Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle.

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.

Stable Picodisc Assemblies from Saposin Proteins and Branched Detergents.

Methods for maintaining membrane proteins in their native state after removal from the lipid bilayer are essential for the study of this important class of biomacromolecules. Common solubilization strategies range from the use of detergents to more complex systems that involve a polypeptide working in concert with lipids or detergents, such as nanodiscs, picodiscs, and peptidiscs, in which an engineered protein or synthetic peptide surrounds the membrane protein along with a lipid sheath. Picodiscs employ the protein saposin A, which naturally functions to facilitate lipid degradation in the lysozome. Saposin A-amphiphile complexes therefore tend to be most stable at acidic pH, which is not optimal for most membrane protein applications. In search of new picodisc assemblies, we have explored pairings of saposin A or other saposin proteins with a range of detergents, and we have identified a number of combinations that spontaneously co-assemble at neutral pH. The resulting picodiscs are stable for weeks and have been characterized by size-exclusion chromatography, native mass spectrometry, and small angle X-ray scattering. The new assemblies are formed by double-tail detergents rather than more traditional single-tail detergents; the double-tail detergents can be seen as structurally intermediate between single-tail detergents and common lipids. In addition to saposin A, an engineered variant of saposin B (designated saposin BW) forms picodisc assemblies. These findings provide a framework for future efforts to solubilize membrane proteins with multiple picodisc systems that were previously unknown.

Retention of Native Quaternary Structure in Racemic Melittin Crystals.

Racemic crystallography has been used to elucidate the secondary and tertiary structures of peptides and small proteins that are recalcitrant to conventional crystallization. It is unclear, however, whether racemic crystallography can capture native quaternary structure, which could be disrupted by heterochiral associations. We are exploring the use of racemic crystallography to characterize the self-assembly behavior of membrane-associated peptides, very few of which have been crystallized. We report a racemic crystal structure of the membrane-active peptide melittin; the new structure allows comparison with a previously reported crystal structure of L-melittin. The tetrameric assembly observed in crystalline L-melittin has been proposed to represent the tetrameric state detected in solution for this peptide. This tetrameric assembly is precisely reproduced in the racemic crystal, which strengthens the conclusion that the tetramer is biologically relevant. More broadly, these findings suggest that racemic crystallography can provide insight on native quaternary structure.