GLTPs are soluble proteins discovered initially in the membrane-free cytosolic extract of bovine spleen, and later in a wide variety of tissues, that selectively accelerate the transfer of glycolipids between membranes6–8
. The 23–24-kDa GLTPs display absolute specificity for glycolipids9
and are highly conserved among mammals10
, with some orthologues implicated in apoptosis11,12
. Although specific folding motifs and lipid-binding domains have been characterized in other lipid transfer/binding proteins13–19
, this is not true of GLTP. We have solved the structure of apo-GLTP crystal from human skin fibroblasts at 1.65 Å resolution and the corresponding lactosylceramide–GLTP complex co-crystal at 1.95 Å resolution (see Methods
and Supplementary Table S1
). The novel protein folding of apo-GLTP () is organized as a two-layer arrangement of α-helices, with helices 1, 2, 6 and 7 forming the front layer, and helices 3, 8, 4 and 5 belonging to the back layer (Supplementary Fig. S1
), with two long α-helices, 8 and 4, coiling around each other to form a superhelix. Such a sandwich topology generates a lactosylceramide-binding site between the α-helical layers in the lactosylceramide–GLTP complex (; Supplementary Fig. S2a, b
Figure 1 Overall structures of apo-GLTP and the lactosylceramide–GLTP complex. a, Stereo view of the apo-protein in a ribbon representation. Four α-helices on the front face are coloured grey, and four other α-helices on the back face are (more ...)
Lactosylceramide () binding to GLTP is achieved through recognition and anchoring of the hydrophilic headgroup to the protein surface, and through accommodation of the hydrophobic lipid chains within the interior of the protein. The lactosyl headgroup is anchored within the GLTP recognition centre, located on the protein surface, through a network of hydrogen-bonding (with Asp 48, Asn 52 and Lys 55 of α2, and Tyr 207 towards the carboxy terminus) and hydrophobic (Trp 96 of α4 stacks over the glucose ring, whereas Leu 92 contacts the galactose ring) interactions (; Supplementary Fig. S3
), analogous to carbohydrate–lectin interactions20,21
. The ceramide amide group is oriented through a pair of hydrogen bonds (with negatively charged Asp 48 and positively charged His 140), whose alignment is facilitated by hydrophobic contacts (with Val 209) of the initial three-carbon ceramide segment (; Supplementary Fig. S3
). The importance of individual recognition events within the headgroup recognition centre was probed by point mutation studies, as monitored by glycolipid intervesicular transfer assays with radiolabelled glycolipid (Supplementary Fig. S4
). The experimental data support the key role of Trp 96, His 140, Asp 48 and Asn 52 for recognition of the glycosyl and ceramide amide moieties in the complex. Mutations W96A and H140L resulted in almost complete inactivation (residual activity 1–3%), whereas mutations D48V and N52I resulted in significant inactivation (residual activity about 15%) (, top). The extent of inactivation is different for the W96F mutation (residual activity 63%) relative to its W96A counterpart (residual activity 1%), highlighting the importance of aromatic ring stacking over the glucose ring to recognition (, top). By contrast, Tyr 207 and Lys 55, which form single intermolecular hydrogen bonds, seem to be much less crucial for recognition, because the mutations Y207L and K55I retain practically full (90–97%) activity (, top). We have verified the integrity of our GLTP headgroup mutants for unanticipated structural alterations. The crystal structure of the D48V mutant shows only local rearrangements (Supplementary Fig. S5
), whereas circular dichroism (CD) spectra of other mutants indicate minimal perturbation of a-helical global folds (far-ultraviolet CD; Supplementary Fig. S6
, top) and of aromatic amino acid environments (near-ultraviolet CD; Supplementary Fig. S7
Figure 2 Intermolecular interactions in the lactosylceramide–GLTP complex. a, The headgroup recognition centre residues interacting with the two sugars and the ceramide amide group of bound lactosylceramide. Hydrogen bonds are shown by dashed lines. The (more ...)
When GLTP acquires lactosylceramide, both hydrocarbon chains of the ceramide moiety become buried within a single completely hydrophobic tunnel, lined by the side chains of nonpolar phenylalanine, leucine, isoleucine and alanine residues, together with a few valine and proline residues (). Residual activities ranging from 50% to 90% were observed when individual phenylalanines were replaced by serines at positions 103, 148 and 183 (, bottom), indicative of a possible additive role in creating the favourable hydrophobic environment for the nonpolar chains. Significant retention of activity (82%) was also found for the I45N mutant (, bottom), located near the entrance to the channel. Replacement of leucines by arginines affected activity differently depending on their location within the channel. Thus, the L165R, mutant located towards the end of the channel, retained 54% of activity, whereas L136R, located in the interior of the channel, retained only 5% of activity (, bottom). We have verified the integrity of the GLTP channel mutants by recording the far-ultraviolet and near-ultraviolet CD spectra (Supplementary Fig S6
and Supplementary Fig S7
, bottom panels).
Neither the hydrophobic tunnel nor lipid chains (a Δ9,10 cis
-double bond occurs halfway down the N
-acyl chain) are straight in the complex ( and ). The N
-acyl chain extends deeper into the hydrophobic tunnel because it is longer than the adjacent sphingoid-base chain (). The terminal segments of both chains become nearly orthogonal with respect to the long axis of the sphingoid base ( and ). Our measured solvent-accessible volume of about 315 Å3
for the lipid-binding tunnel in the complex is much smaller than reported values for other lipid-binding pockets19
, indicative of a very tight fit between the inserted portion of the lipid chains, with an estimated volume of about 330 Å3
, and the walls of the tunnel. The measured solvent-accessible volume is further reduced to 170 Å3
in apo-GLTP, so that this residual space can no longer accommodate lipid chains.
Our structure of the lactosylceramide–GLTP complex (1.95 Å) can be compared with the structures of the ganglioside GM2–CD1b (2.8 Å)16
and sulphated-galactosylceramide–CD1a (2.15 Å)17
complexes. The recognition specificity associated with the network of hydrogen-bonding interactions involving the glycosyl and ceramide moieties distinguishes the GLTP complex () from the corresponding CD1a complex, which involved far fewer hydrogen-bonding recognition contacts17
. Further, whereas the dual-chain lipid inserts into a common hydrophobic tunnel in the GLTP complex (), the sphingoid-base and N
-acyl chains insert into separate interconnecting hydrophobic pockets in the CD1a complex17
. It therefore seems that unique glycolipid–protein interactions are associated with distinct functional events, namely glycolipid transfer in the GLTP complex in contrast to glycolipid presentation in the CD1b16
major histocompatibility complexes.
Superposition of the apo-GLTP and lactosylceramide-bound GLTP structures reveals essentially no difference in the headgroup recognition centre but provides clear evidence of localized conformational differences related to the hydrophobic channel (). The differences, positioned along the front layer of GLTP, are associated with interhelical loops α1–α2 and α6–α7, helix α6 and the amino terminus of helix α2 (). The conformational consequences of glycolipid acquisition are: first, bending of the α2-helix; second, rearrangement of the α1–α2 loop, accompanied by the appearance of a new 310
helix near the N terminus of the α2 helix; third, outward displacement of the α6 helix by 2.7 Å; and fourth, shortening of the C terminus of the α6 helix that is compensated for by formation of a new 310
helix ( and ; Supplementary Fig S1
, Supplementary Fig S2c
and Supplementary Fig S8c
). Acquisition of glycolipid by GLTP results in amino acids Pro 44, Ile 45, Val 41, Phe 42, Leu 37 and Ile 147, Phe 148, Ala 151, Leu 152, associated with separate regions, moving farther apart with respect to each other (), while the side chains of Phe 148 and Leu 152 rotate around their Cα–Cβ bonds to move from an inside-facing orientation in apo-GLTP () to a swung-out orientation in lactosylceramide-bound GLTP (). The net result of all movements and rearrangements is an opening and expansion of a hydrophobic tunnel () when GLTP acquires the lactosylceramide molecule from the membrane.
Figure 3 Comparison of apo-GLTP and lactosylceramide-bound GLTP. a, Superposition of apo-GLTP and lactosylceramide-bound GLTP structures, with similarly positioned α-helices shown in a cylinder representation, while 310 helices and loop segments are coloured (more ...)
It is difficult to imagine penetration of the lipid chains into the protein interior via an ‘end-on digging or tunnelling mechanism’ (pushing the hydrophobic walls apart to get the terminal methyl groups buried deeper and deeper). Rather, the GLTP structure most probably provides a cleft-like gate, which could open and close to let the lipid chains in and out. Indeed, the crystallographic B
-factor distribution reveals local conformational mobility within the lactosylceramide-bound GLTP (; highest B
-factors in red). A comparative analysis for apo-GLTP and lactosylceramide-bound GLTP structures suggests that the loops α1–α2 and α5–α6, helix 6 and possibly helix 7 could form the lipid ‘gate’ in GLTP. The proper orientation of the glycolipid recognition centre and the ‘gate’ region with respect to the membrane surface might be aided by Trp 142 as well as Tyr 81, Tyr 153, Tyr 157 and Tyr 207, nearby residues known to interact favourably with membrane interfaces22,23
. The GLTP-membrane interface could potentially provide a favourable environment for protein conformational changes that enhance the binding and desorption of glycolipid amphiphiles from the membrane into GLTP.
To our knowledge, the results on apo-GLTP and lactosylceramide-bound GLTP are the first demonstration of a two-layered α-helical motif functioning to accommodate a glycolipid by the creation and expansion of a ‘moulded-to-fit’ hydrophobic tunnel () that does not pre-exist in the free protein (). This architecture differs from all previously reported motifs of lipid-binding and lipid-transfer proteins, which are either α-helical with extensive disulphide crosslinking or contain extensive β-structure. Thus, GLTP represents a new emerging family of sphingolipid-binding/transfer proteins. Our results provide a potential framework for understanding how GLTPs, with their structurally conservative and conformationally flexible segments, acquire and release membrane glycolipids during lipid transport and presentation processes.