Characterizing solution surface loop conformational flexibility of the GM2 activator protein.

GM2AP has a ß-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

Crystal structure analysis of phosphatidylcholine-GM2-activator product complexes: evidence for hydrolase activity.

GM2-activator protein (GM2AP) is a lysosomal lipid transfer protein with important biological roles in ganglioside catabolism, phospholipid metabolism, and T-cell activation. Previous studies of crystal structures of GM2AP complexed with the physiological ligand GM2 and platelet activating factor (PAF) have shown binding at two specific locations within the spacious apolar pocket and an ordering effect of endogenous resident lipids. To investigate the structural basis of phospholipid binding further, GM2AP was cocrystallized with phosphatidylcholine (PC), known to interact with GM2AP. Analysis of three crystal forms revealed binding of single chain lipids and fatty acids only and surprisingly not intact PC. The regions of best defined electron density are consistent with the presence of lyso-PC and oleic acid, which constitute deacylation products of PC. Their acyl tails are in stacking contact with shorter, less well-defined stretches of electron density that may represent resident fatty acids. The GM2AP associated hydrolytic activity that generates lyso-PC was further confirmed by mass spectrometry and enzymatic assays. In addition, we report the structures of (i) mutant Y137S, assessing the role of Tyr137 in lipid transfer via the hydrophobic cleft, and (ii) apo-mouse GM2AP, revealing a hydrophobic pocket with a constricted opening. Our structural results provide new insights into the biological functions of GM2AP. The combined effect of hydrolytic and lipid transfer properties has profound implications in cellular signaling.

Evidence for lipid packaging in the crystal structure of the GM2-activator complex with platelet activating factor.

GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2A crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement of two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals' contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.