Heterogeneous substrate binding in a glutamate transporter homologue

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family, including kinetic models of transport. Recent studies have revealed transport rate heterogeneity in an archaeal glutamate transporter homologue GltPh, inconsistent with simple kinetic models. The structural and mechanistic determinants of this heterogeneity remain undefined. In a mutant form of GltPh, we demonstrate substrate binding heterogeneity in the outward-facing state, modulated by temperature and salts. We observe similar trends in wild-type GltPh that correlate with changes in the transport rate. Extensive cryo-EM analysis of the fully bound mutant GltPh provides multiple potential explanations of heterogeneous substrate binding. At equilibrium, we show subtle differences in tilts of protomers in the outward-facing state and configurations of the substrate-binding pocket. Within seconds of substrate binding, we observe perturbed helical packing of the extracellular half of the substrate-binding domain. Some or all of these may contribute to the heterogeneity observed in binding and transport.

Rare by Natural Selection: Tunable Disulfide-bonded Supramolecular Antimicrobials Peptides

Short helical antimicrobial peptides forming inter-molecular disulfide bonds are selected against in nature, and were utilized here to design switchable antimicrobials via the formation of functional supramolecular fibrils. Specifically, using the available structural information on the stable fibril-forming human LL-37(17-29), we designed cysteine mutations and demonstrated position-dependent controllable antibacterial activity, mediated by their disulfide-dependent self-assembly into ordered fibrils, which proved sensitive to reducing conditions. The crystal structure of the LL-37(17-29) bearing a I24C substitution, located in a critical structural position, revealed disulfide-bonded dimers that further assembled into a fibrillar structure of densely packed helices. The native and mutant peptides both featured a fibril surface with zigzagged hydrophobic and positively charged belts, which likely underlie interactions with bacterial membranes. Yet, they differed in their helical packing arrangement, which corresponded with different levels of activity, with only the mutant being susceptible to reducing conditions. The presented findings promise to advance the design of novel antimicrobials resistant to harsh conditions for coating of surfaces susceptible to pathogens.

Unusual stoichiometry, band structure and band filling in conducting enantiopure radical cation salts of TM-BEDT-TTF showing helical packing of the donors

Electrocrystallization of tetramethyl-bis(ethylenedithio)-tetrathiafulvalene (TM-BEDT-TTF) (1) as pure (S,S,S,S) and (R,R,R,R) enantiomers in the presence of (n-Bu4N)2(Mo6O19) and chloroform or bromoform afforded a series of four isostructural enantiopure radical cation salts...

Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.

Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones

Type VI secretion systems facilitate the delivery of antibacterial effector proteins between neighbouring Gram-negative bacteria. A subset of these effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across the target cell membrane. However, the abundance and distribution of these TMD-containing effectors has remained unknown. Here we report the discovery of prePAAR, a conserved motif found in over 6,000 putative TMD-containing effectors. Based on their differing sizes and number of TMDs these effectors fall into two distinct classes that are unified by their requirement for a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR functions to facilitate proper folding of the downstream PAAR domain, which is required for effector interaction with the T6SS spike. Taken together, our findings define the mechanism of chaperone-assisted secretion of a widespread family of T6SS membrane protein effectors.

Exploring Protein Fold Space

The model of protein folding proposed by Ptitsyn and colleagues involves the accretion of secondary structures around a nucleus. As developed by Efimov, this model also provides a useful way to view the relationships among structures. Although somewhat eclipsed by later databases based on the pairwise comparison of structures, Efimov’s approach provides a guide for the more automatic comparison of proteins based on an encoding of their topology as a string. Being restricted to layers of secondary structures based on beta sheets, this too has limitations which are partly overcome by moving to a more generalised secondary structure lattice that can encompass both open and closed (barrel) sheets as well as helical packing of the type encoded by Murzin and Finkelstein on small polyhedra. Regular (crystalline) lattices, such as close-packed hexagonals, were found to be too limited so pseudo-latticses were investigated including those found in quasicrystals and the Bernal tetrahedron-based lattice that he used to represent liquid water. The Bernal lattice was considered best and used to generate model protein structures. These were much more numerous than those seen in Nature, posing the open question of why this might be.

From achiral to helical bilayer self-assemblies of a 1,3,5-triazine-2,4,6-triphenol-grafted polyanionic cluster: countercation and solvent modulation

An organic-component grafted polyanionic cluster performs assembly structures from regular head to tail bilayer to inverse helical packing upon solvent polarity and counterions.