Summary The OmpF porin from the Escherichia coli outer membrane folds into a trimer of β-barrel, each forming a wide aqueous pore allowing the passage of ions and small solutes. A long loop (L3) carrying multiple acidic residues folds into the β-barrel pore to form a narrow “constriction zone”. A strong and highly conserved charge asymmetry is observed at the constriction zone, with multiple basic residues attached to the wall of the β-barrel (Lys16, Arg42, Arg82 and Arg132) on one side, and multiple acidic residues of L3 (Asp107, Asp113, Glu117, Asp121, Asp126, Asp127) on the other side. Several computational studies have suggested that a strong transverse electric field could exist at the constriction zone as a result of such charge asymmetry, giving rise to separate permeation pathways for cations and anions. To examine this question, OmpF was expressed, purified and crystallized in the P63 space group and two different data sets were obtained at 2.6 Å and 3.0 Å resolution with K+ and Rb+, respectively. The Rb+ soaked crystals were collected at the rubidium anomalous wavelength of 0.8149 Å and cation positions were determined. A PEG molecule was observed in the pore region for both the K+ and Rb+ soaked crystals, where it is interacting with the loop L3. The results reveal the separate pathways of anions and cations across the constriction zone of the OmpF pore.

The anti-termination protein, HutP, regulates the gene expression of the hut (histidine utilization) operon of Bacillus subtilis, by destabilizing the hut terminator RNA located upstream of the coding region encoding l-histidine degradation enzymes. On the basis of biochemical, in vivo and X-ray structural analyses, we now report that HutP uses its dual RNA-binding surfaces to access two XAG-rich regions (sites I and II) within the terminator RNA to mediate the destabilization process. In this process, HutP initiates destabilization at the 5′-end of its mRNA by binding to the first XAG-rich region (site I) and then accesses the second XAG-rich region (site II), located downstream of the stable G-C-rich segment of the terminator stem. By this action, HutP appears to disrupt the G-C-rich terminator stem, and thus prevents premature termination of transcription in the RNA segment preceding the regions encoding for the histidine degradation enzymes.

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to 106. The discriminating ability of this mini aptamer is calculated as more than 1,000 fold. The equilibrium dissociation constant (K(d)) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.

OmpF, a multiionic porin from Escherichia coli, is a useful protypical model system for addressing general questions about electrostatic interactions in the confinement of an aqueous molecular pore. Here, favorable anion locations in the OmpF pore were mapped by anomalous X-ray scattering of Br− ions from four different crystal structures and compared with Mg2+ sites and Rb+ sites from a previous anomalous diffraction study to provide a complete picture of cation and anion transfer paths along the OmpF channel. By comparing structures with various crystallization conditions, we find that anions bind in discrete clusters along the entire length of the OmpF pore, whereas cations find conserved binding sites with the extracellular, surface-exposed loops. Results from molecular dynamics simulations are consistent with the experimental data and help highlight the critical residues that preferentially contact either cations or anions during permeation. Analysis of these results provides new insights into the molecular mechanisms that determine ion selectivity in OmpF porin.

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