Phylogenetic Profiles Reveal Structural and Functional Determinants of Lipid-binding

Abstract

One of the major challenges in the genomic era is annotating structure/function to the vast quantities of sequence information now available. Indeed, most of the protein sequence database lacks comprehensive annotation, even when experimental evidence exists. Further, within structurally resolved and functionally annotated protein domains, additional functionalities contained in these domains are not apparent. To add further complication, small changes in the amino-acid sequence can lead to profound changes in both structure and function, underscoring the need for rapid and reliable methods to analyze these types of data. Phylogenetic profiles provide a quantitative method that can relate the structural and functional properties of proteins, as well as their evolutionary relationships. Using all of the structurally resolved Src-Homology-2 (SH2) domains, we demonstrate that knowledge-bases can be used to create single-amino acid phylogenetic profiles which reliably annotate lipid-binding. Indeed, these measures isolate the known phosphotyrosine and hydrophobic pockets as integral to lipid-binding function. In addition, we determined that the SH2 domain of Tec family kinases bind to lipids with varying affinity and specificity. Simulating mutations in Bruton's tyrosine kinase (BTK) that cause X-Linked Agammaglobulinemia (XLA) predict that these mutations alter lipid-binding, which we confirm experimentally. In light of these results, we propose that XLA-causing mutations in the SH3-SH2 domain of BTK alter lipid-binding, which could play a causative role in the XLA-phenotype. Overall, our study suggests that the number of lipid-binding proteins is drastically underestimated and, with further development, phylogenetic profiles can provide a method for rapidly increasing the functional annotation of protein sequences.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins

“…Lastly, it should be noted that our results on lipid binding sites of SH2 domains are at odds with previous studies suggesting that some SH2 domains bind lipids in their pY pockets (Hong et al, 2009; Rameh et al, 1995; Tokonzaba et al, 2006). Although the origin of this discrepancy is not fully understood, one can speculate that some SH2 domains without ACPs may be able to bind a lipid(s) in their pY pockets given that the shape and electrostatic properties of pY pockets vary significantly among SH2 domains.…”

“…We thus suspected that either or both of these cationic sites might serve as a binding site for anionic lipids including PtdIns Ps . Previous studies suggested that the pY pocket might be the main site for lipid binding (Hong et al, 2009; Rameh et al, 1995; Tokonzaba et al, 2006). We found, however, mutation of single or multiple cationic residues in the pY pockets of 5 randomly selected SH2 domains did not cause significant decreases in vesicle binding (Fig.…”

Phosphotyrosine recognition domains: the typical, the atypical and the versatile

“…X-chromosome-linked agammaglobulinemia (XLA) is an example of a disease where mutations are present in the SH2 domain of the Bruton's tyrosine kinase (BTK), and many of these mutations are located on the pTyr-ligand binding site, including the indispensable arginine of the pTyr-binding pocket as well as BG loop residues [16,173]. Moreover, Hong et al demonstrated that the BTK SH2 domain binds to phospholipids and the XLA-causing mutations alter lipid binding selectivity [174]. In another example, multiple point mutations within the transcription factor STAT3 SH2 domain have been identified and linked to large granular lymphocytic leukemia and the hyper-IgE syndrome [175,176].…”

The BRCA1 Tumor Suppressor Binds to Inositol 1,4,5-Trisphosphate Receptors to Stimulate Apoptotic Calcium Release

“…We hypothesized that recruitment of BRCA1 to the ER may be mediated by an intrinsic lipid binding activity in the BRCA1 protein. We identified a potential lipid binding domain within the C-terminal tandem BRCT domain of BRCA1 using Adaptive-BLAST (24,25). Specifically, we found that amino acid residues 1664 -1696 within the first BRCT repeat have a strong potential for lipid binding on the basis of homology to fatty acid binding proteins ( Fig.…”