The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell–cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers—but the basis for CpE’s selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin–enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin–CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

function of LDs. While much work has focused on peripheral and integral membrane proteins, the mechanism by which LD binding proteins recognize and target to LDs is still poorly understood (1). This is especially true for dedicated LD binding proteins of the perilipin family, i.e., perilipin 1 through 5, that function in the biogenesis and metabolism (lipolysis) of LDs. No work has directly investigated the interaction of a perilipin family member with a phospholipid monolayer interface. In order to address how this family of proteins interacts with lipid interfaces, we investigated the interaction of perilipin 3 with phospholipid monolayers at the air-buffer interface. We chose perilipin 3 because it is found in the cytosol as well as on the LD surface, and because previous work has characterized the structure and LD association of the protein (1-3).In addition to providing cellular energy, LDs take part in many other cellular functions, including signal transduction, formation of new cellular membranes, hormone synthesis, and lipid trafficking (4-8). Under certain physiological conditions, LDs have been found to act as storehouses for several different types of enzymes and proteins, including histones (9, 10), and they also facilitate virus replication (11-13). An understanding of how proteins Lipid droplets (LDs) are dynamic cell organelles that carry out a multitude of cellular functions vital for life, and protein-lipid interactions are crucial to the structure and

Neutral lipid transport in mammals is complicated involving many types of apolipoprotein. The exchangeable apolipoproteins mediate the transfer of hydrophobic lipids between tissues and particles, and bind to cell surface receptors. Amphipathic α-helices form a common structural motif that facilitates their lipid binding and exchangeability. ApoLp-III, the only exchangeable apolipoprotein found in insects, is a model amphipathic α-helix bundle protein and its three dimensional structure and function mimics that of the mammalian proteins apoE and apoAI. Even the intracellular exchangeable lipid droplet protein TIP47/perilipin 3 contains an α-helix bundle domain with high structural similarity to that of apoE and apoLp-III. Here, we investigated the interaction of apoLp-III from Locusta migratoria with lipid monolayers. Consistent with earlier work we find that insertion of apoLp-III into fluid lipid monolayers is highest for diacylglycerol. We observe a preference for saturated and more highly ordered lipids, suggesting a new mode of interaction for amphipathic α-helix bundles. X-ray reflectivity shows that apoLp-III unfolds at a hydrophobic interface and flexible loops connecting the amphipathic α-helices stay in solution. X-ray diffraction indicates that apoLp-III insertion into diacylglycerol monolayers induces additional ordering of saturated acyl-chains. These results thus shed important new insight into the protein-lipid interactions of a model exchangeable apolipoprotein with significant implications for its mammalian counterparts.

The interactions between proteins and biological membranes play an important role in many aspects of biochemistry. Thus, the ability to monitor the structural dynamics of membrane proteins is of great interest. In general, hydrophobic peptides are disordered and tend to aggregate in aqueous environments. For example, the amyloid-b (Ab) peptide, a major component of the insoluble plaques associated with Alzheimer's disease, is intrinsically disordered under physiological conditions. However, Ab adopts a-helical structure in membrane mimicking environments. This is not surprising as the hydrophobic region derives from the transmembrane (TM) region of the amyloid precursor protein. More interesting is the fact that low concentrations of organic solvents or surfactants promote aggregation and formation of b-sheet structure. The ability to simultaneously monitor lipid association and study its effect on the secondary structure of amyloidogenic proteins would be of great interest. Recent studies have shown a significantly enhanced amide I mode in the deep-UV resonance Raman (dUVRR) spectra of transmembrane proteins is a marker for lipid association. Positively charged hydrophobic peptides, including the hydrophobic Ab(25-40) fragment of Ab, spontaneously insert into anionic lipid bilayers. The application of dUVRR spectroscopy to monitor lipid-association, insertion and folding of these peptides will be presented.

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