Limits...
Distantly related lipocalins share two conserved clusters of hydrophobic residues: use in homology modeling.

Adam B, Charloteaux B, Beaufays J, Vanhamme L, Godfroid E, Brasseur R, Lins L - BMC Struct. Biol. (2008)

Bottom Line: Under the 30% identity threshold, alignment methods do not correctly assign and align proteins.The only safe way to assign a sequence to that family is by experimental determination.This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Biophysique Moléculaire et Numérique, Faculté Universitaire des Sciences Agronomiques de Gembloux, Gembloux, Belgium. adam.b@fsagx.ac.be

ABSTRACT

Background: Lipocalins are widely distributed in nature and are found in bacteria, plants, arthropoda and vertebra. In hematophagous arthropods, they are implicated in the successful accomplishment of the blood meal, interfering with platelet aggregation, blood coagulation and inflammation and in the transmission of disease parasites such as Trypanosoma cruzi and Borrelia burgdorferi. The pairwise sequence identity is low among this family, often below 30%, despite a well conserved tertiary structure. Under the 30% identity threshold, alignment methods do not correctly assign and align proteins. The only safe way to assign a sequence to that family is by experimental determination. However, these procedures are long and costly and cannot always be applied. A way to circumvent the experimental approach is sequence and structure analyze. To further help in that task, the residues implicated in the stabilisation of the lipocalin fold were determined. This was done by analyzing the conserved interactions for ten lipocalins having a maximum pairwise identity of 28% and various functions.

Results: It was determined that two hydrophobic clusters of residues are conserved by analysing the ten lipocalin structures and sequences. One cluster is internal to the barrel, involving all strands and the 310 helix. The other is external, involving four strands and the helix lying parallel to the barrel surface. These clusters are also present in RaHBP2, a unusual "outlier" lipocalin from tick Rhipicephalus appendiculatus. This information was used to assess assignment of LIR2 a protein from Ixodes ricinus and to build a 3D model that helps to predict function. FTIR data support the lipocalin fold for this protein.

Conclusion: By sequence and structural analyzes, two conserved clusters of hydrophobic residues in interactions have been identified in lipocalins. Since the residues implicated are not conserved for function, they should provide the minimal subset necessary to confer the lipocalin fold. This information has been used to assign LIR2 to lipocalins and to investigate its structure/function relationship. This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins.

Show MeSH

Related in: MedlinePlus

ClustalW (A) and refined (B) alignment of LIR2 and 1QFT. The secondary structure of 1QFT and the prediction for LIR2 (PROF) are shown. helix is in black (text in white) and β in gray. The numbering of 1QFT refers to that of Figure 1. Positions at the extremities of the secondary structure elements are numbered to facilitate the reading. Residues interacting with histamine in the structures of 1QFT are underlined. Those belonging to the H site are bold. Boxes indicate examples of realigned regions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2254393&req=5

Figure 4: ClustalW (A) and refined (B) alignment of LIR2 and 1QFT. The secondary structure of 1QFT and the prediction for LIR2 (PROF) are shown. helix is in black (text in white) and β in gray. The numbering of 1QFT refers to that of Figure 1. Positions at the extremities of the secondary structure elements are numbered to facilitate the reading. Residues interacting with histamine in the structures of 1QFT are underlined. Those belonging to the H site are bold. Boxes indicate examples of realigned regions.

Mentions: LIR2 is a protein from Ixodes ricinus. PSI-BLAST was used to scan the PDB to find a homologous protein [24]. The only structure found after 4 iterations with an E value inferior to the threshold was that of RaHBP2 (1QFT). LIR2 has an identity around 15% with 1QFT and no lipocalin recognition motifs. The ClustalW alignment between LIR2 and 1QFT is shown in Figure 4. As for the PSI-BLAST alignment, some aberrations are noticed. The secondary structure of LIR 2 (predicted with the PROF method [25]) does not correspond to that of 1QFT in the N-ter region. Furthermore, the region of LIR2 corresponding to H1 (in 1QFT) contains three prolines, that do not favor the helical conformation. In the region corresponding to βA, position 48 (referring to the lipocalin alignment of Figure 1) does not correspond to an aromatic amino acid in LIR2. This residue is aromatic for all lipocalins including 1QFT; several mutational studies have notably demonstrated the importance of that residue in the lipocalin structure stability [26-28]. Position 49 is not bulky and hydrophobic as in the other lipocalins. The region corresponding to βB is not predicted as β. Position 80 corresponds to an Arg, while being a hydrophobic residue in lipocalins. The cysteine from βB, implicated in a disulfide bridge between the C-ter part and βB in 1QFT, is also not conserved.


Distantly related lipocalins share two conserved clusters of hydrophobic residues: use in homology modeling.

Adam B, Charloteaux B, Beaufays J, Vanhamme L, Godfroid E, Brasseur R, Lins L - BMC Struct. Biol. (2008)

ClustalW (A) and refined (B) alignment of LIR2 and 1QFT. The secondary structure of 1QFT and the prediction for LIR2 (PROF) are shown. helix is in black (text in white) and β in gray. The numbering of 1QFT refers to that of Figure 1. Positions at the extremities of the secondary structure elements are numbered to facilitate the reading. Residues interacting with histamine in the structures of 1QFT are underlined. Those belonging to the H site are bold. Boxes indicate examples of realigned regions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254393&req=5

Figure 4: ClustalW (A) and refined (B) alignment of LIR2 and 1QFT. The secondary structure of 1QFT and the prediction for LIR2 (PROF) are shown. helix is in black (text in white) and β in gray. The numbering of 1QFT refers to that of Figure 1. Positions at the extremities of the secondary structure elements are numbered to facilitate the reading. Residues interacting with histamine in the structures of 1QFT are underlined. Those belonging to the H site are bold. Boxes indicate examples of realigned regions.
Mentions: LIR2 is a protein from Ixodes ricinus. PSI-BLAST was used to scan the PDB to find a homologous protein [24]. The only structure found after 4 iterations with an E value inferior to the threshold was that of RaHBP2 (1QFT). LIR2 has an identity around 15% with 1QFT and no lipocalin recognition motifs. The ClustalW alignment between LIR2 and 1QFT is shown in Figure 4. As for the PSI-BLAST alignment, some aberrations are noticed. The secondary structure of LIR 2 (predicted with the PROF method [25]) does not correspond to that of 1QFT in the N-ter region. Furthermore, the region of LIR2 corresponding to H1 (in 1QFT) contains three prolines, that do not favor the helical conformation. In the region corresponding to βA, position 48 (referring to the lipocalin alignment of Figure 1) does not correspond to an aromatic amino acid in LIR2. This residue is aromatic for all lipocalins including 1QFT; several mutational studies have notably demonstrated the importance of that residue in the lipocalin structure stability [26-28]. Position 49 is not bulky and hydrophobic as in the other lipocalins. The region corresponding to βB is not predicted as β. Position 80 corresponds to an Arg, while being a hydrophobic residue in lipocalins. The cysteine from βB, implicated in a disulfide bridge between the C-ter part and βB in 1QFT, is also not conserved.

Bottom Line: Under the 30% identity threshold, alignment methods do not correctly assign and align proteins.The only safe way to assign a sequence to that family is by experimental determination.This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Biophysique Moléculaire et Numérique, Faculté Universitaire des Sciences Agronomiques de Gembloux, Gembloux, Belgium. adam.b@fsagx.ac.be

ABSTRACT

Background: Lipocalins are widely distributed in nature and are found in bacteria, plants, arthropoda and vertebra. In hematophagous arthropods, they are implicated in the successful accomplishment of the blood meal, interfering with platelet aggregation, blood coagulation and inflammation and in the transmission of disease parasites such as Trypanosoma cruzi and Borrelia burgdorferi. The pairwise sequence identity is low among this family, often below 30%, despite a well conserved tertiary structure. Under the 30% identity threshold, alignment methods do not correctly assign and align proteins. The only safe way to assign a sequence to that family is by experimental determination. However, these procedures are long and costly and cannot always be applied. A way to circumvent the experimental approach is sequence and structure analyze. To further help in that task, the residues implicated in the stabilisation of the lipocalin fold were determined. This was done by analyzing the conserved interactions for ten lipocalins having a maximum pairwise identity of 28% and various functions.

Results: It was determined that two hydrophobic clusters of residues are conserved by analysing the ten lipocalin structures and sequences. One cluster is internal to the barrel, involving all strands and the 310 helix. The other is external, involving four strands and the helix lying parallel to the barrel surface. These clusters are also present in RaHBP2, a unusual "outlier" lipocalin from tick Rhipicephalus appendiculatus. This information was used to assess assignment of LIR2 a protein from Ixodes ricinus and to build a 3D model that helps to predict function. FTIR data support the lipocalin fold for this protein.

Conclusion: By sequence and structural analyzes, two conserved clusters of hydrophobic residues in interactions have been identified in lipocalins. Since the residues implicated are not conserved for function, they should provide the minimal subset necessary to confer the lipocalin fold. This information has been used to assign LIR2 to lipocalins and to investigate its structure/function relationship. This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins.

Show MeSH
Related in: MedlinePlus