Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics.

Arunima Chaudhuri,Priyanka Agiru,Amitabha Chattopadhyay,Anna Rydström,Hirak Chakraborty,James C S Ho,Catharina Svanborg,Xavier Prasanna,Durba Sengupta

doi:10.1038/srep35015

Abstract

Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.

Highlights

The efficacy of HAMLET as a selective killer of tumor cells has been documented in vitro and in vivo in several animal models and clinical studies[34,35,36,37,38]
We verified the formation of the complex by turbidity analysis, detecting a significant reduction in the opacity of the oleic acid (OA) sample upon addition of Bovine α-lactalbumin (BLA) (Fig. 1a), a characteristic feature earlier observed upon formation of protein-lipid complexes[18,26]
We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using coarse-grained and atomistic molecular dynamics simulation

Summary

Introduction

The efficacy of HAMLET as a selective killer of tumor cells has been documented in vitro and in vivo in several animal models and clinical studies[34,35,36,37,38]. Vesicles, rapid membrane insertion of HAMLET, triggered membrane tubulation[40]. These studies have not explored effects of the protein-OA complex on membrane lipids in detail or interactions of defined protein domains with the lipid bilayer. By using the voltage-sensitive membrane bound fluorescent membrane probe, 4-(2-(6-(dioctylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt (di-8-ANEPPS) combined with coarse-grain (CG) MD simulations, we show that there is a preferential interaction of the BLA-OA complex with negatively charged lipids. Similar results were obtained in MD simulations for BLA alone and residues involved in the interactions were mapped, suggesting that the specificity arises from the molecular interactions of the protein. Our integrated approach, combining experiments with simulations, to monitor the interaction of BLA-OA complex with membranes, provide holistic insights into lipid-protein interaction governing the affinity of BLA-OA for membranes

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Discussion

Conclusion