A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA.

Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM-focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors.

Double-stranded DNA viruses use ATP-powered molecular motors to package their genomic DNA. To ensure efficient genome encapsidation, these motors regulate functional transitions between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes oligomerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination. Taken together, these results provide insight into the evolution of functional transitions in viral dsDNA packaging motors.

Structure and dynamics of the fish eye lens protein, γM7-crystallin.

The vertebrate eye lens contains high concentrations of crystallins. The dense lenses of fish are particularly abundant in a class called γM-crystallin whose members are characterized by an unusually high methionine content and partial loss of the four tryptophan residues conserved in all γ-crystallins from mammals which are proposed to contribute to protection from UV-damage. Here, we present the structure and dynamics of γM7-crystallin from zebrafish (Danio rerio). The solution structure shares the typical two-domain, four-Greek-key motif arrangement of other γ-crystallins, with the major difference noted in the final loop of the N-terminal domain, spanning residues 65-72. This is likely due to the absence of the conserved tryptophans. Many of the methionine residues are exposed on the surface but are mostly well-ordered and frequently have contacts with aromatic side chains. This may contribute to the specialized surface properties of these proteins that exist under high molecular crowding in the fish lens. NMR relaxation data show increased backbone conformational motions in the loop regions of γM7 compared to those of mouse γS-crystallin and show that fast internal motion of the interdomain linker in γ-crystallins correlates with linker length. Unfolding studies monitored by tryptophan fluorescence confirm results from mutant mouse γS-crystallin and show that unfolding of a ßγ-crystallin domain likely starts from unfolding of the variable loop containing the more fluorescently quenched tryptophan residue, resulting in a native-like unfolding intermediate.

Characterization of a transient unfolding intermediate in a core mutant of γS-crystallin.

In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-Crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows the adoption of a native fold but renders the protein susceptible to temperature- and concentration-dependent aggregation, including fibril formation. Hydrogen/deuterium exchange and denaturant unfolding studies of this mutant protein (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here, we used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea, Opj forms a stable and partially unfolded entity that is characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill T(2)-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold can explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein, allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light-scattering centers in the cataractous lens.

A single destabilizing mutation (F9S) promotes concerted unfolding of an entire globular domain in gammaS-crystallin.

Conformational change and aggregation of native proteins are associated with many serious age-related and neurological diseases. gammaS-Crystallin is a highly stable, abundant structural component of vertebrate eye lens. A single F9S mutation in the N-terminal domain of mouse gammaS-crystallin causes the severe Opj cataract, with disruption of cellular organization and appearance of fibrillar structures in the lens. Although the mutant protein has a near-native fold at room temperature, significant increases in hydrogen/deuterium exchange rates were observed by NMR for all the well-protected beta-sheet core residues throughout the entire N-terminal domain of the mutant protein, resulting in up to a 3.5-kcal/mol reduction in the free energy of the folding/unfolding equilibrium. No difference was detected for the C-terminal domain. At a higher temperature, this effect further increases to allow for a much more uniform exchange rate among the N-terminal core residues and those of the least well-structured surface loops. This suggests a concerted unfolding intermediate of the N-terminal domain, while the C-terminal domain stays intact. Increasing concentrations of guanidinium chloride produced two transitions for the Opj mutant, with an unfolding intermediate at approximately 1 M guanidinium chloride. The consequence of this partial unfolding, whether by elevated temperature or by denaturant, is the formation of thioflavin T staining aggregates, which demonstrated fibril-like morphology by atomic force microscopy. Seeding with the already unfolded protein enhanced the formation of fibrils. The Opj mutant protein provides a model for stress-related unfolding of an essentially normally folded protein and production of aggregates with some of the characteristics of amyloid fibrils.

Solution structure of RCL, a novel 2'-deoxyribonucleoside 5'-monophosphate N-glycosidase.

RCL is an enzyme that catalyzes the N-glycosidic bond cleavage of purine 2'-deoxyribonucleoside 5'-monophosphates, a novel enzymatic reaction reported only recently. In this work, we determined the solution structure by multidimensional NMR and provide a structural framework to elucidate its mechanism with computational simulation. RCL is a symmetric homodimer, with each monomer consisting of a five-stranded parallel beta-sheet sandwiched between five alpha-helices. Three of the helices form the dimer interface, allowing two monomers to pack side by side. The overall architecture featuring a Rossmann fold is topologically similar to that of deoxyribosyltransferases, with major differences observed in the putative substrate binding pocket and the C-terminal tail. The latter is seemingly flexible and projecting away from the core structure in RCL, but loops back and is positioned at the bottom of the neighboring active site in the transferases. This difference may bear functional implications in the context of nucleobase recognition involving the C-terminal carboxyl group, which is only required in the reverse reaction by the transferases. It was also noticed that residues around the putative active site show significant conformational variation, suggesting that protein dynamics may play an important role in the enzymatic function of apo-RCL. Overall, the work provides invaluable insight into the mechanism of a novel N-glycosidase from the structural point of view, which in turn will allow rational anti-tumor and anti-angiogenesis drug design.

Keratin 2e: a marker for murine nipple epidermis.

Mesenchyme-derived signals influence the unique keratinization and appendage formation programs in specialized skin regions. Interactions between primary mammary mesenchyme and epidermal cells result in the formation of the nipple; however, it is unclear whether this represents a site of regionally specialized epidermis. We profiled the ultrastructure and keratin expression of the murine nipple, and the ventral skin of the K14-parathyroid hormone-related protein (PTHrP) transgenic mouse, which models nipple formation. We found the murine nipple and ventral K14-PTHrP epidermis display expanded suprabasal and granular layers, as well as a thickened cornified layer compared to ventral skin of wild-type littermates. We also observed increased levels of filaggrin in extracts from the ventral epidermis of the K14-PTHrP mouse when compared to that of wild-type littermates. Keratin 2e, previously reported to be expressed in various specialized epidermal sites in the mouse, is expressed in the nipple and the ventral skin of the K14-PTHrP mouse. Keratinocytes grown from the ventral epidermis of the K14-PTHrP mouse or wild-type littermates exhibited identical expression of epidermal markers in vitro, suggesting that the modulated differentiation profile observed in the nipple or the ventral K14-PTHrP skin was dependent on interactions with fibroblasts. The lack of appendages, altered stratification pattern and expression of a specialized keratin suggests that the murine nipple is an example of regionally specialized epidermis.