Membrane attachment and structure models of lipid storage droplet protein 1 Penghui Lin a , Xiao Chen b , Hem Moktan a , Estela L. Arrese b , Lian Duan a , Liying Wang a,c , Jose L. Soulages b , Donghua H. Zhou a, ⁎ a Department of Physics, 230 L Henry Bellmon Research Center, Oklahoma State University, Stillwater, OK 74078, USA b Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA c State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Center for Magnetic Resonance, Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China abstract article info Article history: Received 23 August 2013 Received in revised form 29 November 2013 Accepted 5 December 2013 Available online 12 December 2013 Keywords: Lipid storage droplet protein Triglyceride lipolysis Proton spin diffusion Magic-angle spinning Solid-state NMR MD simulation Neutral lipid triglycerides, a main reserve for fat and energy, are stored in organelles called lipid droplets. The storage and release of triglycerides are actively regulated by several proteins specific to the droplet surface, one of which in insects is PLIN1. PLIN1 plays a key role in the activation of triglyceride hydrolysis upon phosphor- ylation. However, the structure of PLIN1 and its relation to functions remain elusive due to its insolubility and crystallization difficulty. Here we report the first solid-state NMR study on the Drosophila melanogaster PLIN1 in combination with molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a trans-model agreed with experi- mental spectra. In this model, lipids from the bottom leaflet were very close to the surface in the region enclosed by membrane attachment motif. This may imply that in real lipid droplet, triglyceride molecules might be brought close to the surface by the same mechanism, ready to leave the droplet in the event of lipolysis. Juxtapo- sition of triglyceride lipase structure to the trans-model suggested a possible interaction of a conserved segment with the lipase by electrostatic interactions, opening the lipase lid to expose the catalytic center. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Animals store most of excess energy in the form of neutral lipid tri- glycerides for later use as metabolic fuel. The hydrophobicity of triglyc- erides allows them to be densely packed into lipid droplets, providing an energy density 10 times that of hydrated proteins and carbohydrates [1]. The lipid droplets are composed of a triglyceride core surrounded by a monolayer of phospholipids and a variety of proteins [2]. Utilization of the stored triglycerides requires enzymatic breakdown (lipolysis) by lipases, while the surface layer of the droplet controls the accessibility of lipases to the stored triglycerides. Among the proteins surrounding the lipid droplet surface, proteins in the PAT family (named after three earliest members) have raised great interest in recent years. The PAT family consists of the mammalian perilipin, ADRP, TIP47, S3-12, and OXPAT, as well as insect lipid storage droplet protein 1 (Lsd1) and 2 (Lsd2) [3]. Perilipin, ADRP, and Lsd1 constitutively attach to the lipid droplets, and they maintain fat storage and regulation of lipolysis. TIP47, S3-12, OXPAT, and arguably Lsd2 bind reversibly to the droplets; hypothetically they are responsible for the packaging of newly synthe- sized triglycerides into lipid droplets [3]. A new nomenclature has been recently proposed for the PAT-family of proteins [4]. Accordingly, from now on we will refer Lsd1 and Lsd2 as PLIN1 and PLIN2, respectively. Mammals and insects share significant conservation in the molecular mechanism of lipid droplet metabolism, highlighting the tremendous potential of using genetic technical advantages of insects to discover novel features of lipid homeostasis [3]. Studies of fruit fly models have established a correlation between triglyceride accumulation and the level of PLIN2 expression [5]. PLIN1 is found exclusively associated with lipid droplets [6]. It dynamically interacts with lipid droplet to con- trol access of lipase to triglycerides thus regulates the lipids homeosta- sis. In contrast to mouse perilipin, which protects triglycerides from hydrolysis [7,8], depletion of PLIN1 leads to adult-onset obesity [9] while overexpression of PLIN1 induces lipid droplet to shrink and Biochimica et Biophysica Acta 1838 (2014) 874–881 Abbreviations: Lsd1, lipid storage droplet protein 1; PAT family, perilipin, ADRP, TIP 47 family; MD, molecular dynamics; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; MAS, magic-angle spinning; CP, cross polarization; DARR, dipolar-assisted rotational resonance ⁎ Corresponding author. Tel.: +1 405 744 3277. E-mail address: donghua@okstate.edu (D.H. Zhou). 0005-2736/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamem.2013.12.003 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem