Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

Real-Time Biosynthetic Reaction Monitoring Informs the Mechanism of Action of Antibiotics.

The rapid spread of drug-resistant pathogens and the declining discovery of new antibiotics have created a global health crisis and heightened interest in the search for novel antibiotics. Beyond their discovery, elucidating mechanisms of action has necessitated new approaches, especially for antibiotics that interact with lipidic substrates and membrane proteins. Here, we develop a methodology for real-time reaction monitoring of the activities of two bacterial membrane phosphatases, UppP and PgpB. We then show how we can inhibit their activities using existing and newly discovered antibiotics such as bacitracin and teixobactin. Additionally, we found that the UppP dimer is stabilized by phosphatidylethanolamine, which, unexpectedly, enhanced the speed of substrate processing. Overall, our results demonstrate the potential of native mass spectrometry for real-time biosynthetic reaction monitoring of membrane enzymes, as well as their in situ inhibition and cofactor binding, to inform the mode of action of emerging antibiotics.

How Clavulanic Acid Inhibits Serine ß-Lactamases.

Clavulanic acid is a medicinally important inhibitor of serine ß-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC­MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.

Formation of Lipid-Bilayer Nanodiscs by Diisobutylene/Maleic Acid (DIBMA) Copolymer.

Membrane proteins usually need to be extracted from their native environment and separated from other membrane components for in-depth in vitro characterization. The use of styrene/maleic acid (SMA) copolymers to solubilize membrane proteins and their surrounding lipids into bilayer nanodiscs is an attractive approach toward this goal. We have recently shown that a diisobutylene/maleic acid (DIBMA) copolymer similarly solubilizes model and cellular membranes but, unlike SMA(3:1), has a mild impact on lipid acyl-chain order and thermotropic phase behavior. Here, we used fluorescence spectroscopy, small-angle X-ray scattering, size-exclusion chromatography, dynamic light scattering, and 31P nuclear magnetic resonance spectroscopy to examine the self-association of DIBMA and its membrane-solubilization properties against lipids differing in acyl-chain length and saturation. Although DIBMA is less hydrophobic than commonly used SMA(3:1) and SMA(2:1) copolymers, it efficiently formed lipid-bilayer nanodiscs that decreased in size with increasing polymer/lipid ratio while maintaining the overall thickness of the membrane. DIBMA fractions of different molar masses were similarly efficient in solubilizing a saturated lipid. Coulomb screening at elevated ionic strength or reduced charge density on the polymer at low pH enhanced the solubilization efficiency of DIBMA. The free-energy penalty for transferring phospholipids from vesicular bilayers into nanodiscs became more unfavorable with increasing acyl-chain length and unsaturation. Altogether, these findings provide a rational framework for using DIBMA in membrane-protein research by shedding light on the effects of polymer and lipid properties as well as experimental conditions on membrane solubilization.

Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer.

Once removed from their natural environment, membrane proteins depend on membrane-mimetic systems to retain their native structures and functions. To this end, lipid-bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane-protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl-chain order, does not interfere with optical spectroscopy in the far-UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations.

Mass spectrometry of intact membrane proteins: shifting towards a more native-like context.

Integral membrane proteins are involved in a plethora of biological processes including cellular signalling, molecular transport, and catalysis. Many of these functions are mediated by non-covalent interactions with other proteins, substrates, metabolites, and surrounding lipids. Uncovering such interactions and deciphering their effect on protein activity is essential for understanding the regulatory mechanisms underlying integral membrane protein function. However, the detection of such dynamic complexes has proven to be challenging using traditional approaches in structural biology. Native mass spectrometry has emerged as a powerful technique for the structural characterisation of membrane proteins and their complexes, enabling the detection and identification of protein-binding partners. In this review, we discuss recent native mass spectrometry-based studies that have characterised non-covalent interactions of membrane proteins in the presence of detergents or membrane mimetics. We additionally highlight recent progress towards the study of membrane proteins within native membranes and provide our perspective on how these could be combined with recent developments in instrumentation to investigate increasingly complex biomolecular systems.

Structural basis of telomeric nucleosome recognition by shelterin factor TRF1.

Shelterin and nucleosomes are the key players that organize mammalian chromosome ends into the protective telomere caps. However, how they interact with each other at telomeres remains unknown. We report cryo-electron microscopy structures of a human telomeric nucleosome both unbound and bound to the shelterin factor TRF1. Our structures reveal that TRF1 binds unwrapped nucleosomal DNA ends by engaging both the nucleosomal DNA and the histone octamer. Unexpectedly, TRF1 binding shifts the register of the nucleosomal DNA by 1 bp. We discovered that phosphorylation of the TRF1 C terminus and a noncanomical DNA binding surface on TRF1 are critical for its association with telomeric nucleosomes. These insights into shelterin-chromatin interactions have crucial implications for understanding telomeric chromatin organization and other roles of shelterin at telomeres including replication and transcription.

Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein N-acyltransferase.

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein N-acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo-electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt's substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.

Peptidoglycan biosynthesis is driven by lipid transfer along enzyme-substrate affinity gradients.

Maintenance of bacterial cell shape and resistance to osmotic stress by the peptidoglycan (PG) renders PG biosynthetic enzymes and precursors attractive targets for combating bacterial infections. Here, by applying native mass spectrometry, we elucidate the effects of lipid substrates on the PG membrane enzymes MraY, MurG, and MurJ. We show that dimerization of MraY is coupled with binding of the carrier lipid substrate undecaprenyl phosphate (C55-P). Further, we demonstrate the use of native MS for biosynthetic reaction monitoring and find that the passage of substrates and products is controlled by the relative binding affinities of the different membrane enzymes. Overall, we provide a molecular view of how PG membrane enzymes convey lipid precursors through favourable binding events and highlight possible opportunities for intervention.

Lipids mediate supramolecular outer membrane protein assembly in bacteria.

ß Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric ß barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.

Insights into SusCD-mediated glycan import by a prominent gut symbiont.

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the ß2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.

Thermodynamics of nanodisc formation mediated by styrene/maleic acid (2:1) copolymer.

Styrene/maleic acid copolymers (SMA) have recently attracted great interest for in vitro studies of membrane proteins, as they self-insert into and fragment biological membranes to form polymer-bounded nanodiscs that provide a native-like lipid-bilayer environment. SMA copolymers are available in different styrene/maleic acid ratios and chain lengths and, thus, possess different charge densities, hydrophobicities, and solubilisation properties. Here, we studied the equilibrium solubilisation properties of the most commonly used copolymer, SMA(2:1), by monitoring the formation of nanodiscs from phospholipid vesicles using 31P nuclear magnetic resonance spectroscopy, dynamic light scattering, and differential scanning calorimetry. Comparison of SMA(2:1) phase diagrams with those of SMA(3:1) and diisobutylene/maleic acid (DIBMA) revealed that, on a mass concentration scale, SMA(2:1) is the most efficient membrane solubiliser, despite its relatively mild effects on the thermotropic phase behaviour of solubilised lipids. In contrast with previous kinetic studies, our equilibrium experiments demonstrate that the solubilisation of phospholipid bilayers by SMA(2:1) is most efficient at moderately alkaline pH values. This pH dependence was also observed for the solubilisation of native Escherichia coli membranes, for which SMA(2:1) again turned out to be the most powerful solubiliser in terms of the total amounts of membrane proteins extracted.