The structure of unliganded sterol carrier protein 2 from Yarrowia lipolytica unveils a mechanism for binding site occlusion

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Highlights

  • SCP2 binding cavity expands about six times to accommodate bulky hydrophobic ligands.

  • Concerted movements of α helices bring about the expansion of the cavity.

  • Unliganded binding cavity follows fundamental packing principles of protein structure.

  • Large, apparently empty SCP2 cavities are likely to be occupied by undetected ligands.

Abstract

Isolated or as a part of multidomain proteins, Sterol Carrier Protein 2 (SCP2) exhibits high affinity and broad specificity for different lipidic and hydrophobic compounds. A wealth of structural information on SCP2 domains in all forms of life is currently available; however, many aspects of its ligand binding activity are poorly understood. ylSCP2 is a well-characterized single domain SCP2 from the yeast Yarrowia lipolytica. Herein, we report the X-ray structure of unliganded ylSCP2 refined to 2.0 Å resolution. Comparison with the previously solved liganded ylSCP2 structure unveiled a novel mechanism for binding site occlusion. The liganded ylSCP2 binding site is a large cavity with a volume of more than 800 Å3. In unliganded ylSCP2 the binding site is reduced to about 140 Å3. The obliteration is caused by a swing movement of the C-terminal α helix 5 and a subtle compaction of helices 2–4. Previous pairwise comparisons were between homologous SCP2 domains with a uncertain binding status. The reported unliganded ylSCP2 structure allows for the first time a fully controlled comparative analysis of the conformational effects of ligand occupation dispelling several doubts regarding the architecture of SCP2 binding site.

Introduction

As a single domain or as part of a large variety of multidomain proteins, Sterol Carrier Protein 2 (SCP2) can be found in all forms of life, and it is characterized by its high affinity and broad specificity for lipids and hydrophobic compounds (Burgardt et al., 2017, Noland et al., 1980). Known cellular functions of SCP2 include nonvesicular lipid transport and storage, membrane remodeling, lipid mediated signaling, isoprenoid and cholesterol metabolism, steroidogenesis, peroxisomal oxidation of branched and long-chain fatty acids, and cholesterol uptake (Falomir Lockhart et al., 2009, Hillard et al., 2017, Schroeder et al., 2007, Seedorf et al., 1998, Singarapu et al., 2016, Wüstner and Solanko, 2015, Zheng et al., 2008, Stolowich et al., 2002, Zhang et al., 2014).

The first crystal structure of a SCP2 domain was described in 2000 (Choinowski et al. 2000), and over the years, a wealth of structural information on SCP2 domains in the three domains of life has become available and was extensively reviewed (Burgardt et al., 2017). SCP2 domains are dome-shaped structures, with a five-stranded, mixed β sheet floor covered by four α helices (Perez De Berti et al., 2013) defining a large inner binding cavity.

The SCP2 binding cavity is singular in many respects. First, it is entirely lined with aliphatic and aromatic side chains. Second, it promiscuously accommodates one or more bulky hydrophobic ligands (typically >200 Å3 each). Third, it is very variable in size and shape across the SCP2 family. Fourth, it appears empty and desolvated in most X-ray structures, and in the few cases where the presence of a ligand was reported, a common pattern of its interaction with the cavity could not be defined. Lastly, a comparison of different SCP2 domains shows that the concerted movement of α-helical secondary structure elements permits the expansion and reshaping of the binding site adapting the cavity for different hydrophobic ligands and binding situations (Burgardt et al., 2017).

The singularity of the SCP2 binding site poses several interesting questions. Perhaps the more general one is what is the nature of the interaction of the ligands with the protein cavity. In this regard, the heavy atoms defining the internal void comprise almost invariably carbon atoms, in such a way that oxygen and nitrogen atoms are excluded from interacting with ligand atoms. Moreover, since SCP2 ligands only have in common bulkiness and a hydrophobic character, their binding cannot be properly described by a lock-and-key fit mediated by specific noncovalent interactions (Burgardt et al., 2017, Gianotti et al., 2018).

Another interesting question is why water molecules are undetected inside the large binding cavities of the many SCP2 solved structures. This is in striking contrast with the much more studied case of fatty acid-binding protein (FABP), in which the similarly sized cavity is filled with a significant number of organized water molecules (Howard et al., 2016). Answering this question will be important because water molecules play a crucial role in binding and determine the energetic of the process in general. Namely, there is an important energy term associated to the removal of a ligand from bulk solvent and also in the confinement of solvent to a limited cavity. In addition, if present in the binding site, water must be expelled out to allow binding.

Most of the reported SCP2 domain X-ray structures do not show a bound ligand in its binding cavity (Burgardt et al., 2017). However, none of these SCP2 structures was reportedly delipidated before crystallization. Since special precautions need to be taken to effectively remove bound lipids from lipid-binding proteins (Dyer et al., 2003, Matsuoka et al., 2015), the apparent emptiness of the binding sites seems to be more the result of weak electron density of highly mobile or heterogeneous ligands than a genuine absence of them. Thus, the question arises as to what is the structure of SCP2 certifiably free of ligands. Answering this question is essential to advance in the knowledge of the binding mechanism of SCP2 domains, particularly to gain insight into the transfer process of hydrophobic ligands to and from membranes and cognate lipid binding proteins (Gianotti et al., 2018, Falomir Lockhart et al., 2009).

To answer the above questions, we undertook the structural characterization of unliganded ylSCP2, hereafter referred to as apo ylSCP2, which is a well-studied stand-alone SCP2 domain from the yeast Yarrowia lipolytica (Burgardt et al., 2009, Dell’Angelica et al., 1996, Falomir Lockhart et al., 2009, Ferreyra et al., 2006, Gianotti et al., 2018, Perez De Berti et al., 2013). We have previously described the X-ray structure of liganded ylSCP2 with bound palmitic acid (hereafter holo ylSCP2) (Perez De Berti et al., 2013)⁠, which offers the opportunity for a direct comparison between the apo and holo form of the same SCP2 domain.

Herein, we report the X-ray structure of apo ylSCP2 refined to 2.0 Å resolution. The comparison with holo ylSCP2 at similar resolution unveiled a novel mechanism for binding site occlusion.

Section snippets

Protein expression and purification

Recombinant ylSCP2 (UniProtKB ID: P80547) was expressed in Escherichia coli BL21(DE3) cells transformed with the plasmid pYLSCP2 (Ferreyra et al., 2006, Gebhard et al., 2006), and ylSCP2 expression was induced with 1 mM IPTG.

The preparation of the apo forms of lipid binding proteins is a hard practical problem, because proteins purified from natural sources or from heterologous recombinant expression systems by conventional protocols are contaminated with endogenous lipids (see for instance the

Overall structure of apo ylSCP2

The structure of apo ylSCP2 was solved by molecular replacement and refined to 2.0 Å resolution. ylSCP2 crystals belong to the trigonal space group P3121, and the asymmetric unit contains a homodimer which corresponds to the biological unit of the protein in solution (Fig. 1, panel A). Except for the C-terminal helix (residues 109–127), both monomers exhibit very similar backbone structure (RMSD = 0.59 Å).

It is important to note that the refinement process yielded moderately high values for R/R

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by grants from Universidad Nacional de Quilmes and CONICET, Argentina. We are grateful for the access to the PROXIMA-2A beamline at the SOLEIL Synchrotron, France. The sponsors had no involvement in the study design, in the collection, analysis and interpretation of data, in the writing of the report; and in the decision to submit the article for publication.

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