Annular anionic lipids stabilize the integrin αIIbß3 transmembrane complex.

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbß3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg(995))-ß3(Asp(723)) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg(995)) contacts with ß3(Asp(723)) but paradoxically do not diminish the contribution of αIIb(Arg(995))-ß3(Asp(723)) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbß3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.

Characterization of membrane protein interactions by isothermal titration calorimetry.

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbß3 TM complex affinity, to study the consequences of helix-helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbß3 proceeded with a free energy change of -4.61±0.04kcal/mol at bicelle conditions where the sampling of random helix-helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of >1kcal/mol was obtained from helix-helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbß3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbß3 association and dissociation rates of 4500±100s(-1) and 2.1±0.1s(-1), respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.

Analysis of co-assembly and co-localization of ameloblastin and amelogenin.

Epithelially-derived ameloblasts secrete extracellular matrix proteins including amelogenin, enamelin, and ameloblastin. Complex intermolecular interactions among these proteins are believed to be important in controlling enamel formation. Here we provide in vitro and in vivo evidence of co-assembly and co-localization of ameloblastin with amelogenin using both biophysical and immunohistochemical methods. We performed co-localization studies using immunofluorescence confocal microscopy with paraffin-embedded tissue sections from mandibular molars of mice at 1, 5, and 8 days of age. Commercially-available ameloblastin antibody (M300) against mouse ameloblastin residues 107-407 and an antibody against full-length recombinant mouse (rM179) amelogenin were used. Ameloblastin-M300 clearly reacted along the secretory face of ameloblasts from days 1-8. Quantitative co-localization was analyzed (QCA) in several configurations by choosing appropriate regions of interest (ROIs). Analysis of ROIs along the secretory face of ameloblasts revealed that at day 1, very high percentages of both the ameloblastin and amelogenin co-localized. At day 8 along the ameloblast cells the percentage of co-localization remained high for the ameloblastin whereas co-localization percentage was reduced for amelogenin. Analysis of the entire thickness on day 8 revealed no significant co-localization of amelogenin and ameloblastin. With the progress of amelogenesis and ameloblastin degradation, there was a segregation of ameloblastin and co-localization with the C-terminal region decreased. CD spectra indicated that structural changes in ameloblastin occurred upon addition of amelogenin. Our data suggest that amelogenin-ameloblastin complexes may be the functional entities at the early stage of enamel mineralization.

Insight into α-synuclein plasticity and misfolding from differential micelle binding.

Misfolded species of the 140-residue protein α-synuclein (αS) are implicated in the demise of dopaminergic neurons, resulting in fatal neurodegeneration. The intrinsically unstructured protein binds curved synaptic vesicle membranes in helical conformations but misfolds into amyloid fibrils via ß-sheet interactions. Breaks in helical αS conformation may offer a pathway to transition from helical to sheet conformation. Here, we explore the evolution of broken αS helix conformations formed in complex with SDS and SLAS micelles by molecular dynamics simulations. The population distribution of experimentally observed αS conformations is related to the spatial concentration of intrinsic micelle shape perturbations. For the success of micelle-induced αS folding, we posit the length of the first helical segment formed, which controls micelle ellipticity, to be a key determinant. The degree of micelle curvature relates to the arrangement and segmental motions of helical secondary structure elements. A criterion for assessing the reproduction of such intermediate time scale protein dynamics is introduced by comparing the sampling of experimental and simulated spin label distributions. Finally, at the sites of breaks in the elongated, marginally stable αS helix, vulnerability to forming a transient, intramolecular ß-sheet is identified. Upon subsequent intermolecular ß-sheet pairing, pathological αS amyloid formation from initial helical conformation is thus achievable.

Conformations, dynamics and interactions of di-, tri- and pentamannoside with mannose binding lectin: a molecular dynamics study.

The binding of serum mannose-binding protein A (MBP-A) to high mannose N-linked glycoproteins, present on the surface of microorganism, activates the complement system. It is very important to explore the overall conformations of these ligands in the binding site of the MBP-A, which is very much dependent on the conformation of the manno-di-, tri- and the penta-saccharides that represent the component structures of these high-mannose type oligosaccharides. Herein, we report the possible conformations of α-(1→6)-linked dimannoside, benzyl-substituted trimannoside and core pentamannoside of the N-linked glycan in the binding site of MBP-A, with the help of molecular dynamics simulations. The results indicate that for all three ligands in addition to the non-reducing terminal mannose moiety the reducing moieties also interact with protein. Binding free energy calculations also indicate that the benzyl-substituted trisaccharide has higher affinity in comparison to the methyl substituted one. We have also found some conformers of the pentasaccharide, which have higher binding affinity than the monosaccharide.

Molecular modeling and NMR studies of benzyl substituted mannosyl trisaccharide binding to two mannose-specific lectins: Allium sativam agglutinin I and Concanavalin A.

The interaction of trimannoside, α-benzyl 3, 6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside, 1 with ASAI (Allium sativam agglutinin I, garlic lectin) was studied to reveal the conformational preferences of this ligand in bound-state and detailed binding mode at atomic level. The binding phenomenon was then compared with another well-known mannose-binding lectin, ConA (Concanavalin A). Structural studies of the ligand in free state were done using NMR spectroscopy and Molecular Dynamics simulations. It is found that the substituted-trimannoside can undergo conformational transitions in solution, with one major and one minor conformation per glycosidic linkage (α 1→3 and α 1→6). On the other hand in the bound-state only one of the two major conformations was significantly populated. The role of phenyl ring in the binding process was explored. An extended binding site was observed for the trimannoside in ASAI utilizing the aromatic substituent, which is not seen in ConA. Binding data from difference absorption spectroscopy supported this fact that the binding of benzyl-substituted ligand is tighter with ASAI than ConA. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 952-967, 2010.

Conformational behavior of alpha-D-mannopyranosyl-(1-->6)-alpha,beta-D-mannose complexed with two mannose-binding plant lectins, Allium sativam agglutinin I and concanavalin A, using NMR and molecular modeling techniques.

Herein, we report the intrinsic conformational preferences of alpha-d-Manp-(1-->6)-alpha,beta-d-Manp, (1) in the free state and as two (ASAI and ConA) lectin-bound forms. NMR spectroscopy and molecular dynamics techniques are used as 3D-structural determination tools. In free form disaccharide 1 displays a fair amount of conformational freedom, with one major (phi/psi 95 +/- 30 degrees/195 +/- 20 degrees and one minor (95 +/- 30 degrees/70 +/- 20 degrees) conformations around the glycosidic linkage and around the omega angle, both the gg and gt rotamers are almost equally populated. This is a first report of a three-dimensional structure of 1 bound with ASAI. Both lectins recognize a major phi/psi 95 +/- 30 degrees/200 +/- 30 degrees conformer with the ligand showing more flexibility in the binding site of ConA. Comparison of the mode of binding of the two lectins explains the differences in observed specificities.