Structural basis of ion - substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY

The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen induced-fit mechanism. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY's induced-fit mechanism is a result of the sodium-dependent formation of the substrate binding site.

Xtrapol8: automatic elucidation of low-occupancy intermediate-states in crystallographic studies

Unstable states studied in kinetic, time-resolved and ligand-based crystallography are often characterized by a low occupancy, hindering structure determination by conventional methods. To automatically extract such structures, we developed Xtrapol8, a program which (i) applies various flavors of Bayesian-statistics weighting to generate the most informative Fourier difference maps; (ii) determines the occupancy of the intermediate state; (iii) calculates various types of extrapolated structure factors, and (iv) refines the corresponding structures.

Surface Engineering of Top7 to Facilitate Structure Determination

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

[Sulfonylbis(bromomethylene)]dibenzene

The title compound, C14H12Br2O2S, crystallizes as the meso isomer of a diastereoisomeric pair. This structure determination was key to determining that the 1,3 elimination of bromine by triphenylphosphine occurs with inversion of the configuration at each of the two chiral carbon atoms. In the crystal, the molecules are linked by weak C—H...O and C—H...Br hydrogen bonds.