Introduction
Mitochondrial uncoupling protein 1 (UCP1), which is expressed in brown adipose tissue (BAT), is a critical regulator of adaptive non-shivering thermogenesis via the biological function of proton leak (1–4). UCP1, a transmembrane protein, is located at the inner mitochondrial inner membrane, and uncouples the oxidative phosphorylation to produce heat through decreasing the proton gradients (1,2). Free fatty acids are the established activators that reduce the transmembrane potential via UCP1 and increase UCP1-associated oxygen consumptions (5). Chouchani et al (6) identified the sulfenylation of UCP1 regulated by mitochondrial reactive oxygen species in 2016. Uncoupling proteins (UCPs) expressed in adipose cells, skeletal muscle and macrophages participate in fatty acid metabolism (7,8). In addition, UCPs have been associated with metabolic diseases, including obesity and diabetes (9,10).
Subcellular compartments from yeast to mammalian cells contain numerous proteins and lipids. Phosphatidylcholine and phosphatidylethanolamine are major mitochondrial phospholipids (~80%). High cardiolipin content (10–15%) is found in mitochondrial membranes and its concentration is implicated in human health and disease (11,12). Protein-lipid interactions are crucial for protein stability (12). Cardiolipin directly interacts with ADP/ATP translocase (ANT) at the mitochondrial inner membrane (13), and in vitro reconstituted experiments have demonstrated the binding of six molecules of cardiolipin per ANT dimer (14). The exact associations of UCP1 with mitochondrial inner membrane lipids still remain unknown. In the present study, bioinformatics was used to identify the lipid-binding α-helix regions of UCP1. The results may provide novel insight into UCP1-lipid binding.
Results and Discussion
Mitochondrial UCP1 is specifically expressed in mammalian BAT. Thus far, to our knowledge, its high-resolution structure has not been determined. Reportedly, UCP1 as a mitochondrial transmembrane protein interacts with mitochondrial lipids, which considerably affects its biological function (23). In the current study, the putative lipid-binding α-helices of UCP1 were determined by bioinformatics. The HeliQuest algorithm has successfully predicted the lipid-binding sites of membrane proteins previously (16). The overall HeliQuest-calculated scores (mean hydrophobicity, hydrophobic moment and net charges) are presented in Fig. 1A. Following further discrimination factor-based analyses, a total of 8 lipid-binding α-helix regions of UCP1 were identified (37AKVRLQIQGEGQASS TIR54, 56KGVLGTITTLAKTEGLPK73, 75YSGLPAGIQRQISF ASLR92, 134TEVVKVRMQAQSHLHGIK151, 166TTESLSTLWK GTTPNLMR183, 235VVKTRFINSLPGQYPSVP252, 260TKEGPT AFFKGFVASFLR277 and 283VIMFVCFEQLKKELMKSR300). These predicted results may be considered acceptable among the range of strategies available to calculate the discrimination factor. The helical wheel plots of lipid-binding α-helix regions of UCP1 are shown in Fig. 1B. Helical wheel plots with an 18 amino acid window are considered to depict the best α-helices (16). As shown in Fig. 1B, the identified lipid-binding α-helix regions displayed the amphiphilic properties. The amino acid sequences of the lipid-binding α-helix regions of UCP1 exhibited no high sequence identities with other known lipid-binding domains (C1, C2, PH, FYVE, PX, ENTH, ANTH, BAR, FERM, PDZ or tubby domains), indicating that these lipid-binding regions were the novel lipid-binding sites. Of these lipid-binding regions of UCP1, the fourth region (134TEVVKVRMQAQSHLHGIK151) was a highly confident lipid-binding peptide based on the subsequent screening scores of HeliQuest. The peptide with 100% sequence identity was indicated to be exclusively found in mice and rats by BLAST, and may be a crucial interacting sequence for UCP1-lipid associations and UCP1 stability. Furthermore, there were some sequence similarities among these novel lipid-binding α-helix regions despite the lack of peptides with 100% sequence identity. For example, the above-mentioned fourth lipid-binding protein region had a 47% sequence similarity to 14NDRTLRRM RKVVNIINAME33 (Escherichia coli SecA) as predicted by HeliQuest (24).
To assess the 3D structure of UCP1, acceptable templates were searched for with high sequence identity. Their sequence alignment is presented in Fig. 2A. Functional BAT and UCP1 have been identified and characterized in rodents and human (25,26). In silico analysis of mouse UCP1 is performed since rodents generally work as important validated targets prior to clinical trials, and BAT with UCP1 expression is easily identifiable in small mammals including mice (26,27). The crystal structures of Bos taurus and Saccharomyces cerevisiae ANT carriers (PDB IDs: 2c3e and 4c9g) (28,29) were used to build a final homology model of UCP1. An available NMR structure of UCP2 with high sequence identity to UCP1 was not selected due to its low resolution (30). The PROCHECK program was used to evaluate the reliability and reasonability of the model, with >95% residues in most-favored/additionally allowed/generously allowed regions. Surprisingly, the 8 lipid-binding α-helix regions were mostly present outside the six main helix bundles of UCP1 (Fig. 2B), suggesting that the interactions of lipid-binding α-helices belonged to nonannular protein-lipid interactions (31). Previous observations have demonstrated that mutations of certain residues significantly influence the biological function of UCP1 (9). Of these sites, P79, R84, R92, E135, M141, H146, H148, E168, R183, 262EGPTAFFKG270 and R277 are located at the lipid-binding α-helix regions (9), and these sites associated with the lipid-binding α-helix regions may involve the biological function of UCP1. It is hypothesized that mutations alter protein functions via the conformational disturbances theoretically caused by the physicochemical properties of amino acid residues (32–34). Another possible complementary explanation is that these mutations of membrane proteins may disturb the protein-lipid interactions, thus causing the protein instability or conformational abnormalities.
Cardiolipin acts as an insulator and stabilizes mitochondrial membrane proteins, including mitochondrial respirasome (35). As shown in Fig. 3, the putative lipid-binding α-helix regions of UCP1 in the current UCP1-lipid docking model appeared to mainly associate with three cardiolipins; an interaction eluded to previously (23). LigPlot+ analysis demonstrated a variety of hydrophobic and hydrophilic interactions between UCP1 and cardiolipins (Fig. 3B); indeed cardiolipin might be a key regulator of optimal biological activity of many membrane proteins (36). An example is cardiolipin-binding cytochrome c oxidase. Of these cardiolipin-related interactions determined by LigPlot+, the main interacting contributors of UCP1 appeared to be 75YSGLPAGIQRQISFASLR92, 166TTE SLSTLWKGTTPNLMR183, 235VVKTRFINSLPGQYP SVP252 and 260TKEGPTAFFKGFVASFLR277. The lysine residues of cardiolipin-binding cytochrome c seem to be crucial for their recognition and biological activities (37). In the current docking model, two lysines (K175 and K269) were revealed to bind to cardiolipins. The interactions may be implicated in the same function related to the cytochrome c-cardiolipin complex. However, the exploration of these interactions between UCP1 and cardiolipins still requires high-resolution or standard biophysical techniques. Previous studies have demonstrated that cardiolipin may be a potential target for therapies against Parkinson's disease; the current results indicate that it may be a promising therapeutic target in UCP1-associated diseases.
Protein-lipid interactions contribute to the structural stability and biological function of transmembrane proteins. We herein analyzed the lipid-binding α-helix regions of mitochondrial UCP1, using bioinformatic approaches due to the limitations of experimental techniques in this field. A total of 8 plausible lipid-binding α-helix regions of UCP1 were predicted based on the characteristics of protein-lipid interactions. These interaction regions also mapped onto the key functional regions of UCP1, suggesting that these lipid-binding regions are involved in the biological function of UCP1. The UCP1-lipid docking model indicate that cardiolipin is a crucial interacting molecule and may maintain optimal biological function. The findings support the presence of specific lipid-binding α-helices in UCP1 that may represent the therapeutic targets for UCP1-associated diseases.