In Gram-negative bacteria, many proteins are anchored to the inner or the outer membrane via a lipid moiety. This group of proteins is called lipoproteins. They are important for the viability of the cell as they fulfil a variety of different functions 1. Some are stress sensors, others participate in the biogenesis and the integrity of the cell envelope, while some contribute to the bacterial virulence.
With the help of our collaborator Dr. Bogdan I. Iorga, we were able to show that half of the outer membrane lipoproteins of E. coli display a long N-terminal intrinsically disordered region composed of more than 22 residues. Intrigued by the presence of these unstructured linkers, we decided to study their role in the targeting of the lipoproteins of E. coli.
The current paradigm is that the sorting of lipoproteins between the inner and outer membranes depends on the identity of two amino acids (the “Lol sorting signal”) located at the N-terminus of the protein, next to the lipidated cysteine residue 2. Our results challenge this widely accepted model by showing that correct trafficking by the Lol lipoprotein sorting system and targeting to the outer membrane also depend on the N-terminal intrinsically disordered linkers. We determined that the length of the linker is important, but the sequence is not, on the condition that it cannot fold into a defined secondary structure. Focusing on three structurally unrelated outer membrane lipoproteins (RcsF, Pal and NlpD), we showed the functional relevance of our findings. Additionally, we demonstrated that N-terminal disordered segments are required for optimal processing of lipoproteins by the Lol system: decreasing the load of the Lol system by deleting the gene encoding Lpp, the most abundant lipoprotein in E. coli, rescues outer membrane targeting of lipoproteins variants devoid of their N-terminal linker. Finally, our results also suggest that unstructured linkers might be used by lipoproteins to cross the outer membrane and reach the cell surface.
Altogether, our results provide important mechanistic insights into the biogenesis of lipoproteins, which are attracting a lot of attention due to their crucial cellular functions and potential as targets for new antibacterials.
Excitingly, our work also provides direct evidence that evolution can select protein intrinsic disorder by function. Most proteins present segments that are intrinsically disordered (it is estimated that 30-50% of eukaryotic proteins contain regions that do not adopt a defined secondary structure in vitro 3). However, demonstrating that these unstructured regions are functionally important in vivo has been challenging. By showing that displaying an N-terminal disordered segment downstream of the Lol signal is required for the correct sorting of some lipoproteins, the current investigation provides crucial functional evidence that disorder matters.
To find out about our results and the role of disorder in lipoprotein processing, you can find our full publication by using the following link:
1. Szewczyk, J. & Collet, J.F. The Journey of Lipoproteins Through the Cell: One Birthplace, Multiple Destinations. Adv Microb Physiol 69, 1-50 (2016).
2. Okuda, S. & Tokuda, H. Lipoprotein sorting in bacteria. Annu Rev Microbiol 65, 239-59 (2011).
3. Bardwell, J.C. & Jakob, U. Conditional disorder in chaperone action. Trends Biochem Sci 37, 517-25 (2012).