Coordination of bilayer properties by an inward-rectifier K+ channel is a cooperative process driven by protein-lipid interaction.

Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is difficult to assess due to reciprocal effects between membrane proteins, individual lipids, and membrane architecture. Using solid-state NMR (SSNMR) we previously showed that KirBac1.1, a bacterial Inward-Rectifier K+ channel, nucleates bilayer ordering and microdomain formation through tethering anionic lipids. Conversely, these lipids cooperatively bind cationic residues to activate the channel and initiate K+ flux. The mechanistic details governing the relationship between cooperative lipid loading and bilayer ordering are, however, unknown. To investigate, we generated KirBac1.1 samples with different concentrations of 13C-lableded phosphatidyl glycerol (PG) lipids and acquired a full suite of SSNMR 1D temperature series experiments using the ordered all-trans (AT) and disordered trans-gauche (TG) acyl conformations as markers of bilayer dynamics. We observed increased AT ordered signal, decreased TG disordered signal, and increased bilayer melting temperature with increased PG concentration. Further, we identified cooperativity between ordering and direct binding of PG lipids, indicating KirBac1.1-driven bilayer ordering and microdomain formation is a classically cooperative Hill-type process driven by and predicated upon direct binding of PG lipids. Our results provide unique mechanistic insight into how proteins and lipids in tandem contribute to supramolecular bilayer heterogeneity in the lipid membrane.

Cooperative Gating of a K+ Channel by Unmodified Biological Anionic Lipids Viewed by Solid-State NMR Spectroscopy.

Lipids adhere to membrane proteins to stimulate or suppress molecular and ionic transport and signal transduction. Yet, the molecular details of lipid-protein interaction and their functional impact are poorly characterized. Here we combine NMR, coarse-grained molecular dynamics (CGMD), and functional assays to reveal classic cooperativity in the binding and subsequent activation of a bacterial inward rectifier potassium (Kir) channel by phosphatidylglycerol (PG), a common component of many membranes. Past studies of lipid activation of Kir channels focused primarily on phosphatidylinositol bisphosphate, a relatively rare signaling lipid that is tightly regulated in space and time. We use solid-state NMR to quantify the binding of unmodified 13C-PG to the K+ channel KirBac1.1 in liposomes. This specific lipid-protein interaction has a dissociation constant (Kd) of ∼7 mol percentage PG (ΧPG) with positive cooperativity (n = 3.8) and approaches saturation near 20% ΧPG. Liposomal flux assays show that K+ flux also increases with PG in a cooperative manner with an EC50 of ∼20% ΧPG, within the physiological range. Further quantitative fitting of these data reveals that PG acts as a partial (80%) agonist with fivefold K+ flux amplification. Comparisons of NMR chemical shift perturbation and CGMD simulations at different ΧPG confirm the direct interaction of PG with key residues, several of which would not be accessible to lipid headgroups in the closed state of the channel. Allosteric regulation by a common lipid is directly relevant to the activation mechanisms of several human ion channels. This study highlights the role of concentration-dependent lipid-protein interactions and tightly controlled protein allostery in the activation and regulation of ion channels.

Cardiolipin occupancy profiles of YidC paralogs reveal the significance of respective TM2 helix residues in determining paralog-specific phenotypes.

YidC belongs to an evolutionarily conserved family of insertases, YidC/Oxa1/Alb3, in bacteria, mitochondria, and chloroplasts, respectively. Unlike Gram-negative bacteria, Gram-positives including Streptococcus mutans harbor two paralogs of YidC. The mechanism for paralog-specific phenotypes of bacterial YidC1 versus YidC2 has been partially attributed to the differences in their cytoplasmic domains. However, we previously identified a W138R gain-of-function mutation in the YidC1 transmembrane helix 2. YidC1W138R mostly phenocopied YidC2, yet the mechanism remained unknown. Primary sequence comparison of streptococcal YidCs led us to identify and mutate the YidC1W138 analog, YidC2S152 to W/A, which resulted in a loss of YidC2- and acquisition of YidC1-like phenotype. The predicted lipid-facing side chains of YidC1W138/YidC2S152 led us to propose a role for membrane phospholipids in specific-residue dependent phenotypes of S. mutans YidC paralogs. Cardiolipin (CL), a prevalent phospholipid in the S. mutans cytoplasmic membrane during acid stress, is encoded by a single gene, cls. We show a concerted mechanism for cardiolipin and YidC2 under acid stress based on similarly increased promoter activities and similar elimination phenotypes. Using coarse grain molecular dynamics simulations with the Martini2.2 Forcefield, YidC1 and YidC2 wild-type and mutant interactions with CL were assessed in silico. We observed substantially increased CL interaction in dimeric versus monomeric proteins, and variable CL occupancy in YidC1 and YidC2 mutant constructs that mimicked characteristics of the other wild-type paralog. Hence, paralog-specific amino acid- CL interactions contribute to YidC1 and YidC2-associated phenotypes that can be exchanged by point mutation at positions 138 or 152, respectively.

Strategies for acquisition of resonance assignment spectra of highly dynamic membrane proteins: a GPCR case study.

In protein nuclear magnetic resonance (NMR), chemical shift assignment provides a wealth of information. However, acquisition of high-quality solid-state NMR spectra depends on protein-specific dynamics. For membrane proteins, bilayer heterogeneity further complicates this observation. Since the efficiency of cross-polarization transfer is strongly entwined with protein dynamics, optimal temperatures for spectral sensitivity and resolution will depend not only on inherent protein dynamics, but temperature-dependent phase properties of the bilayer environment. We acquired 1-, 2-, and 3D homo- and heteronuclear experiments of the chemokine receptor CCR3 in a 7:3 phosphatidylcholine:cholesterol lipid environment. 1D direct polarization, cross polarization (CP), and T2' experiments indicate sample temperatures below - 25 °C facilitate higher CP enhancement and longer-lived transverse relaxation times. T1rho experiments indicate intermediate timescales are minimized below a sample temperature of - 20 °C. 2D DCP NCA experiments indicated optimal CP efficiency and resolution at a sample temperature of - 30 °C, corroborated by linewidth analysis in 3D NCACX at - 30 °C compared to - 5 °C. This optimal temperature is concluded to be directly related the lipid phase transition, measured to be between - 20 and 15 °C based on rINEPT signal of all-trans and trans-gauche lipid acyl conformations. Our results have critical implications in acquisition of SSNMR membrane protein assignment spectra, as we hypothesize that different lipid compositions with different phase transition properties influence protein dynamics and therefore the optimal acquisition temperature.

Colossal Anisotropic Thermal Expansion in a Diazo-Functionalized Compound with Switchable Solid-State Behavior.

Achieving substantial anisotropic thermal expansion (TE) in solid-state materials is challenging as most materials undergo volumetric expansion upon heating. Here, we describe colossal, anisotropic TE in crystals of an organic compound functionalized with two azo groups. Interestingly, the material exhibits distinct and switchable TE behaviors within different temperature regions. At high temperature, two-dimensional, area zero TE and colossal, positive linear TE (α=211 MK-1 ) are attained due to dynamic motion, while at low temperature, moderate positive TE occurs in all directions. Investigation of the solid-state motion showed the change in enthalpy and entropy are quite different in the two temperature regions and solid-state NMR experiments support motion in the solid. Cycling experiments demonstrate that the solid-state motions and TE behaviors are completely reversible. These results reveal strategies for designing significant anisotropic and switchable behaviors in solid-state materials.

Cholesterol Biases the Conformational Landscape of the Chemokine Receptor CCR3: A MAS SSNMR-Filtered Molecular Dynamics Study.

Cholesterol directs the pathway of ligand-induced G protein-coupled receptor (GPCR) signal transduction. The GPCR C-C motif chemokine receptor 3 (CCR3) is the principal chemotactic receptor for eosinophils, with roles in cancer metastasis and autoinflammatory conditions. Recently, we discovered a direct correlation between bilayer cholesterol and increased agonist-triggered CCR3 signal transduction. However, the allosteric molecular mechanism escalating ligand affinity and G protein coupling is unknown. To study cholesterol-guided CCR3 conformational selection, we implement comparative, objective measurement of protein architectures by scoring shifts (COMPASS) to grade model structures from molecular dynamics simulations. In this workflow, we scored predicted chemical shifts against 2-dimensional solid-state NMR 13C-13C correlation spectra of U-15N,13C-CCR3 samples prepared with and without cholesterol. Our analysis of trajectory model structures uncovers that cholesterol induces site-specific conformational restraint of extracellular loop (ECL) 2 and conserved motion in transmembrane helices and ECL3 not observed in simulations of bilayers with only phosphatidylcholine lipids. PyLipID analysis implicates direct cholesterol agency in CCR3 conformational selection and dynamics. Residue-residue contact scoring shows that cholesterol biases the conformational selection of the orthosteric pocket involving Y411.39, Y1133.32, and E2877.39. Lastly, we observe contact remodeling in activation pathway residues centered on the initial transmission switch, Na+ pocket, and R3.50 in the DRY motif. Our observations have unique implications for understanding of CCR3 ligand recognition and specificity and provide mechanistic insight into how cholesterol functions as an allosteric regulator of CCR3 signal transduction.

Mutational Insight into Allosteric Regulation of Kir Channel Activity.

Potassium (K+) channels are regulated in part by allosteric communication between the helical bundle crossing, or inner gate, and the selectivity filter, or outer gate. This network is triggered by gating stimuli. In concert, there is an allosteric network which is a conjugated set of interactions which correlate long-range structural rearrangements necessary for channel function. Inward-rectifier K+ (Kir) channels favor inward K+ conductance, are ligand-gated, and help establish resting membrane potentials. KirBac1.1 is a bacterial Kir (KirBac) channel homologous to human Kir (hKir) channels. Additionally, KirBac1.1 is gated by the anionic phospholipid ligand phosphatidylglycerol (PG). In this study, we use site-directed mutagenesis to investigate residues involved in the KirBac1.1 gating mechanism and allosteric network we previously proposed using detailed solid-state NMR (SSNMR) measurements. Using fluorescence-based K+ and sodium (Na+) flux assays, we identified channel mutants with impaired function that do not alter selectivity of the channel. In tandem, we performed coarse grain molecular dynamics simulations, observing changes in PG-KirBac1.1 interactions correlated with mutant channel activity and contacts between the two transmembrane helices and pore helix tied to this behavior. Lipid affinity is closely tied to the proximity of two tryptophan residues on neighboring subunits which lure anionic lipids to a cationic pocket formed by a cluster of arginine residues. Thus, these simulations establish a structural and functional basis for the role of each mutated site in the proposed allosteric network. The experimental and simulated data provide insight into key functional residues involved in gating and lipid allostery of K+ channels. Our findings also have direct implications on the physiology of hKir channels due to conservation of many of the residues identified in this work from KirBac1.1.

Spectroscopic signatures of bilayer ordering in native biological membranes.

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (Lo) and liquid-disordered (Ld) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, Lo microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In 13C-13C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes.

Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling.

Cholesterol as an allosteric modulator of G protein-coupled receptor (GPCR) function is well documented. This quintessential mammalian lipid facilitates receptor-ligand interactions and multimerization states. Functionally, this introduces a complicated mechanism for the homeostatic modulation of GPCR signaling. Chemokine receptors are Class A GPCRs responsible for immune cell trafficking through the binding of endogenous peptide ligands. CCR3 is a CC motif chemokine receptor expressed by eosinophils and basophils. It traffics these cells by transducing the signal stimulated by the CC motif chemokine primary messengers 11, 24, and 26. These behaviors are close to the human immunoresponse. Thus, CCR3 is implicated in cancer metastasis and inflammatory conditions. However, there is a paucity of experimental evidence linking the functional states of CCR3 to the molecular mechanisms of cholesterol-receptor cooperativity. In this vein, we present a means to combine codon harmonization and a maltose-binding protein fusion tag to produce CCR3 from E. coli. This technique yields ∼2.6 mg of functional GPCR per liter of minimal media. We leveraged this protein production capability to investigate the effects of cholesterol on CCR3 function in vitro. We found that affinity for the endogenous ligand CCL11 increases in a dose-dependent manner with cholesterol concentration in both styrene:maleic acid lipid particles (SMALPs) and proteoliposomes. This heightened receptor activation directly translates to increased signal transduction as measured by the GTPase activity of the bound G-protein α inhibitory subunit 3 (Gα i3). This work represents a critical step forward in understanding the role of cholesterol-GPCR allostery in regulation of signal transduction.

In Silico Identification of Cholesterol Binding Motifs in the Chemokine Receptor CCR3.

CC motif chemokine receptor 3 (CCR3) is a Class A G protein-coupled receptor (GPCR) mainly responsible for the cellular trafficking of eosinophils. As such, it plays key roles in inflammatory conditions, such as asthma and arthritis, and the metastasis of many deadly forms of cancer. However, little is known about how CCR3 functionally interacts with its bilayer environment. Here, we investigate cholesterol binding sites in silico through Coarse-Grained Molecular Dynamics (MD) and Pylipid analysis using an extensively validated homology model based on the crystal structure of CCR5. These simulations identified several cholesterol binding sites containing Cholesterol Recognition/Interaction Amino Acid Consensus motif (CRAC) and its inversion CARC motifs in CCR3. One such site, a CARC site in TM1, in conjunction with aliphatic residues in TM7, emerged as a candidate for future investigation based on the cholesterol residency time within the binding pocket. This site forms the core of a cholesterol binding site previously observed in computational studies of CCR2 and CCR5. Most importantly, these cholesterol binding sites are conserved in other chemokine receptors and may provide clues to cholesterol regulation mechanisms in this subfamily of Class A GPCRs.

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization.

The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K+ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K+ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional 'fully codon optimized' construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional.