Solid-State NMR Investigations of the MHC II Transmembrane Domains: Topological Equilibria and Lipid Interactions.

The major histocompatibility complex class II (MHC II) membrane proteins are key players in the adaptive immune response. An aberrant function of these molecules is associated with a large number of autoimmune diseases such as diabetes type I and chronic inflammatory diseases. The MHC class II is assembled from DQ alpha 1 and DQ beta 1 which come together as a heterodimer through GXXXG-mediated protein-protein interactions and a highly specific protein-sphingomyelin-C18 interaction motif located on DQA1. This association can have important consequences in regulating the function of these membrane proteins. Here, we investigated the structure and topology of the DQA1 and DQB1 transmembrane helical domains by CD-, oriented 2H and 15N solid-state NMR spectroscopies. The spectra at peptide-to-lipid ratios of 0.5 to 2 mol% are indicative of a topological equilibrium involving a helix crossing the membrane with a tilt angle of about 20° and another transmembrane topology with around 30° tilt. The latter is probably representing a dimer. Furthermore, at the lowest peptide-to-lipid ratio, a third polypeptide population becomes obvious. Interestingly, the DQB1 and to a lesser extent the DQA1 transmembrane helical domains exhibit a strong fatty acyl chain disordering effect on the inner segments of the 2H-labelled palmitoyl chain of POPC bilayers. This phosphatidylcholine disordering requires the presence of sphingomyelin-C18 suggesting that the ensemble of transmembrane polypeptide and sphingolipid exerts positive curvature strain.

Effect of lipid saturation on the topology and oligomeric state of helical membrane polypeptides.

Natural liquid crystalline membranes are made up of many different lipids carrying a mixture of saturated and unsaturated fatty acyl chains. Whereas in the past considerable attention has been paid to cholesterol content, the phospholipid head groups and the membrane surface charge the detailed fatty acyl composition was often considered less important. However, recent investigations indicate that the detailed fatty acyl chain composition has pronounced effects on the oligomerization of the transmembrane helical anchoring domains of the MHC II receptor or the membrane alignment of the cationic antimicrobial peptide PGLa. In contrast the antimicrobial peptides magainin 2 and alamethicin are less susceptible to lipid saturation. Using histidine-rich LAH4 designer peptides the high energetic contributions of lipid saturation in stabilizing transmembrane helical alignments are quantitatively evaluated. These observations can have important implications for the biological regulation of membrane proteins and should be taken into considerations during biophysical or structural experiments.

Lipid saturation and head group composition have a pronounced influence on the membrane insertion equilibrium of amphipathic helical polypeptides.

The histidine-rich peptides of the LAH4 family were designed using cationic antimicrobial peptides such as magainin and PGLa as templates. The LAH4 amphipathic helical sequences exhibit a multitude of interesting biological properties such as antimicrobial activity, cell penetration of a large variety of cargo and lentiviral transduction enhancement. The parent peptide associates with lipid bilayers where it changes from an orientation along the membrane interface into a transmembrane configuration in a pH-dependent manner. Here we show that LAH4 adopts a transmembrane configuration in fully saturated DMPC membranes already at pH 3.5, i.e. much below the pKa of the histidines whereas the transition pH in POPC correlates closely with histidine neutralization. In contrast in POPG membranes the in-planar configuration is stabilized by about one pH unit. The differences in pH can be converted into energetic contributions for the in-plane to transmembrane transition equilibrium, where the shift in the transition pH due to lipid saturation corresponds to energies which are otherwise obtained by the exchange of several cationic with hydrophobic residues. A similar dependence on lipid saturation has also been observed when the PGLa and magainin antimicrobial peptides interact within lipid bilayers suggesting that the quantitative evaluation presented in this paper also applies to other membrane polypeptides.

Solid-State NMR Approaches to Study Protein Structure and Protein-Lipid Interactions.

Solid-state NMR spectroscopy has been developed for the investigation of membrane-associated polypeptides and remains one of the few techniques to reveal high-resolution structural information in liquid-disordered phospholipid bilayers. In particular, oriented samples have been used to investigate the structure, dynamics and topology of membrane polypeptides. Much of the previous solid-state NMR work has been developed and performed on peptides but the technique is constantly expanding towards larger membrane proteins. Here, a number of protocols are presented describing among other the reconstitution of membrane proteins into oriented membranes, monitoring membrane alignment by 31P solid-state NMR spectroscopy, investigations of the protein by one- and two-dimensional 15N solid-state NMR and measurements of the lipid order parameters using 2H solid-state NMR spectroscopy. Using such methods solid-state NMR spectroscopy has revealed a detailed picture of the ensemble of both lipids and proteins and their mutual interdependence in the bilayer environment.

Structure, Topology, and Dynamics of Membrane-Inserted Polypeptides and Lipids by Solid-State NMR Spectroscopy: Investigations of the Transmembrane Domains of the DQ Beta-1 Subunit of the MHC II Receptor and of the COP I Protein p24.

MHC class II receptors carry important function in adaptive immunity and their malfunctioning is associated with diabetes type I, chronic inflammatory diseases and other autoimmune diseases. The protein assembles from the DQ alpha-1 and DQ beta-1 subunits where the transmembrane domains of these type I membrane proteins have been shown to be involved in homo- and heterodimer formation. Furthermore, the DQ alpha 1 chain carries a sequence motif that has been first identified in the context of p24, a protein involved in the formation of COPI vesicles of the intracellular transport machinery, to specifically interact with sphingomyelin-C18 (SM-C18). Here we investigated the membrane interactions and dynamics of DQ beta-1 in liquid crystalline POPC phospholipid bilayers by oriented 15N solid-state NMR spectroscopy. The 15N resonances are indicative of a helical tilt angle of the membrane anchor sequence around 20°. Two populations can be distinguished by their differential dynamics probably corresponding the DQ beta-1 mono- and homodimer. Whereas, this equilibrium is hardly affected by the addition of 5 mole% SM-C18 a single population is visible in DMPC lipid bilayers suggesting that the lipid saturation is an important parameter. Furthermore, the DQ alpha-1, DQ beta-1 and p24 transmembrane helical domains were reconstituted into POPC or POPC/SM-C18 lipid bilayers where the fatty acyl chain of either the phosphatidylcholine or of the sphingolipid have been deuterated. Interestingly in the presence of both sphingolipid and polypeptide a strong decrease in the innermost membrane order of the POPC palmitoyl chain is observed, an effect that is strongest for DQ beta-1. In contrast, for the first time the polypeptide interactions were monitored by deuteration of the stearoyl chain of SM-C18. The resulting 2H solid-state NMR spectra show an increase in order for p24 and DQ alpha-1 which both carry the SM recognition motif. Thereby the data are suggestive that SM-C18 together with the transmembrane domains form structures imposing positive curvature strain on the surrounding POPC lipids. This effect is attenuated when SM-C18 is recognized by the protein.

Solid-state NMR structural investigations of peptide-based nanodiscs and of transmembrane helices in bicellar arrangements.

The membrane topology of the peptide 18A, a derivative of apolipoprotein A-I, is investigated in structural detail. Apolipoprotein A-I is the dominant protein component of high density lipoproteins with important functions in cholesterol metabolism. 18A (Ac-DWLKA FYDKV AEKLK EAF- NH2) was designed to mimic the structure of tandem domains of class A amphipathic helices and has served as a lead peptide for biomedical applications. At low peptide-to-lipid ratios 18A partitions into phosphatidylcholine membranes with helix topologies parallel to the membrane surface, an alignment that is maintained when disc-like bicelles form at higher peptide-to-lipid ratios. Notably, the bicelles interact cooperatively with the magnetic field of the NMR spectrometer, thus the bilayer normal is oriented perpendicular to the magnetic field direction. A set of peptides that totals four 15N or 2H labelled positions of 18A allowed the accurate analysis of tilt and azimuthal angles relative to the membrane surface under different conditions. The topology agrees with a double belt arrangement forming a rim that covers the hydrophobic fatty acyl chains of the bicelles. In another set of experiments, it was shown that POPC nanodiscs prepared in the presence of diisobutylene/maleic acid (DIBMA) polymers can also be made to align in the magnetic field. Finally, the transmembrane domains of the DQ alpha-1 and DQ beta-1 subunits of the major histocomptability complex (MHC) class II have been prepared and reconstituted into magnetically oriented bicelles for NMR structural analysis.