The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.

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Membrane proteins and lipids coevolved to yield unique coregulatory mechanisms. Inward-rectifier K+ (Kir) channels are often activated by anionic lipids endemic to their native membranes and require accessible water along their K+ conductance pathway. To better understand Kir channel activation, we target multiple mutants of the Kir channel KirBac1.1 via solid-state nuclear magnetic resonance (SSNMR) spectroscopy, potassium efflux assays, and Förster resonance energy transfer (FRET) measurements. In the I131C stability mutant (SM), we observe an open-active channel in the presence of anionic lipids with greater activity upon addition of cardiolipin (CL). The introduction of three R to Q mutations (R49/151/153Q (triple Q mutant, TQ)) renders the protein inactive within the same activating lipid environment. Our SSNMR experiments reveal a stark reduction of lipid–protein interactions in the TQ mutant explaining the dramatic loss of channel activity. Water-edited SSNMR experiments further determined the TQ mutant possesses greater overall solvent exposure in comparison to wild-type but with reduced water accessibility along the ion conduction pathway, consistent with the closed state of the channel. These experiments also suggest water is proximal to the selectivity filter of KirBac1.1 in the open-activated state but that it may not directly enter the selectivity filter. Our findings suggest lipid binding initiates a concerted rotation of the cytoplasmic domain subunits, which is stabilized by multiple intersubunit salt bridges. This action buries ionic side chains away from the bulk water, while allowing water greater access to the K+ conduction pathway. This work highlights universal membrane protein motifs, including lipid–protein interactions, domain rearrangement, and water-mediated diffusion mechanisms.

KirBac1.1 is a prokaryotic inward-rectifier K þ channel from Burkholderia pseudomallei. It shares the common inward-rectifier K þ channel fold with eukaryotic channels, including conserved lipid-binding pockets. Here, we show that KirBac1.1 changes the phase properties and dynamics of the surrounding bilayer. KirBac1.1 was reconstituted into vesicles composed of 13 C-enriched biological lipids. Two-dimensional liquid-state and solid-state NMR experiments were used to assign lipid 1 H and 13 C chemical shifts as a function of lipid identity and conformational degrees of freedom. A solid-state NMR temperature series reveals that KirBac1.1 lowers the primary thermotropic phase transition of Escherichia coli lipid membranes while introducing both fluidity and internal lipid order into the fluid phases. In B. thailandensis liposomes, the bacteriohopanetetrol hopanoid, and potentially ornithine lipids, introduce a similar primary lipid-phase transition and liquid-ordered properties. Adding KirBac1.1 to B. thailandensis lipids increases B. thailandensis lipid fluidity while preserving internal lipid order. This synergistic effect of KirBac1.1 in bacteriohopanetetrol-rich membranes has implications for bilayer dynamic structure. If membrane proteins can anneal lipid translational degrees of freedom while preserving internal order, it could offer an explanation to the nature of liquid-ordered protein-lipid organization in vivo.

Cholesterol oligomers reside in multiple membrane protein X-ray crystal structures. Yet, there is no direct link between these oligomers and a biological function. Here we present the structural and functional details of a cholesterol dimer that stabilizes the inactivated state of an inward-rectifier potassium channel KirBac1.1. K + efflux assays confirm that high cholesterol concentration reduces K + conductance. We then determine the structure of the cholesterol-Kir-Bac1.1 complex using Xplor-NIH simulated annealing calculations driven by solid-state NMR distance measurements. These calculations identified an α-α cholesterol dimer docked to a cleft formed by adjacent subunits of the homotetrameric protein. We compare these results to coarse grain molecular dynamics simulations. This is one of the first examples of a cholesterol oligomer performing a distinct biological function and structural characterization of a conserved promiscuous lipid binding region.

The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming β-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid β-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms.

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New linkages for covalent organic frameworks (COFs) have been continuously pursued by chemists as they serve as the structure and property foundation for the materials. Developing new reaction types or modifying known linkages have been the only two methods to create new COF linkages. Herein, we report a novel strategy that uses H3PO3 as a bifunctional catalyst to achieve amine-linked COFs from readily available amine and aldehyde linkers. The acidic proton of H3PO3 catalyzes the imine framework formation, which is then in situ reduced to the amine COF by the reductive P–H moiety. The amine-linked COF outperforms its imine analogue in promoting Knoevenagel condensation because of the more basic sites and higher stability.