Fellow: Luana Ferrara
The increasing of multidrug resistance (MDR) amongst bacteria is a global concern for public health. MDR is most serious in Gram negative bacteria due to their additional outer membrane, whose low permeability makes the influx of antibiotics more difficult. Embedded in the outer membrane, water filled channels, known as porins, represent a “gate” through which small hydrophilic molecules such as sugar, drugs and chemicals can enter the cell. As a resistance mechanism, porins can be down-regulated and/or mutated when bacteria are pressured by antibiotics. A better understanding of porins structure will help us to clarify how they interact with antibiotics and hence accelerate the design of new drugs.
My goals are to determine the structure of MOMP and Omp50 porins from the pathogen Campylobacter jejuni and Omp35, Omp36 and Omp37 porins from the pathogen Enterobacter aerogenes. To do so, all the proteins have been expressed in a suitable system, a purification protocol have been optimized and the purified proteins have been crystallized. MOMP, Omp35, Omp36 and Omp37 have been successfully solved at 2.9, 2.8, 2.4 and 2.5 Å, respectively. The structures will serve as entry for all-atom modeling of the antibiotic entry.
MOMP has been purified from the native Campylobacter jejuni. Culture preparation and purification were performed in Prof. Jean-Marie Pages‘s lab (secondments). The purified protein was then successfully crystallized and the structure solved at 2.89Å resolution. The structure shows that MOMP is a trimeric 18-stranded porin with the typical β-barrel character (Fig.1). MOMP structure was also solved using the protein overexpressed in a E. coli system. Comparison of the two structures did not show any significant difference. Also, conductance profiles in planar membrane of both native and recombinant MOMP are the same (native MOMP G= 2.3±0.3 nS; recombinant MOMP G=2.2±0.2 nS) (secondment at Jacobs University). The structure has identified a Ca2+ bound at the constriction zone, which molecular dynamics and single channel experiments have revealed to be important for stability. Modelling of the transit of ciprofloxacin, an antibiotic of choice for treating Campylobacter infection, through the pore of MOMP reveals a trajectory that is dependent upon the metal ion.
Figure 1. MOMP trimer view from the outside the cell. The molecule of calcium is colored in blue (left). A detailed view of the amino acids involved in calcium binding site same orientation as 1d. The Fo-Fc and 2Fo-Fc electron density maps at 5σ and 2σ respectively shown the final refined coordinates. The phases for calculation of the map were based on a model that had never included the metal ion (right).
Omp35, Omp36 and Omp37 have been cloned and purified. All three porins have been crystallized and solved at 2.8, 2.4 and 2.5 Å, respectively. The structures show that Omp35, Omp36 and Omp37 are trimeric 16-stranded porin with the typical β-barrel character (Fig. 2).
Figure 2. Omp35, Omp36 and Omp37 view from the side (top) and view from outside the cell (bottom).