Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-β-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.

Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them into a highly confined environment. We show that the enzymatic activity of a model enzyme, horseradish peroxidase (HRP), can be accurately controlled by relaxing its confinement within the cubic phases' water channels, when the aqueous channel diameters are systematically swollen with varying amount of hydration-enhancing sugar ester. The in-meso activity and kinetics of HRP are then systematically investigated by UV-vis spectroscopy, as a function of the size of the aqueous mesophase channels. The enzymatic activity of HRP increases with the swelling of the water channels. In swollen mesophases with water channel diameter larger than the HRP size, the enzymatic activity is more than double that measured in standard mesophases, approaching again the enzymatic activity of free HRP in bulk water. We also show that the physically-entrapped enzymes in the mesophases exhibit a restricted-diffusion-induced initial lag period and report the first observation of in-meso enzymatic kinetics significantly deviating from the normal Michaelis-Menten behaviour observed in free solutions, with deviations vanishing when enzyme confinement is released by swelling the mesophase.

Summary Cotton is white gold across the globe and composed of fiber cells derived from the outer integument of cotton ovules. Fiber elongation uses sucrose as a direct carbon source. The molecular mechanism transcriptionally controlling sucrose transport from ovules into the elongating fibers remains elusive. In this study the involvement of GhMYB212 in the regulation of sucrose transportion into expanding fibers was investigated. GhMYB212 RNAi plants (GhMYB212i) accumulated less sucrose and glucose in developing fibers, and had shorter fibers and a lower lint index. RNA‐seq and protein−DNA binding assays revealed that GhMYB212 was closely linked to the pathways of sucrose and starch transportation and metabolism, directly controling the expression of a sucrose transporter gene GhSWEET12. GhSWEET12 RNAi plants (GhSWEET12i) possessed similar fiber phenotypes to those of GhMYB212i. Exogenous sucrose supplementation in ovule cultures did not rescue the shorter fiber phenotype of GhMYB212i and GhSWEET12i. This finding supported the idea that the attenuated rate of sucrose transport from the outer seed coat into the fibers is responsible for the retardation of fiber elongation. Current investigations support the idea that GhMYB212 functions as the main regulator of fiber elongation by controlling the expression of GhSWEET12, and therefore it is important to study cell expansion and sugar transportation during seed development.