp

- Adapters: Aps, Crk Proto-oncogen, Gbr2, Gbr7, Gbr10, Mona

- Dna Bindings: Cbl, STAT1,STAT3b, STAThomolog

- Others: Eat2, PLCgamma, Sap

Types of proteins with SH2

INDEX

• Introduction

• General traits of SH2 domain

• Proteins with SH2 domain

• Study of conservation– Sequence – Structure – Phylogenetic

Sequence conservation

• ClustalW Multiple sequence alignment between the kinases

– Low conservation of the residues– High conservation of the characteristics

Arg

pTyr

Phosphate

Nitrogen (ARG)

3,45A

Polar pocket

But why one SH2 domain does not bind to any PTyr ?

Hydrophobic pocket

Importance of the hydrophobic pocket

Thr 215

PTyr-X-Asn

Src Kinase

Trp

Ptyr- Val- Asn- Val

Grb2

INDEX

• Introduction

• General traits of SH2 domain

• Proteins with SH2 domain

• Study of conservation– Sequence – Structure – Phylogenetic

Structural alignment using STAMP

Reading in matrix file kinases.mat...Doing cluster analysis...Cluster: 1 ( hck & lck ) Sc 7.90 RMS 1.01 Len 105 nfit 96 See file kinases.1 for the alignment and transformationsCluster: 2 ( blk & hck lck ) Sc 8.40 RMS 1.32 Len 108 nfit 96 See file kinases.2 for the alignment and transformationsCluster: 3 ( cskk & blk hck lck ) Sc 7.96 RMS 0.95 Len 107 nfit 85 See file kinases.3 for the alignment and transformationsCluster: 4 ( brk & cskk blk hck lck ) Sc 7.97 RMS 1.31 Len 106 nfit 84 See file kinases.4 for the alignment and transformationsCluster: 5 ( potk & brk cskk blk hck lck ) Sc 2.11 RMS 3.52 Len 163 nfit 24 See file kinases.5 for the alignment and transformationsCluster: 6 ( itk & potk brk cskk blk hck lck ) Sc 2.74 RMS 4.67 Len 187 nfit 25 See file kinases.6 for the alignment and transformationsCluster: 7 ( syk & itk potk brk cskk blk hck lck ) Sc 5.44 RMS 2.11 Len 191 nfit 24 See file kinases.7 for the alignment and transformationsCluster: 8 ( pi3k & syk itk potk brk cskk blk hck lck ) Sc 2.55 RMS 2.50 Len 245 nfit 13 See file kinases.8 for the alignment and transformationsCluster: 9 ( fyn & pi3k syk itk potk brk cskk blk hck lck ) Sc 3.05 RMS 1.71 Len 289 nfit 16 See file kinases.9 for the alignment and transformationsCluster: 10 ( zap & fyn pi3k syk itk potk brk cskk blk hck

lck ) Sc 0.31 RMS 100.00 Len 661 nfit 1 LOW SCORE See file kinases.10 for the alignment and transformations

With the hole protein !!!

The solution

• Cut manually ALL the PDB files

selecting only the

SH2 domain ???

A LOT OF WORK!!!

We used our bioinformatic knowledge!!!

• Do a Phyton script to cut the PDB files given a starting and ending residue numbers

• Get the FASTA sequences of the resulting PDBs

But…where we can find the start/end residues?

http://sanger.ac.uk/software/Pfam

cutPDB.py

Aminoacid2simbol function

NO LOW SCORES!!!

Doing cluster analysis...Cluster: 1 ( lck & src ) Sc 9.14 RMS 0.80 Len 83 nfit 80 See file sh2.1 for the alignment and transformationsCluster: 2 ( zap & zap2 ) Sc 8.94 RMS 1.02 Len 78 nfit 75 See file sh2.2 for the alignment and transformationsCluster: 3 ( cskk & zap zap2 ) Sc 8.88 RMS 0.85 Len 79 nfit 65 See file sh2.3 for the alignment and transformationsCluster: 4 ( syk & cskk zap zap2 ) Sc 8.57 RMS 1.17 Len 80 nfit 63 See file sh2.4 for the alignment and transformationsCluster: 5 ( lck src & syk cskk zap zap2 ) Sc 8.52 RMS 0.82 Len 85 nfit 61 See file sh2.5 for the alignment and transformationsCluster: 6 ( itk & lck src syk cskk zap zap2 ) Sc 8.07 RMS 1.25 Len 89 nfit 60 See file sh2.6 for the alignment and transformationsCluster: 7 ( fyn & itk lck src syk cskk

zap zap2 ) Sc 6.05 RMS 1.07 Len 89 nfit 63 See file sh2.7 for the alignment and transformations

Scstructural similarity

funcional and/or

evolutionary relationships

Pij

5.5-9.8 high almost always greater than 6.0 genuine

2.5 - 5.5 more distantlydo not always

imply4.0 - 6.0 50%

less than 2.0 little overall less than 4.0 no

Doing cluster analysis...Cluster: 1 ( lck & src ) Sc 9.14 RMS 0.80 Len 83 nfit 80 See file sh2.1 for the alignment and transformationsCluster: 2 ( zap & zap2 ) Sc 8.94 RMS 1.02 Len 78 nfit 75 See file sh2.2 for the alignment and transformationsCluster: 3 ( cskk & zap zap2 ) Sc 8.88 RMS 0.85 Len 79 nfit 65 See file sh2.3 for the alignment and transformationsCluster: 4 ( syk & cskk zap zap2 ) Sc 8.57 RMS 1.17 Len 80 nfit 63 See file sh2.4 for the alignment and transformationsCluster: 5 ( lck src & syk cskk zap zap2 ) Sc 8.52 RMS 0.82 Len 85 nfit 61 See file sh2.5 for the alignment and transformationsCluster: 6 ( itk & lck src syk cskk zap zap2 ) Sc 8.07 RMS 1.25 Len 89 nfit 60 See file sh2.6 for the alignment and transformationsCluster: 7 ( fyn & itk lck src syk cskk

zap zap2 ) Sc 6.05 RMS 1.07 Len 89 nfit 63 See file sh2.7 for the alignment and transformations

Structural alignment

Secondary structure highly conserverd

High variability in the loops

Structural alignment of all the SH2 domains

Rmsd = 1,68

Sc= 3,27

Advanced options of STAMP

- Guide the program to do the superposition giving an initial alignment.– 1-. ClustalW of all the sequences.– 2-. > alignfit – f sh2.align –d sh2.domains –out sh2.trans

> stamp –l sh2.trans –prefix sh2Stamp

We can’t do it :- alignfit ?

- other .trans files didn’t work.

INDEX

• Introduction

• General traits of SH2 domain

• Proteins with SH2 domain

• Study of conservation– Sequence – Structure – Phylogenetic

Phylogenetic analysis

• Compare the same protein between species (c-src)

Good alignment !!!

Is the Arginin conserved through the evolution ?

Which Arginin is the one that interacts directly with pTyr ?

The Arginin is very conserved between the species due to its critical function

References - Bradshaw JM, Waksman G

Molecular Recognition by SH2 domains

- Bhushan Nagar, Oliver Hantschel, Markus Seeliger, Jason M. Davies, William I. Weis, Giulio Superti-Furga and John Kuriyan

Organization of the SH3-SH2 Unit in Active and Inactive Forms of the c-Abl Tyrosine Kinase

- The SRC kinases. http://jkweb.mcb.berkeley.edu (Berkeley Univeristy)

That’s All

Any questions?