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Dario CORRADA1, Pasqualina D’URSI1,2, Sara BOTTI3, Aldo LUPERINI4, Luciano MILANESI1, Ermanna ROVIDA1

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

• 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

SI SIalic acid immuno alic acid immunoGL GLobulin

• bulin LEC

LECtins tins

Human SIGLECs

Sialoadhesin Sialoadhesin

 Specifically expressed on tissue macrophages  Endocytic receptor for PRRSV  Potential mediator of the trans-infection of T-cells by HIV-1  Short cytoplasmic tail, no direct internalization

(Siglec (Siglec-

• 1, Sn)

1, Sn)

Protein/protein interaction?

Sialoadhesin Sialoadhesin

• ~ 1600 residues
• 17 Ig-like domains
• High sequence identity among mammals (over 70% )
• Crystallographic structures avalaible only for N-term domain

Ig Ig-

• like domains

like domains

Bork P, The Immunoglobulin Fold: Strctural Classification, Sequence Patterns and Common Core – J. Mol. Biol., (1994) 242 309-320

V-set

Variable C’ and C’’ strand often part of GFC b-sheet Variable position of A strand

C1-set

Constant  C’ strand  D strand

C2-set

Constant  C’ strand  D strand

I-set

Constant, between C2-set e V-set  C’ strand  D 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:

44-51 38-43 54-64

C↔C

31-40 7-17

I-set

25-30 7-17

C2-set

40-50 ~13

C1-set W↔C (n) C↔W (m) 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

BLASTp Search Sequence Alignment Secondary Structure Prediction Restraint list Homology Modelling Alignment of Sequence to Structure

Protein Sequence

Protein Data Bank Homologue in PDB? Threading

3D Models

Domain Assignment Domains sequence AB BC … CD Superposition

Ensemble Model

NO YES

Protein Sequence

Secondary Structure Prediction Template Search

Model Building

Restraints List Alignment of Sequence to Structure

AB Ensemble Model BC CD … Superpose and Fit Models

Secondary Structure Secondary Structure Profile Profile

Jpred Jpred PSIpred PSIpred PROF PROF PHDsec PHDsec SSpro SSpro

Cluster of C-set domains (res 142-1643)

Consensus Secondary Structure Prediction Consensus Secondary Structure Prediction

Secondary Structure Secondary Structure Profile Profile

PFAM/SMART annotation PFAM/SMART annotation Additional Strands Additional Strands Strands not well defined in the profile Strands not well defined in the profile

Protein Sequence

Secondary Structure Prediction Template Search

Model Building

Restraints List Alignment of Sequence to Structure

AB Ensemble Model BC CD … Superpose and Fit Models

Template Search Template Search

Sn sequence Sn sequence

SWTVSSPETVQGIKGS CLIIPCTFGFPANVEVP IWYYDYSGKRLVVSHS VENHFQGRALLLGQVE

…

• Eval. ≤ 10-5

%Id > 30 BLASTp BLASTp

Protein Protein Data Data Bank Bank

No structures available to cover the full-length sequence

Homology Homology Modelling Modelling

SnD01 SnD01-

• 02

02

Threading Threading

SnD03 SnD03-

• 04

04 SnD05 SnD05-

• 06

06 SnD07 SnD07-

• 08

08

… searching for portion of sequence

NO YES

Templates

SnD01/02 Model SnD01/02 Model

Ig like Ig like V V-

• set

set Ig like Ig like C C-

• set

set N N-

• term

term C C-

• term

term

Pig Sialoadhesin Pig Sialoadhesin

Ig like Ig like V V-

• set

set N N-

• term

term C C-

• term

term

Mouse Sialoadhesin (1QFO) Mouse Sialoadhesin (1QFO) 66.7 %id

Ig like Ig like V V-

• set

set Ig like Ig like C C-

• set

set N N-

• term

term C C-

• term

term

Human SIGLEC Human SIGLEC-

• 5 (2ZG3)

5 (2ZG3) 28.8 %id

Among all SIGLECs 2ZG3* is the

• nly X-ray structure which has a

Ig-like domain besides the N- terminal one (Ig-like V-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 Ig-like C2-set type -strand C’ ? Template Search Template Search ICAM-2 D02 Ig-like C2-set type

SnD01/02 Model SnD01/02 Model

Templates

Ig like Ig like V V-

• set

set Ig like Ig like C C-

• set

set N N-

• term

term C C-

• term

term

Pig Sialoadhesin Pig Sialoadhesin

Ig like Ig like V V-

• set

set N N-

• term

term C C-

• term

term

Mouse Sialoadhesin (1QFO) Mouse Sialoadhesin (1QFO)

Ig like Ig like V V-

• set

set Ig like Ig like C C-

• set

set N N-

• term

term C C-

• term

term

Human SIGLEC Human SIGLEC-

• 5 (2ZG3)

5 (2ZG3)

Ig like Ig like C C-

• set

set N N-

• term

term C C-

• term

term

Human ICAM Human ICAM-

• 2 (1ZXQ)

2 (1ZXQ)

Threading Threading

Query Seq Query Seq

SWTVSSPETVQGIKGS CLIIPCTFGFPANVEVP IWYYDYSGKRLVVSHS VENHFQGRALLLGQVE …

GenThreader

Alignment Alignment

>P1;5fd1 structureX AFVVTDNCIKCKVGPN* >P1;1fdx sequence AYVINDSC--IAS—IYA*

3D Jury Rank INUB SAM-T02 3D-PSSM FUGUE

SnD4 SnD4 SnD5 SnD5 SnD3 SnD3 SnD8 SnD8 SnD6 SnD6 SnD7 SnD7 SnD4 SnD4 SnD3 SnD3 SnD5 SnD5 SnD6 SnD6 SnD8 SnD8 SnD7 SnD7 SnD8 SnD8 SnD4 SnD4 SnD5 SnD5 SnD6 SnD6 SnD7 SnD7 SnD3 SnD3 SnD8 SnD8 SnD4 SnD4 SnD5 SnD5 SnD6 SnD6 SnD7 SnD7 SnD3 SnD3

PDB a PDB b PDB c PDB d PDB e PDB f Query Sequences Query Sequences Top Rank Top Rank Templates Templates

Threading Results Threading Results

NO gaps NO gaps inside inside predicted predicted strands strands

Templates Templates

1RHF 2C5D 1RHF 2C5D 1E4J 2ZG3 1QFO

TEMPLATE

Human TYR03 receptor Human TYR03 receptor

Threading SnD07/SnD08

Human TYR03 receptor Human TYR03 receptor

Threading SnD05/SnD06

Human Fc-γ receptor

Threading SnD03/SnD04

Human SIGLEC-5 Mouse sialoadhesin

Homology modelling SnD01/SnD02 METHOD QUERY SEQUENCE

Protein Sequence

Secondary Structure Prediction Template Search

Model Building

Restraints List Alignment of Sequence to Structure

AB Ensemble Model BC CD … Superpose and Fit Models

Model Building Model Building

• 18756

Model250 … …

• 19649

Model003

• 19716

Model002

• 19903

Model001

29-32 strand 37;167 disulfide 38-44 strand … >P1;5fd1 structureX TDNCIKCKVG…* >P1;1fdx sequence NDSC--IAS—I…*

• 18679

Model50 … …

• 19801

Model003

• 19869

Model02

• 20107

Model01

Raw Job

DOPE Ranking DOPE Ranking

Loop Refinement

DOPE Ranking DOPE Ranking

Validation (ProCheck)

MODELLER 9

Energy Profiles Energy Profiles

SnD01/02 SnD01/02 SnD05/06 SnD05/06 SnD03/04 SnD03/04 SnD07/08 SnD07/08

Sippl MJ, Recognition of Errors in Three-Dimensional Structures of Proteins – Proteins (1993) 17 355-362

ProSa scoring function ProSa scoring function

Structure Assesment Structure Assesment

SnD01/02 SnD01/02 SnD03/04 SnD03/04 SnD05/06 SnD05/06

Not allowed residues correspond to position at boundaries of loop regions

SnD07/08 SnD07/08

Protein Sequence

Secondary Structure Prediction Template Search

Model Building

Restraints List Alignment of Sequence to Structure

AB Ensemble Model BC CD … Superpose and Fit Models

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

How the models could be How the models could be spatially oriented each other? spatially oriented each other?

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

Joining The Models Joining The Models

Domains C/D Domains C/D

Nterm Nterm-

• YSGKRL

YSGKRLVV…

Domains B/C Domains B/C

…EVPHGITYSGKRL PHGITYSGKRLVV…

Domains A/B Domains A/B

…EVPHGIT PHGIT-

• Cterm

Cterm HINGE REGION HINGE REGION PHGITYSGKRL PHGITYSGKRL A/B …EVPHGIT--------… HINGE …--PHGITYSGKRL--… C/D …-------YSGKRLVV…

Model SnD01/02 Model SnD05/06 Hinge SnD06/07 Model SnD07/08 Hinge SnD04/05 Model SnD03/04 Hinge SnD02/03

Variants Variants’ ’ Position Position

The ensemble model covers The ensemble model covers 15/35 15/35 total variants. total variants.

V141M G240D R293H P402R A468T N579S T583A L552I A526V L547R Q525R H661G G524R V680A R641H

Surface Analysis Surface Analysis

R641H G240D P402R L547R G524R Q525R V680A

7 variants 7 variants are facing the putative are facing the putative (predicted) (predicted) cavities cavities

G240D G240D

 Across SnD02 and SnD03  Aspartic introduces a negative charge  Mutation may introduce salt bridge with lysine K239?

K239 K239 G240D G240D

P402R P402R

 Set on top of the hydrophobic interface between SnD03 and SnD04  It introduces a positive charged bulky side chain  It reduces accessibility to the cleft

L547R L547R

 Nearby the structural core of SnD06 (strand C)  It introduces a positive charge  It reduces accessibility to the cleft

Conclusions Conclusions

 The ensemble model suggests hints of Sialoadhesin’s overall fold and surface properties. Some variants are likely to alter protein surfaces in terms of steric hindrance and electrostatic properties.

These evidences may drive experimental work for mapping regions involved in protein/protein interactions. Interpretation of the possible effects of mutations should be confirmed in vivo or in vitro data. This would help to correlate alterations of biological activity with the structural consequences of the variation.

Acknowledgements Acknowledgements

This work has been supported by:

• EGEE-III Enabling Grids for E-sciencE (INFSO-

RI-222667)

• Interdepartmental CNR-BIOINFORMATICS

network, FIRB LITBIO (RBLA0332RH) and (RBPR05ZK2Z)

Thank you for the attention