(342bc) Molecular Dynamic Investigations of E. coli and V. Cholerae Fadl Homologs
AIChE Annual Meeting
Tuesday, November 9, 2021 - 3:30pm to 5:00pm
Using the singularly expressed E. coli FadL protein as template, we performed a Basic Local Alignment Search Tool (BLAST) search to find homologs in V. cholerae. We found 17 unique proteins across the three virulent serotypes and the three most prevalent FadL homologs (NP_233248, NP_230687, and NP_230688) were chosen for further study. We then used I-TASSER5.1 standalone to fold the protein sequences and generate 3D structural models of each of the selected V. cholerae FadL homologs. The generated structures were compared to the known X-ray crystal structure of E. coli FadL (RCSB ID: 1T16) and several key structures outlined by van den Berg3 were identified (Figure 1). The L3 loop, L4 loop, S3 kink, and N terminal hatch domain were found to be aligned, but much of the E. coliâs characteristic loop structures showed deviations between the homologs. To better simulate cell conditions, each structure was inserted into a model V. cholerae outer membrane [outer leaflet of V. cholerae LPS (with 15 O-antigen units) and an inner leaflet of 67% phosphatidylethanolamine (PE) and 33% phosphatidylglycerol (PG)] using CHARMM-GUI Membrane Builder4. Each trial of the four FadL/membrane systems were equilibrated for a minimum of 250 ns. A large array of FAs with various unsaturation ranging from 16:1 to 22:6 as well as the original detergent molecules found in the 1T16 X-ray crystal structure were chosen as ligands to be docked; these ligands are: Lauryldimethylamine oxide (LDAO), Palmitoleic acid (16:1), Î±-Linolenic acid (18:3Î±), Î³-Linolenic acid (18:3Î³), Dihomo-Î³-linolenic acid (20:3), Arachidonic acid (20:4), Eicosapentaenoic acid (20:5), Tetraethylene glycol monooctyl ether (C8E4), and Docosahexaenoic acid (22:6).
The docking simulations predicted 25,000 possible ligand conformations spread over 250 unique protein conformations. These conformations were compiled and examined for recurrences in certain areas of each protein. Several sites were identified as common docking locations and were analyzed. In the case of the crystal structure 1T16, docking appeared to follow the convention of placing the ligands in the low affinity binding site, the high affinity binding site, and the S3 kink as outlined by van den Berg3. Specifically, the majority of docking of the high affinity binding site was restricted to the lower chain FAs, which E. coli is known to uptake, while the higher chain FAs were found in the low affinity binding site and the S3 kink. In the case of the membrane equilibrated FadL, only the smallest ligand, LDAO, was found to bind to the high affinity site. This implies that the high affinity binding site was not open, and is instead activated/opened through some selective uptake mechanism. In the case of NP_230687 and NP_230688 (similar V. cholerae homologs), the uptake channels appear to be non-selective until the N terminal hatch domain. These channels seem to be nonselective in regards to FA length and unsaturation, supporting the fact that V. cholerae serotypes have been shown to uptake longer chained and polyunsaturated FAs2. The channels end at the N terminus hatch domain right before the S3 kink. This is similar to the proposed N terminal hatch for E. coli proposed by van den Berg3. It is thought that E. coli has a N terminus selection mechanism near the S3 kink, and this highly conserved structure found in each homolog appears perform that same function for both V. cholerae homologs NP_230687 and NP_230688. Strangely, for V. cholerae homolog NP_233248, there was no discernible channel and binding did not occur within the FadLâs Î² barrel. This could be due to a conformational structure mechanism that was not initiated by general equilibration, but for the other homologs tested, this did not seem to be the case.