Structural and functional characterizations of the C-terminal domains of CzcD proteins.

Zinc is a potent antimicrobial component of the innate immune response at the host-pathogen interface. Bacteria subvert or resist host zinc insults by metal efflux pathways that include cation diffusion facilitator (CDF) proteins. The structural and functional examination of this protein class has been limited, with only the structures of the zinc transporter YiiP proteins from E. coli and Shewanella oneidensis described to date. Here, we determine the metal binding properties, solution quaternary structures and three dimensional architectures of the C-terminal domains of the metal transporter CzcD proteins from Cupriavidus metallidurans, Pseudomonas aeruginosa and Thermotoga maritima. We reveal significant diversity in the metal-binding properties and structures of these proteins and discover a potential novel mechanism for metal-promoted dimerization for the Cupriavidus metallidurans and Pseudomonas aeruginosa proteins.

The purification of the σFpvI/FpvR20 and σPvdS/FpvR20 protein complexes is facilitated at room temperature.

Bacteria contain sigma (σ) factors that control gene expression in response to various environmental stimuli. The alternative sigma factors σFpvI and σPvdS bind specifically to the antisigma factor FpvR. These proteins are an essential component of the pyoverdine-based system for iron uptake in Pseudomonas aeruginosa. Due to the uniqueness of this system, where the activities of both the σFpvI and σPvdS sigma factors are regulated by the same antisigma factor, the interactions between the antisigma protein FpvR20 and the σFpvI and σPvdS proteins have been widely studied in vivo. However, difficulties in obtaining soluble, recombinant preparations of the σFpvI and σPvdS proteins have limited their biochemical and structural characterizations. In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His6-σFpvI/FpvR1-67, His6-σFpvI/FpvR1-89, His6-σPvdS/FpvR1-67 and His6-σPvdS/FpvR1-89 protein complexes (where FpvR1-67 and FpvR1-89 are truncated versions of FpvR20) at high purities and concentrations, appropriate for biophysical analyses by circular dichroism spectroscopy and analytical ultracentrifugation. These results showed the proteins to be folded in solution and led to the determination of the affinities of the protein-protein interactions within the His6-σFpvI/FpvR1-67 and His6-σPvdS/FpvR1-67 complexes. A comparison of these values with those previously reported for the His6-σFpvI/FpvR1-89 and His6-σPvdS/FpvR1-89 complexes is made.

Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins.

Members of the Drosophila behavior human splicing (DBHS) protein family are nuclear proteins implicated in many layers of nuclear functions, including RNA biogenesis as well as DNA repair. Definitive of the DBHS protein family, the conserved DBHS domain provides a dimerization platform that is critical for the structural integrity and function of these proteins. The three human DBHS proteins, splicing factor proline- and glutamine-rich (SFPQ), paraspeckle component 1 (PSPC1), and non-POU domain-containing octamer-binding protein (NONO), form either homo- or heterodimers; however, the relative affinity and mechanistic details of preferential heterodimerization are yet to be deciphered. Here we report the crystal structure of a SFPQ/PSPC1 heterodimer to 2.3-Å resolution and analyzed the subtle structural differences between the SFPQ/PSPC1 heterodimer and the previously characterized SFPQ homodimer. Analytical ultracentrifugation to estimate the dimerization equilibrium of the SFPQ-containing dimers revealed that the SFPQ-containing dimers dissociate at low micromolar concentrations and that the heterodimers have higher affinities than the homodimer. Moreover, we observed that the apparent dissociation constant for the SFPQ/PSPC1 heterodimer was over 6-fold lower than that of the SFPQ/NONO heterodimer. We propose that these differences in dimerization affinity may represent a potential mechanism by which PSPC1 at a lower relative cellular abundance can outcompete NONO to heterodimerize with SFPQ.

Structural basis of interprotein electron transfer in bacterial sulfite oxidation.

Interprotein electron transfer underpins the essential processes of life and relies on the formation of specific, yet transient protein-protein interactions. In biological systems, the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs), which interact with an electron acceptor for catalytic turnover. Here, we report the structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the SorT/SorU interaction show the complex is dynamic in solution, and that the proteins interact with Kd = 13.5 ± 0.8 µM. The crystal structures of the oxidized SorT and SorU, both in isolation and in complex, reveal the interface to be remarkably electrostatic, with an unusually large number of direct hydrogen bonding interactions. The assembly of the complex is accompanied by an adjustment in the structure of SorU, and conformational sampling provides a mechanism for dissociation of the SorT/SorU assembly.