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The identification of protein domains that mediate functional interactions between Rab-GTPases and RabGAPs using 3D protein modeling

View Article: PubMed Central - PubMed

ABSTRACT

Currently, time-consuming serial in vitro experimentation involving immunocytochemistry or radiolabeled materials is required to identify which of the numerous Rab-GTPases (Rab) and Rab-GTPase activating proteins (RabGAP) are capable of functional interactions. These interactions are essential for numerous cellular functions, and in silico methods of reducing in vitro trial and error would accelerate the pace of research in cell biology. We have utilized a combination of three-dimensional protein modeling and protein bioinformatics to identify domains present in Rab proteins that are predictive of their functional interaction with a specific RabGAP. The RabF2 and RabSF1 domains appear to play functional roles in mediating the interaction between Rabs and RabGAPs. Moreover, the RabSF1 domain can be used to make in silico predictions of functional Rab/RabGAP pairs. This method is expected to be a broadly applicable tool for predicting protein–protein interactions where existing crystal structures for homologs of the proteins of interest are available.

No MeSH data available.


Hierarchical clustering analyses demonstrate that RabSF1 domain sequence variations correlate with known Rab-Rab-GTPase activating proteins interaction pairs.Notes: Hierarchical clustering analyses were performed using the unweighted pair group method with arithmetic mean algorithm using (A) whole Rab protein sequence, (B) RabF2 domain only, or the (C) RabSF1 domain only. The RabSF1 domains from Rabs capable of interacting with the same RabGAP were consistently placed on the same branches. Hierarchical clustering analyses performed using the Neighbor-Joining method produced similar results (data not shown).
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f5-aabc-10-047: Hierarchical clustering analyses demonstrate that RabSF1 domain sequence variations correlate with known Rab-Rab-GTPase activating proteins interaction pairs.Notes: Hierarchical clustering analyses were performed using the unweighted pair group method with arithmetic mean algorithm using (A) whole Rab protein sequence, (B) RabF2 domain only, or the (C) RabSF1 domain only. The RabSF1 domains from Rabs capable of interacting with the same RabGAP were consistently placed on the same branches. Hierarchical clustering analyses performed using the Neighbor-Joining method produced similar results (data not shown).

Mentions: In an attempt to confirm the above-mentioned results, Rab11 and Rab23 were queried for the presence of any other recognized functional domains known to the CDD (as implemented in PhyreInvestigator). This analysis identified 20 structural motifs or function regions present in each protein (Table 4). Of the features mentioned in Table 4, only nine are characteristic of Rab proteins.5,16 Although the expected Rab domains were identified, their existence has not yet been correlated with a specific function. Multiple sequence alignments of the Rabs revealed the presence of amino acid substitutions within the Rab-specific domain signatures (Figure 4). These variations occurred in patterns that mimicked the ability of the Rab proteins in question to bind to Evi5 or Evi5L. In particular, among Rabs capable of interacting with the same RabGAP, binding appeared to be associated with a high degree of homology between the RabF1, RabF2, RabF3, RabF4, RabF5, RabSF1, and RabSF4 domains. When comparing Rabs that cannot interact with the same RabGAP, RabF2, RabF5, RabSF1, and RabSF2 show diminished homology. Among these two groups of Rab-family domains, the RabSF1 and RabF2 domains are both highly similar between Rabs interacting with the same RabGAP and dissimilar between Rabs interacting with the different RabGAPs. This line of inquiry was expanded by exploring whether the RabSF1 and/or RabF2 domains could be used to infer RabGAP-binding preferences. Alignments of whole protein or the RabF2 and RabSF1 domains revealed that, unlike alignments of the whole Rab proteins, alignments of the RabSF1 domain could be used to reconstruct a tree of Rab proteins that could interact with the same RabGAP (Figure 5).


The identification of protein domains that mediate functional interactions between Rab-GTPases and RabGAPs using 3D protein modeling
Hierarchical clustering analyses demonstrate that RabSF1 domain sequence variations correlate with known Rab-Rab-GTPase activating proteins interaction pairs.Notes: Hierarchical clustering analyses were performed using the unweighted pair group method with arithmetic mean algorithm using (A) whole Rab protein sequence, (B) RabF2 domain only, or the (C) RabSF1 domain only. The RabSF1 domains from Rabs capable of interacting with the same RabGAP were consistently placed on the same branches. Hierarchical clustering analyses performed using the Neighbor-Joining method produced similar results (data not shown).
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5391865&req=5

f5-aabc-10-047: Hierarchical clustering analyses demonstrate that RabSF1 domain sequence variations correlate with known Rab-Rab-GTPase activating proteins interaction pairs.Notes: Hierarchical clustering analyses were performed using the unweighted pair group method with arithmetic mean algorithm using (A) whole Rab protein sequence, (B) RabF2 domain only, or the (C) RabSF1 domain only. The RabSF1 domains from Rabs capable of interacting with the same RabGAP were consistently placed on the same branches. Hierarchical clustering analyses performed using the Neighbor-Joining method produced similar results (data not shown).
Mentions: In an attempt to confirm the above-mentioned results, Rab11 and Rab23 were queried for the presence of any other recognized functional domains known to the CDD (as implemented in PhyreInvestigator). This analysis identified 20 structural motifs or function regions present in each protein (Table 4). Of the features mentioned in Table 4, only nine are characteristic of Rab proteins.5,16 Although the expected Rab domains were identified, their existence has not yet been correlated with a specific function. Multiple sequence alignments of the Rabs revealed the presence of amino acid substitutions within the Rab-specific domain signatures (Figure 4). These variations occurred in patterns that mimicked the ability of the Rab proteins in question to bind to Evi5 or Evi5L. In particular, among Rabs capable of interacting with the same RabGAP, binding appeared to be associated with a high degree of homology between the RabF1, RabF2, RabF3, RabF4, RabF5, RabSF1, and RabSF4 domains. When comparing Rabs that cannot interact with the same RabGAP, RabF2, RabF5, RabSF1, and RabSF2 show diminished homology. Among these two groups of Rab-family domains, the RabSF1 and RabF2 domains are both highly similar between Rabs interacting with the same RabGAP and dissimilar between Rabs interacting with the different RabGAPs. This line of inquiry was expanded by exploring whether the RabSF1 and/or RabF2 domains could be used to infer RabGAP-binding preferences. Alignments of whole protein or the RabF2 and RabSF1 domains revealed that, unlike alignments of the whole Rab proteins, alignments of the RabSF1 domain could be used to reconstruct a tree of Rab proteins that could interact with the same RabGAP (Figure 5).

View Article: PubMed Central - PubMed

ABSTRACT

Currently, time-consuming serial in vitro experimentation involving immunocytochemistry or radiolabeled materials is required to identify which of the numerous Rab-GTPases (Rab) and Rab-GTPase activating proteins (RabGAP) are capable of functional interactions. These interactions are essential for numerous cellular functions, and in silico methods of reducing in vitro trial and error would accelerate the pace of research in cell biology. We have utilized a combination of three-dimensional protein modeling and protein bioinformatics to identify domains present in Rab proteins that are predictive of their functional interaction with a specific RabGAP. The RabF2 and RabSF1 domains appear to play functional roles in mediating the interaction between Rabs and RabGAPs. Moreover, the RabSF1 domain can be used to make in silico predictions of functional Rab/RabGAP pairs. This method is expected to be a broadly applicable tool for predicting protein–protein interactions where existing crystal structures for homologs of the proteins of interest are available.

No MeSH data available.