Colin P. Johnson

Ferlins are vesicle trafficking proteins composed of folded C2 domains conjugated by linkers which are largely disordered. Although a role for the for the C2 domains as calcium sensors has been established it remains unclear whether the linkers function beyond acting as passive spacers. We examined the C2A-C2B linker of vertebrate ferlins and found both putative AP2 and SH3 binding short linear motifs (SLiMs) as well as membrane binding sequences for members of the protein family. Specifically for otoferlin we identified an arginine-rich region proximal to a AP2 binding dileucine motif which interacts with negatively charged lipid membranes. Further, the linker region dominated the liposome binding properties of a larger C2A-C2B two-C2 domain segment of otoferlin, suggesting a dominant role in mediating the membrane binding property of the N-terminus. We also found that alternative splicing of the otoferlin C2A-C2B linker adds and additional membrane binding segment and alters the affinity and kinetics of membrane binding. By contrast alternative splicing of the dysferlin linker is not predicted to alter membrane binding but rather alters the number of predicted short linear motifs (SLiMs). In addition we found the otoferlin linker-membrane interaction was sensitive to ionic strength, and simulations suggest positively charged residues including an arginine-rich region mediates binding. We conclude that the C2A-C2B linker of vertebrate ferlins encode both SLiMs which recruit endocytic proteins as well as membrane binding regions that would place the endocytic binding motif proximal to the membrane surface to facilitate endocytosis and synaptic vesicle resupply.

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its β-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its β-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.

Antibiotic biosynthesis by microorganisms is commonly regulated through autoinduction, which allows producers to quickly amplify the production of antibiotics in response to environmental cues. Antibiotic autoinduction generally involves one pathway-specific transcriptional regulator that perceives an antibiotic as a signal and then directly stimulates transcription of the antibiotic biosynthesis genes. Pyoluteorin is an autoregulated antibiotic produced by some Pseudomonas spp. including the soil bacterium Pseudomonas protegens Pf-5. In this study, we show that PltR, a known pathway-specific transcriptional activator of pyoluteorin biosynthesis genes, is necessary but not sufficient for pyoluteorin autoinduction in Pf-5. We found that pyoluteorin is perceived as an inducer by PltZ, a second pathway-specific transcriptional regulator that directly represses the expression of genes encoding a transporter in the pyoluteorin gene cluster. Mutation of pltZ abolished the autoinducing effect of pyoluteorin on the transcription of pyoluteorin biosynthesis genes. Overall, our results support an alternative mechanism of antibiotic autoinduction by which the two pathway-specific transcriptional regulators PltR and PltZ coordinate the autoinduction of pyoluteorin in Pf-5. Possible mechanisms by which PltR and PltZ mediate the autoinduction of pyoluteorin are discussed.

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domai...

C2 domains are the second-most abundant calcium binding module in the proteome. Activity of the muscular dystrophy associated protein dysferlin is dependent on the C2A domain at the N-terminus of the protein, which couples calcium and PI(4,5)P2 binding through an unknown mechanism. Using solution state nuclear magnetic resonance spectroscopy we confirm the phosphoinositide binding site for the domain and find that calcium binding attenuates millisecond to microsecond motions at both in the calcium binding loops and the concave face of the C2A, including a portion of the phosphoinositide binding site. Our results support a model whereby increasing calcium concentrations shift the phosphoinositide binding pocket of C2A into a binding-competent state, allowing for calcium dependent membrane targeting. This model contrasts with the canonical mechanism for C2 domain-phosphoinositide interaction and provides a basis for how pathogenic mutations in the C2A domain result in loss of function and disease.

Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm^–1 related to the C2A domain β-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD₃ and CD₂ symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.