RESEARCH ARTICLE Cholesterol Interaction with the MAGUK Protein Family Member, MPP1, via CRAC and CRAC-Like Motifs: An In Silico Docking Analysis Marcin A. Listowski1, Jacek Leluk2, Sebastian Kraszewski3, Aleksander F. Sikorski1,2* 1 Department of Cytobiochemistry, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland, 2 Department of Molecular Biology, University of Zielona Góra, Zielona Góra, Poland, 3 Department of Biomedical Engineering, Wrocław University of Technology, Wrocław, Poland * [email protected] Abstract OPEN ACCESS Cholesterol is essential for the proper organization of the biological membrane. Therefore, predicting which proteins can bind cholesterol is important in understanding how proteins Citation: Listowski MA, Leluk J, Kraszewski S, participate in lateral membrane organization. In this study, a simple bioinformatics approach Sikorski AF (2015) Cholesterol Interaction with the MAGUK Protein Family Member, MPP1, via CRAC was used to establish whether MPP1, a member of the MAGUK protein family, is capable of and CRAC-Like Motifs: An In Silico Docking Analysis. binding cholesterol. Modelled and experimentally-validated fragment structures were mined PLoS ONE 10(7): e0133141. doi:10.1371/journal. from online resources and searched for CRAC and CRAC-like motifs. Several of these pone.0133141 motifs were found in the primary structure of MPP1, and these were structurally visualized Editor: Robert J Deschenes, College of Medicine, to see whether they localized to the protein surface. Since all of the CRAC and CRAC-like University of South Florida, UNITED STATES motifs were found at the surface of MPP1 domains, in silico docking experiments were per- Received: December 18, 2014 formed to assess the possibility of interaction between CRAC motifs and cholesterol. The Accepted: June 24, 2015 results obtained show that MPP1 can bind cholesterol via CRAC and CRAC-like motifs Published: July 17, 2015 with moderate to high affinity (KI in the nano- to micro-molar range). It was also found that palmitoylation-mimicking mutations (C/F or C/M) did not affect the affinity of MPP1 towards Copyright: © 2015 Listowski et al. This is an open access article distributed under the terms of the cholesterol. Data presented here may help to understand at least one of the molecular Creative Commons Attribution License, which permits mechanisms via which MPP1 affects lateral organization of the membrane. unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are available from Figshare (http://dx.doi.org/10.6084/m9. figshare.1397519). Introduction Funding: This work was supported by NCN (NCN = A specific sequence motif, termed the cholesterol recognition/interaction amino acid consen- Narodowe Centrum Nauki (National Centre for sus (CRAC) [1], is present in many proteins that are known to interact with cholesterol. It con- Research)); and Grant DEC-2012/05/B/NZ/01638 sists of branched apolar amino-acid residues, L and V, followed by any residues in a 1–5 and faculty grant of Department of Biotechnology, segment, then a mandatory aromatic residue Y and, then again, a segment of 1–5 of any resi- University of Wrocław, Poland. Publication cost was supported by Wrocław Center of Biotechnology dues, and ends with K or R. Looseness of the motif definition caused some scepticism about its Program, The Leading National Research Center predictive value [2–3]. However, this motif is present in many proteins that are known to bind (KNOW) for years 2014–2018. cholesterol and, in many cases, the interaction was confirmed by physicochemical approaches. PLOS ONE | DOI:10.1371/journal.pone.0133141 July 17, 2015 1/15 Cholesterol Interaction with MPP1 Competing Interests: The authors have declared Occurrence of the CRAC motif within the transmembrane segment of a protein increases that no competing interests exist. the reliability of prediction of cholesterol interaction with such a protein. The CRAC motif can also be present in proteins that are not integrated with a biomembrane. A good example of this is cytolethal-, distending toxin C of Aggregatibacter actinomycetemcomitans (CdtC). This protein contains a CRAC sequence motif, LIDYKGK, which is exposed at the surface of the protein and binds to liposomes containing cholesterol. Mutation of the Y residue to P results in reduced binding to cholesterol-containing membranes and to target cells, which reduces its toxicity [4]. CRAC motifs have also been found on the surface of viral proteins. HIV-1 fusion protein gp41 contains a CRAC motif, which is localized to the extracellular surface of the mem- brane, adjacent to the transmembrane segment of this protein. It has been suggested that this protein is important for membrane fusion [5–6]. Depletion of cholesterol from infected cell membranes reduces virus release, and released HIV virions show minimal infectivity [7]. HIV- 1 is known to enter the cell through binding to the CD4 receptor [8], which is associated with a cholesterol-rich raft region [9–10]. This raises the possibility that the CRAC motif in the gp41 protein interacts with the cholesterol in the host cell-membrane. Protein interaction with cho- lesterol via the CRAC motif was also demonstrated for human type-1 cannabinoid receptor (CB1R). Point mutation in the CRAC lysine residue K402G resulted in the receptor becoming insensitive to membrane cholesterol [11]. Apart from CRAC, another motif with a very similar sequence, termed CARC or CRAC- like motif, has been proposed [12]. It is sometimes referred to as an “inverted CRAC”. Reverse orientation is not the only feature which distinguishes it from CRAC. Another difference is that a central residue can be either Y or F. Docking studies demonstrated a favorable fit of cho- lesterol to both Y and F containing motifs [12]. Also, in biochemical studies, substitution of the Y residue with an F did not abolish the redistribution of cholesterol [13]. Membrane Palmitoylated Protein 1 (MPP1) is a member of the membrane-associated gua- nylate kinase homologues (MAGUK) protein family. Proteins of this family share similar domains, such as a PDZ domain, SH3 (src-homology-3) motif and a GUK domain (domain homologous to guanylate kinases) [14–16]. It was originally identified as a membrane skeletal protein in erythrocytes, where it stabilizes the spectrin-actin skeleton by interacting with glyco- phorin C and protein 4.1R [17–19]. MPP1 is a major target of palmitoylation in red blood cells. Lack of protein acyltransferase (PAT) activity in these cells causes changes in lateral membrane organization, which is, as we suggest, an impairment of resting-state raft formation. This was revealed by several methods [20]. Moreover, silencing of MPP1 gene expression in human ery- throleukemia (HEL) cells results in similar changes in membrane lateral organization [21]. Since cholesterol is a major component of raft domains, the mechanism of their formation could include interaction between MPP1 protein and cholesterol. However, no interaction of cholesterol with human MPP1 protein has been demonstrated to date. Here we present an in silico analysis of cholesterol interaction with MPP1 protein. The results obtained show that there is a possibility of such an interaction. These results provide a new insight on at least one possible molecular mechanism of resting-state raft formation and function of MPP1. Materials and Methods Identification of CRAC and CRAC-like motifs The sequence of MPP1 was obtained in FASTA format from UniProt (http://www.uniprot.org/ ). A search for CRAC and CRAC-like motifs was then performed with EMBROSS: fuzzpro (http://emboss.bioinformatics.nl/cgi-bin/emboss/fuzzpro). Sequences given as a search pattern PLOS ONE | DOI:10.1371/journal.pone.0133141 July 17, 2015 2/15 Cholesterol Interaction with MPP1 were: [LV]-X(1,5)-Y-X(1,5)-[RK], [RK]-X(1,5)-Y-X(1,5)-[LV], [LV]-X(1,5)-F-X(1,5)-[RK], [RK]-X(1,5)-F-X(1,5)-[LV] [1,12]. Experimental structures For searching existing experimental structures (obtained either by NMR or X-Ray diffraction), The Protein Model Portal (http://www.proteinmodelportal.org/) was used. The complete sequence of human MPP1 protein was entered as a query. Three experimentally validated par- tial-structures of MPP1 protein were obtained and downloaded from Protein Data Bank (http://www.rcsb.org), Namely, the PDZ domain [2ev8] [22], the PDZ domain complexed with glycophorin C [2ejy] [23] and the GUK domain [3ney] were used. The sequence range of these results were then compared with CRAC and CRAC-like motifs in the primary structure of the MPP1 protein. If the sequence range of experimental structures contained cholesterol-binding motifs, these structures were chosen for further processing. Building the full model structure Since there was no single complete protein structure of MPP1 published, to build a full model structure, CASP7, 8, 9 and 10 winning web server, I-TASSER was chosen to assemble a com- plete structural model of MPP1 [24–26]. The full amino-acid sequence of MPP1, obtained from Uniprot (http://www.uniprot.org/), was given as a query. The web server operated at its default setting, that is, no specification of templates was given. Also, no assignment of contact or distance restraints was given. The server was also set to not exclude any homologous or spe- cific templates. In brief, the templates selected by the server corresponded to the three main domains of MPP1, ie. PDZ, SH3 and GUK, as well as the inter-domain regions. Since the full structure of MPP1 remains unknown, the top template which was selected by the server was one for another MAGUK family member, PSD-95, whose structure has been fully resolved. To validate the correctness of the server selection, known fragments of MPP1 (PDZ and GUK domains) were compared to those of PSD-95 by the TM-align online tool [27] and were found to be structurally similar (TM-score 0,72 and 0,76 respectively).