Abstract
Human glycolipid transfer protein (hsGLTP) forms the prototypical GLTP fold and is characterized by a broad transfer selectivity for glycosphingolipids (GSLs). The GLTP mutation D48V near the `portal entrance' of the glycolipid binding site has recently been shown to enhance selectivity for sulfatides (SFs) containing a long acyl chain. Here, nine novel crystal structures of hsGLTP and the SF-selective mutant complexed with short-acyl-chain monoSF and diSF in different crystal forms are reported in order to elucidate the potential functional roles of lipid-mediated homodimerization. In all crystal forms, the hsGLTP-SF complexes displayed homodimeric structures supported by similarly organized intermolecular interactions. The dimerization interface always involved the lipid sphingosine chain, the protein C-terminus (C-end) and α-helices 6 and 2, but the D48V mutant displayed a `locked' dimer conformation compared with the hinge-like flexibility of wild-type dimers. Differences in contact angles, areas and residues at the dimer interfaces in the `flexible' and `locked' dimers revealed a potentially important role of the dimeric structure in the C-end conformation of hsGLTP and in the precise positioning of the key residue of the glycolipid recognition centre, His140. ΔY207 and ΔC-end deletion mutants, in which the C-end is shifted or truncated, showed an almost complete loss of transfer activity. The new structural insights suggest that ligand-dependent reversible dimerization plays a role in the function of human GLTP.
Highlights
Regulated interaction between specific proteins, commonly referred to as reversible dimerization, serves as a fundamental mechanism by which cells are able to control many key processes (Kuriyan & Eisenberg, 2007)
A magnified view of the protein contact area in the dimers is shown in Fig. 1(e) by comparative superimposition of wild-type GLTP (wtGLTP) and D48V-GLTP complexed with 12:0 monoSF, with one monomer superimposed and with the sulfatide molecule omitted for clarity
Our new crystal structures reveal lipiddependent reversible dimerization of human GLTP that could be important for protein function
Summary
Regulated interaction between specific proteins, commonly referred to as reversible dimerization, serves as a fundamental mechanism by which cells are able to control many key processes (Kuriyan & Eisenberg, 2007). In the ‘sphingosine-out’ mode, common to all other (over a dozen tested) GSLs, the sphingosine chain projects outwards and enters the hydrophobic interior of a partner GLTP monomer as part of a lipidmediated homodimeric arrangement Other approaches such as analytical centrifugation, gel-filtration and dynamic light scattering (DLS), which all operate in solution and use different ranges of sample concentrations, show different percentages of GLTP dimerization from negligibly small to almost complete (Malinina et al, 2004; Zhai et al, 2009; Samygina et al, 2011), indicating reversibility of the dimer formation. ÁY207 and ÁC-end, in which the C-end (Val209) is shifted or truncated show an almost complete loss of transfer activity, confirming the important functional role of the C-terminus in hsGLTP
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