Limits...
Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

Alqahtani F, Mahdavi J, Wheldon LM, Vassey M, Pirinccioglu N, Royer PJ, Qarani SM, Morroll S, Stoof J, Holliday ND, Teo SY, Oldfield NJ, Wooldridge KG, Ala'Aldeen DA - Open Biol (2014)

Bottom Line: Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization.Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE.This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

Show MeSH

Related in: MedlinePlus

Molecular modelling of 37LRP interaction with galectin-3. (a) Ribbon diagram of the heterodimer interaction of 37LRP with (a) lactose-liganded or (b) non-lactose-liganded Gal-3. Residues involved in the hypothetical large protein–protein interface are represented with sticks. (c) Ribbon diagram with superimposed molecular surface of the homodimer interaction of 37LRP (red) with a second 37LRP (cyan) as proposed by Jamieson et al. [18]. (d) Fluorescently labelled recombinant Gal-3 and 37LRP were detected in transiently co-transfected COS7 cells via vYFP or mCherry tags. Cells were fixed at 24 h with 4% paraformaldehyde without permeabilization and stained with Hoechst.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4221890&req=5

RSOB140053F1: Molecular modelling of 37LRP interaction with galectin-3. (a) Ribbon diagram of the heterodimer interaction of 37LRP with (a) lactose-liganded or (b) non-lactose-liganded Gal-3. Residues involved in the hypothetical large protein–protein interface are represented with sticks. (c) Ribbon diagram with superimposed molecular surface of the homodimer interaction of 37LRP (red) with a second 37LRP (cyan) as proposed by Jamieson et al. [18]. (d) Fluorescently labelled recombinant Gal-3 and 37LRP were detected in transiently co-transfected COS7 cells via vYFP or mCherry tags. Cells were fixed at 24 h with 4% paraformaldehyde without permeabilization and stained with Hoechst.

Mentions: The proposed interaction of 37LRP and Gal-3 was investigated using the ZDOCK server (zdock.umassmed.edu) and their docking compared with the previously proposed interaction of two 37LRP molecules forming a homodimer [18]. ZDOCK has been reported to achieve high predictive accuracy on protein–protein docking benchmarks for rigid-body cases and consistent success in the international protein–protein docking experiment [37]. The model revealed a large protein–protein interface between the 37LRP and Gal-3 subunits, involving several prominent 37LRP residues, mainly through salt bridges and hydrogen bonds (figure 1). In the proposed complex between 37LRP and lactose-associated Gal-3 (figure 1a), four salt bridges are apparent. They are K166 of 37LRP with D178 and D154 of Gal-3, R80 of 37LRP with D178 of Gal-3, D151 of 37LRP with R151 and K227 of Gal-3, and D44 of 37LRP with K233 of Gal-3. Hydrogen bonds between N81 of 37LRP and N180 of Gal-3, N29 of 37LRP and K233 and E230 of Gal-3, and Y28 of 37LPR and E230 of Gal-3 are also involved in the interaction (figure 1a). A similar interaction mode occurs between 37LRP and non-liganded Gal-3 (figure 1b). Here, K166 in 37LRP also interacts with D178 and D154 of Gal-3, and S233 of Gal-3 forms a hydrogen bond with D44 in 37LRP.Figure 1.


Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

Alqahtani F, Mahdavi J, Wheldon LM, Vassey M, Pirinccioglu N, Royer PJ, Qarani SM, Morroll S, Stoof J, Holliday ND, Teo SY, Oldfield NJ, Wooldridge KG, Ala'Aldeen DA - Open Biol (2014)

Molecular modelling of 37LRP interaction with galectin-3. (a) Ribbon diagram of the heterodimer interaction of 37LRP with (a) lactose-liganded or (b) non-lactose-liganded Gal-3. Residues involved in the hypothetical large protein–protein interface are represented with sticks. (c) Ribbon diagram with superimposed molecular surface of the homodimer interaction of 37LRP (red) with a second 37LRP (cyan) as proposed by Jamieson et al. [18]. (d) Fluorescently labelled recombinant Gal-3 and 37LRP were detected in transiently co-transfected COS7 cells via vYFP or mCherry tags. Cells were fixed at 24 h with 4% paraformaldehyde without permeabilization and stained with Hoechst.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221890&req=5

RSOB140053F1: Molecular modelling of 37LRP interaction with galectin-3. (a) Ribbon diagram of the heterodimer interaction of 37LRP with (a) lactose-liganded or (b) non-lactose-liganded Gal-3. Residues involved in the hypothetical large protein–protein interface are represented with sticks. (c) Ribbon diagram with superimposed molecular surface of the homodimer interaction of 37LRP (red) with a second 37LRP (cyan) as proposed by Jamieson et al. [18]. (d) Fluorescently labelled recombinant Gal-3 and 37LRP were detected in transiently co-transfected COS7 cells via vYFP or mCherry tags. Cells were fixed at 24 h with 4% paraformaldehyde without permeabilization and stained with Hoechst.
Mentions: The proposed interaction of 37LRP and Gal-3 was investigated using the ZDOCK server (zdock.umassmed.edu) and their docking compared with the previously proposed interaction of two 37LRP molecules forming a homodimer [18]. ZDOCK has been reported to achieve high predictive accuracy on protein–protein docking benchmarks for rigid-body cases and consistent success in the international protein–protein docking experiment [37]. The model revealed a large protein–protein interface between the 37LRP and Gal-3 subunits, involving several prominent 37LRP residues, mainly through salt bridges and hydrogen bonds (figure 1). In the proposed complex between 37LRP and lactose-associated Gal-3 (figure 1a), four salt bridges are apparent. They are K166 of 37LRP with D178 and D154 of Gal-3, R80 of 37LRP with D178 of Gal-3, D151 of 37LRP with R151 and K227 of Gal-3, and D44 of 37LRP with K233 of Gal-3. Hydrogen bonds between N81 of 37LRP and N180 of Gal-3, N29 of 37LRP and K233 and E230 of Gal-3, and Y28 of 37LPR and E230 of Gal-3 are also involved in the interaction (figure 1a). A similar interaction mode occurs between 37LRP and non-liganded Gal-3 (figure 1b). Here, K166 in 37LRP also interacts with D178 and D154 of Gal-3, and S233 of Gal-3 forms a hydrogen bond with D44 in 37LRP.Figure 1.

Bottom Line: Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization.Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE.This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

Show MeSH
Related in: MedlinePlus