3D 3D- -Str Str Struct ucture re Pr Predict ction on of of th the 3D 3D Struct ucture re Pr Predict ction on of of th the Mo Modul ular ar Pr Prot otei ein Sialoa oadhe hesin Mo Modul ular ar Pr Prot otei ein Sialoa oadhe hesin Using sing a Mu Multi- -ste ste step Mo Modelling ng Using sing a Mu Multi step Mo Modelling ng Str Strate ategy Str Strate ategy Dario CORRADA 1 , Pasqualina D’URSI 1,2 , Sara BOTTI 3 , Aldo LUPERINI 4 , Luciano MILANESI 1 , Ermanna ROVIDA 1 1 Institute for Biomedical Technologies – National Research Council (ITB-CNR) 2 Consorzio Interuniversitario Lombardo per l'Elaborazione Automatica (CILEA) 3 Parco Tecnologico Padano – Centro Ricerche e Studi Agroalimentari (PTP-CERSA) 4 ENEA TER ENE-SIST
SIGLECs SIGLECs SIalic acid immuno alic acid immunoGL GLobulin obulin LEC LECtins tins SI Type I transmembrane • glycoproteins • Belong to immunoglobulin (Ig) superfamily lectins • Recognize glycans containing sialic acids • 12-14 isoforms N-terminal V-set Ig-like domain, • variable numbers (1~16) of C2- set Ig-like domains Human SIGLECs
Sialoadhesin Sialoadhesin (Siglec- -1, Sn) 1, Sn) (Siglec Specifically expressed on tissue macrophages Endocytic receptor for PRRSV Potential mediator of the Protein/protein interaction ? trans-infection of T-cells by HIV-1 Short cytoplasmic tail, no direct internalization
Sialoadhesin Sialoadhesin • ~ 1600 residues • 17 Ig-like domains • High sequence identity among mammals (over 70% ) • Crystallographic structures avalaible only for N-term domain
Ig- -like domains like domains Ig C2-set I-set Constant Constant, between C’ strand C2-set e V-set D strand C’ strand D strand V-set C1-set Variable Constant C’ and C’’ strand often C’ strand part of GFC b-sheet Bork P, The Immunoglobulin Fold: Strctural D strand Variable position of A Classification, Sequence Patterns and Common Core – J. Mol. Biol., (1994) 242 309-320 strand
Internal alignments Internal alignments Crocker et al. found similarity in length and sequence between even and odd domains of murine Sn; he proposed that sialoadhesin is composed of tandem homologous pairs of domains. Crocker PR, Sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains – EMBO J., (1994) 13 4490-4503 Pig sialoadhesin PIN motif
PIN motif PIN motif • Sequence pattern: C-m-[LFYW]-n-C • Tryptophan packs against the disulfide bridge • Distances between conserved residues are variable: C ↔ C C ↔ W (m) W ↔ C (n) C1-set 54-64 ~13 40-50 C2-set 38-43 7-17 25-30 I-set 44-51 7-17 31-40 Smith DK, Sequence profiles of immunoglobulin and immunoglobulin-like domains – J. Mol. Biol., (1997) 274 530-545
In a project aimed to investigate the genetic components of pig natural immunity segregating in commercial herd, the genetic variability of Sn was studied 35 variants in Sn coding regions
Aims Aims To characterize the spatial position of the mutated residues and to gain new insight into functional aspects of the protein Modelling project to predict the structure of the protein
Protein Sequence Domains sequence Domain Assignment Secondary Structure Prediction BLASTp Search Protein Data Restraint list Bank Ensemble Model Sequence Alignment 3D Models NO Homologue Threading in PDB? AB BC Superposition YES CD Alignment of … Homology Modelling Sequence to Structure
Protein Sequence Secondary Structure Template Search Prediction Alignment of Restraints Sequence to List Structure Model Building AB BC CD … Superpose and Fit Ensemble Models Model
Secondary Structure Secondary Structure Profile Profile Cluster of C-set domains (res 142-1643) Jpred PHDsec Jpred PHDsec SSpro SSpro PROF PROF PSIpred PSIpred Consensus Secondary Structure Prediction Consensus Secondary Structure Prediction
Secondary Structure Secondary Structure Profile Profile Additional PFAM/SMART Additional PFAM/SMART Strands annotation Strands annotation Strands not Strands not well defined well defined in the profile in the profile
Protein Sequence Secondary Structure Template Search Prediction Alignment of Restraints Sequence to List Structure Model Building AB BC CD … Superpose and Fit Ensemble Models Model
Template Search Template Search Sn sequence Sn sequence Threading Threading NO SWTVSSPETVQGIKGS CLIIPCTFGFPANVEVP IWYYDYSGKRLVVSHS VENHFQGRALLLGQVE SnD03- SnD03 -04 04 … SnD05- -06 06 SnD05 SnD07- SnD07 -08 08 BLASTp BLASTp Eval. ≤ 10-5 %Id > 30 Protein Protein Data Data Homology Homology Bank Bank Modelling Modelling YES SnD01- -02 02 SnD01 No structures available to cover the full-length sequence … searching for portion of sequence
SnD01/02 Model SnD01/02 Model Pig Sialoadhesin Pig Sialoadhesin Ig like Ig like Ig like Ig like N- N -term term C- C -term term V- -set set C- -set set Among all SIGLECs 2ZG3* is the V C only X-ray structure which has a Templates Ig-like domain besides the N- terminal one (Ig-like V-set) Mouse Sialoadhesin (1QFO) 66.7 %id Mouse Sialoadhesin (1QFO) Ig like Ig like N N- -term term C C- -term term V- -set set V Human SIGLEC- Human SIGLEC -5 (2ZG3) 5 (2ZG3) 28.8 %id Ig like Ig like Ig like Ig like N- -term term C- -term term N C V- V -set set C- C -set set * Zhuravleva MA, Structral implication of siglec-5-mediated sialoglycan recognition – J. Mol. Biol., (2008) 375 437-447
SnD01/02 Model SnD01/02 Model SnD02 Ig-like C2-set type SIGLEC-5 D02 ICAM-2 D02 Ig-like C2-set type Ig-like C2-set type Template Search Template Search -strand C’ ?
SnD01/02 Model SnD01/02 Model Pig Sialoadhesin Pig Sialoadhesin Ig like Ig like Ig like Ig like N N- -term term C- C -term term V- -set set C- -set set V C Templates Mouse Sialoadhesin (1QFO) Mouse Sialoadhesin (1QFO) Ig like Ig like N- N -term term C- C -term term V- -set set V Human SIGLEC- -5 (2ZG3) 5 (2ZG3) Human SIGLEC Ig like Ig like Ig like Ig like N N- -term term C C- -term term V- -set set C- -set set V C Human ICAM- -2 (1ZXQ) 2 (1ZXQ) Human ICAM Ig like Ig like N- -term term C- -term term N C C- C -set set
Threading Threading Query Seq Query Seq Alignment Alignment SWTVSSPETVQGIKGS >P1;5fd1 CLIIPCTFGFPANVEVP structureX IWYYDYSGKRLVVSHS AFVVTDNCIKCKVGPN* VENHFQGRALLLGQVE >P1;1fdx … sequence AYVINDSC--IAS—IYA* GenThreader SAM-T02 3D Jury 3D-PSSM Rank FUGUE INUB
Threading Results Threading Results Top Rank Top Rank Query Sequences Query Sequences Templates Templates SnD3 SnD4 SnD5 SnD6 SnD7 SnD8 SnD3 SnD4 SnD5 SnD6 SnD7 SnD8 PDB a SnD6 SnD6 SnD7 SnD7 SnD8 SnD8 PDB b SnD3 SnD3 SnD4 SnD4 SnD5 SnD5 PDB c SnD3 SnD3 SnD4 SnD4 SnD3 SnD4 PDB d SnD3 SnD4 NO gaps NO gaps inside inside SnD5 SnD5 predicted predicted SnD5 SnD5 SnD6 SnD6 PDB e SnD6 SnD6 strands strands SnD7 SnD7 SnD7 SnD8 PDB f SnD7 SnD8 SnD8 SnD8
Templates Templates QUERY SEQUENCE METHOD TEMPLATE 1QFO Mouse sialoadhesin Homology SnD01/SnD02 modelling 2ZG3 Human SIGLEC-5 Human Fc- γ receptor SnD03/SnD04 Threading 1E4J 2C5D Human TYR03 receptor SnD05/SnD06 Threading 1RHF Human TYR03 receptor 2C5D Human TYR03 receptor SnD07/SnD08 Threading 1RHF Human TYR03 receptor
Protein Sequence Secondary Structure Template Search Prediction Alignment of Restraints Sequence to List Structure Model Building AB BC CD … Superpose and Fit Ensemble Models Model
Model Building Model Building MODELLER 9 >P1;5fd1 structureX TDNCIKCKVG…* >P1;1fdx sequence NDSC--IAS—I…* Raw Validation Loop (ProCheck) Job Refinement 29-32 strand 37;167 disulfide 38-44 strand … DOPE Ranking DOPE Ranking DOPE Ranking DOPE Ranking Model01 -20107 Model001 -19903 Model02 -19869 Model002 -19716 Model003 -19801 Model003 -19649 … … … … Model50 -18679 Model250 -18756
Energy Profiles Energy Profiles SnD01/02 SnD01/02 SnD03/04 SnD03/04 SnD05/06 SnD05/06 SnD07/08 SnD07/08 Sippl MJ, Recognition of Errors in Three-Dimensional ProSa scoring function ProSa scoring function Structures of Proteins – Proteins (1993) 17 355-362
Structure Assesment Structure Assesment SnD01/02 SnD03/04 SnD01/02 SnD03/04 SnD05/06 SnD07/08 SnD05/06 SnD07/08 Not allowed residues correspond to position at boundaries of loop regions
Protein Sequence Secondary Structure Template Search Prediction Alignment of Restraints Sequence to List Structure Model Building AB BC CD … Superpose and Fit Ensemble Models Model
Joining The Models Joining The Models electron microscopy studies show that mouse Sn doesn’t have a linear arrangement of domains but exhibits an irregular shape Crocker PR, Sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains – EMBO J., (1994) 13 4490-4503 SnD domains could cluster SnD domains could cluster together into a complex folding together into a complex folding How the models could be How the models could be spatially oriented each other? spatially oriented each other?
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