(158y) Structural Modeling Reveals Significant Differences in Fatty Acid Transporter Homologs in Vibrio Cholerae That May Play a Vital Role in Pathogenesis
AIChE Annual Meeting
Tuesday, November 17, 2020 - 8:00am to 8:55am
Vibrio cholerae is an enteric microorganism responsible for the acute intestinal infection known as cholera. Cholera is a well-known threat and has been the source of epidemics and pandemics ranging from the 1800s to this day. A reported 2.3 million cases have been reported in Yemen from 2017-2019. Furthermore, the World Health Organization (WHO) estimates that in endemic countries there are 1.3 to 4.0 million cases of cholera annually, and 21,000â143,00 deaths between 2008 and 2012. While cholera is treatable in many cases with antibiotics and rehydration, current prevention and mitigation strategies leave much to be desired.
One of the more unique and interesting characteristics of V. cholerae is its ability to uptake and utilize a diverse range of long-chain fatty acids (LCFAs) (Giles, et al. Molec. Micro. 2011). The uptake of LCFAs has traditionally been viewed as carbon source acquisition, but more recently researchers have observed the utilization of LCFAs for membrane remodeling and as signaling molecules in the virulence cascade. The long chain fatty acid transport protein FadL is a transmembrane protein that has been determined as the vector for LCFAs to be taken up by Escherichia coli (Van den Berg, et al. Nature 2004). Multiple FadL homologs exist in V. cholerae, and we hypothesize that this additional genetic machinery gives V. cholerae the ability to uptake a wider range of LCFAs than E. coli, as has previously been observed. We further believe that this enhanced uptake ability plays a role in Vcâs ability to cycle between environmental niches. Testing this hypothesis is all the more important due to the fact that similar behavior has been observed in a number of other bacterial pathogens that are hospital-acquired and/or pose a particular threat to the immunocompromised.
Vibrio cholerae FadL homolog amino acid sequences were found using the NCBI websiteâs Basic Local Alignment Search Tool (BLAST) and E. coliâs FadL amino acid sequence. Three of the most prevalent homolog sequences were chosen (accession numbers: NP_230687, NP_230688, and NP_233248), and these sequences were folded into a tertiary structure conformation using the I-TASSER webserver.
The homolog tertiary structures were then visualized with Visual Molecular Dynamic (VMD) software, and comparisons of the Vibrio cholerae FadL homologs and the RCSB database Escherichia coli FadL (PDB ID: 1T16) were made using in-house scripts.
Results and Discussion
The resulting Vibrio cholerae FadL structures conformed to the 14-stranded Î² barrel ideal transmembrane protein, with discernible key structures such as the L3/L4 loops, S3 strand kink, and the N terminus hatch domain (see Figure 1). The S3 kink in particular was highly conserved between the E. coli crystal structure and the V. cholerae FadL homologs, as seen visually when superimposed. This similarity was quantified via root-mean-squared deviation relative to the E. coli FadL, with the V. cholerae homologs (NP_230687, NP_230688, and NP_233248) exhibiting relatively low RMSD values of 1.69, 1.90, and 0.94 Ã respectively. Further analysis showed that the S3 kink is at or close to a
neutral charge across all V. cholerae homologs as well as the E. coli crystal structure. However, there are minor dissimilarities in the location and number of polar and non-polar residues when compared to the crystal structure generated PDB.
Structural similarities are most noticeable near the base of the Î² barrel, with dissimilarities becoming more frequent closer to the extracellular region. This is of particular significance given that LCFAs initially bind to the extracellular L3/L4 loops and enter the channel before diffusing laterally into the outer membrane, rather than being transported into the periplasm. The observed differences in size and structure the extracellular binding loops could explain V. choleraeâs ability to uptake a more diverse spectrum of LCFAs than E. coli, and as such will be the subject of further study.
The N-terminal hatch is thought to be a ligand-gated channel, where the LCFA would bind to the high affinity binding site and cause conformational changes that open the gateway through the S2/S3 pore. Within E. coliâs FadL, the N-terminus is located just below the high affinity binding site, allowing for binding site interactions with the gated channel. The differences in the N-terminal hatch domain are among the most glaring between the V. cholerae homologs and the E. coli FadL crystal structure. Specifically, the Vc N-terminal hatches contain more residues than Ecâs while the Î² barrel sizes are similar. These larger domains are observed to penetrate the S2/S3 pore, wrap around the S3 kink, or pack within the barrel. While it appears all of the Vc N-terminal hatch domains make contact with the high affinity binding site, these structural differences could potentially impact the diffusion of LCFAs into the membrane.
The last of the notable differences is observed in the C-terminus. In E. coli, the C-terminus extends away from the Î² barrel, whereas in the V. cholerae homologs this domain terminates just after the last Î² strand ends. E. coliâs C-terminus is believed to play a role in the transport of LCFAs across the membrane, but its precise function remains unknown. Finally, it should also be mentioned that the LCFA diffusion channels were not observable in the Vibrio cholerae homologs. However, this is thought to be due to the spontaneous conformational changes in the protein structure that guide the LCFA through the channel. If there is no LCFA to be transported, the channel appears to remain closed.
Conclusions and Future Work
This work is the beginning of a comprehensive study of Vibrio choleraeâs fatty acid transport machinery. The tertiary structures of the V. cholerae FadL homologs were successfully generated, and noticeable differences in regions critical for LCFA binding and diffusion were noted when compared to the E. coli crystal structure. These differences could shed light on V. choleraeâs ability to cycle between environmental niches as well as its pathogenesis. Putative LCFA binding sites were identified visually through superimposition, and future work will refine these locations using predictive binding algorithms. Additionally, a quantitative study of the protein structures will be conducted to identify key differences (e.g., polarity, charge, conformation) compared to E. coli FadL as well as amongst homologs. This work is expected to enhance our scientific understanding of the role of LCFA uptake in bacterial pathogenesis, and could have vital implications for disease prevention and mitigation.