Lipid-transfer proteins (LTPs) facilitate the transfer of lipids between membranes. They are widely distributed, being produced by both prokaryotic and eu-karyotic organisms: bacteria, yeasts, plants and animals (Rueckert and Schmidt 1990). LTPs cover a wide spectrum of ligand specificities: FAs, phospholipids, gly-colipids, steroids, acyl-CoA, etc.; in this regard, it must be remarked that whereas some LTPs are highly specific (Roderick et al. 2002), others are non-specific (Tsu-jishita and Hurley 2000; Han et al. 2001). Nowadays, numerous LTPs have been characterized at high resolution, both from plants: wheat (Charvolin et al. 1999), maize (Shin et al. 1995; Han et al. 2001) and rice (Lee et al. 1998; Cheng et al. 2004); and from animals: rabbit (Choinowski et al. 2000), mouse (Romanowski et al. 2002), human (Tsujishita and Hurley 2000; Roderick et al. 2002).
Non-specific LTPs (ns-LTPs) from plants are small (~9 kDa), disulfide-rich, basic proteins, which show no sequence homology with mammalian ns-LTP. Both
Fig.3.17. Ribbon representation ofserum albumin (PDB code: 1GNI). Seven oleic acid molecules are also shown as stick models. Figures were prepared with the program PyMOL
high-resolution crystallographic and NMR studies have revealed that plant ns-LTPs are single-domain proteins, composed of four a-helices and four disulfides andalong C-terminal loop (Fig. 3.18).
Interestingly, this fold was first described for a hydrophobic protein from soybean (Baud et al. 1993). A remarkable structural feature is the presence of a hydrophobic tunnel which is large enough to accommodate a long fatty acyl chain. In this regard, the crystal structures of ns-LTP from maize complexed with palmitate (Shin et al. 1995), and wheat ns-LTP complexed with lyso-myristoyl-phosphatidylcholine (Charvolin et al. 1999) (Figs. 3.18B and a) show that the hydrocarbon chain of the ligand is deeply inserted into the protein cavity and the polar head group is exposed on the protein surface. Nevertheless, the scenario of ligand-binding to the hydrophobic cavity is more complex as NMR results indicate that the existence of the same ligand adopting different orientations is possible (Lerche et al. 1998). Moreover, the solution structure of the ns-LTP from wheat in complex with prostaglandin B2 shows that the ligand is fully buried into the protein (Tassin-Moindrot et al. 2000).
Fig. 3.18. Schematic representation of members of the diverse family of lipid-transfer proteins (LTPs). Non-specific LTPs from plants are composed of four a-helices and four disulfides, and a long C-terminal loop. PDB codes: (a) 1BWO for wheat ns-LTP complexed with two phospholipid molecules; (b) 1FK4, for ns-LTP from maize with stearate bound; (c) 1RZL, for ns-LTP from rice, (d) The ribbon representation of the ns-LTP sterol-carrier protein 2 from rabbit (PDB code: 1C44), and (e) that of the human phosphatidylcholine (PC) transfer protein complexed with two PC molecules (PDB code: 1LN1) are also shown. Figures were prepared with the program PyMOL
The crystal structure of the intracellular ns-LTP from rabbit sterol carrier protein 2 (SCP2) has been solved at 1.8 Á resolution (Choinowski et al. 2000) (Fig. 3.18d). Despite SCP2 having been shown to participate in diverse in vitro functions, which permit defining SCP2 either as a LTP or as a carrier for FAs, fatty acid-CoAs, or sterols such as cholesterol, neither the actual physiological role(s) nor the mechanism of action of SCP2 is yet known. SCP2 is a small (13 kDa), basic protein, highly conserved in different species. It is remarkable that various intracellular proteins, such as peroxisomal D-hydroxyacyl-CoA dehydrogenase and the Caenorhabditis elegans behavioural protein, posses a C-terminal SCP2 domain which would act as a putative lipid-binding domain. SCP2 is composed of a five-stranded P-sheet, which can be divided in two minor antiparallel sheets: strands I-III and IV-V, respectively (Fig. 3.18d). Whereas one side of the sheet is predominantly hydrophobic, the other is highly polar. In addition, this central P-sheet is flanked by five a-helices (Choinowski et al. 2000), the longest one (helix A) being clearly amphipathic with its apolar side packed against the hydrophobic side of the central P-sheet. Functionally, the most remarkable feature of SCP2 is the presence of a hydrophobic tunnel, which may provide a binding site for apolar ligands. Although there is neither sequence homology, nor three-dimensional resemblances between plant nsLTPs (see above) and SCP2, in both cases the hydrophobic tunnel observed has similar dimensions.
A wide diversity of proteins involved in lipid transport and metabolism, signal transduction, and transcriptional regulation possess the so-called START domains (after steroidogenic acute regulatory protein lipid transfer) (Tsujishima and Hurley 2000). In this context, the crystal structure of the START domains from human MLN64 (Tsujishima and Hurley 2000), from mouse cholesterol-regulated protein 4 (Romanowski et al. 2002), and from human phosphatidylcholine transfer protein (PC-TP) (Roderick et al. 2002) have been solved at high resolution. The overall structure of the START domain in all cases is highly conserved: it consists of a curved antiparallel nine-stranded U-shaped P-sheet, flanked by four a-helices (Fig. 3.18e). The N-terminal helix rests against one side of the sheet. As described above for SCP2, the most striking feature is the presence of a hydrophobic tunnel extending nearly the entire length of the protein. The structures of the complexes between PC-TP and dilinoleoyl-PC (DLPC) and PC-TP and palmi-toyl-linoleoyl-PC (PLPC) have revealed the molecular details of the embedded ligand, which in turn explains the exquisite specificity of PC-TP for this kind of lipid (Roderick et al. 2002). The acyl chains of both lipids adopt similar C-shaped conformations and establish numerous hydrophobic contacts both with aliphatic and aromatic side-chains. Additionally, the phosphorylcholine headgroup of the ligand interacts with hydrophilic side-chains; in particular the phosphoryl group interacts with Arg 78 which in turn forms a salt bridge with Asp 82, this motif ArgAsp being conserved in the sequence of many START proteins (Tsujishima and Hurley 2000). Conversely, the quaternary amine of the lipid mainly interacts with aromatic side-chains through cation-rc interactions (Burley and Petsko 1985; Gallivan and Dougherty 1999). This type of interaction has been observed in numerous substrate-binding pockets of diverse enzymes (Bellamy et al. 1989; Sussman et al. 1991), as well as in pore-forming toxins (Mancheño et al. 2003).
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