DESARROLLO DEL PROYECTO

The biosynthesis of OMPs starts, like any other protein, in the cytoplasm. Several proteins, often associated to form multi-protein complexes, are involved in the OMPs assembly [77],[78]. Chaperones, both in the cytoplasm and in the periplasm, keep the precursors of the OMPs (pre-OMPs) unfolded [79],[80], the SEC complex allows the pre-OMPs to pass through the IM and finally the β- barrel assembly machinery, also known as BAM, fold and insert the OMPs in the OM (Figure 1.10)[81].

Figure 1.10: Cartoon representation of the OMPs assembly. The pre-OMPs are

targeted to the IM via the SecAB complex, and reached the periplasm through the secYEG translocone. Here several chaperons bind the pre-OMPS and deliver them to the BAM complex. Figure adapted from [81]

BamA BamB BamD BamC BamE Primary pathway SurA Missfolding

Degrada<on Secondary pathway

SecA SecB Skp DegP SecYEG IM OM

Chapter 1: Structure and function of outer-membrane proteins

The majority of OMPs contains a signal peptide that allows the bacteria to recognize and target the protein to the correct translocon. The signal peptide is a three-part sequence, located at N-terminus of the pre-OMPs and it is usually 18- 20 amino acids long [82]. The three motifs that comprised the signal peptide are identified as n-region (at the N-terminus of the sequence), h-region (composed of hydrophobic amino acids) and c-region (at C-terminus of the sequence, often contains an A-A consensus)[83].

A two proteins complex, SecAB, is responsible to deliver the pre-OMPs to the SEC translocon [84]. SecA is an ATP-powered protein, whose role is to carry the pre- OMPs to the translocon, while the chaperon SecB which bounds the pre-OMPs in specific sequences, usually masked in the folded protein, keeps them unfolded [85].

Although the majority of the proteins cross the IM maintaining an unfolded conformation, fully folded proteins can also translocate through the IM, by using an alternative translocon, known as twin-arginine-translocase (TAT) [86]. In E. coli the TAT system consists of three inner membrane proteins, TatA, TatB and TatC. The signal peptide, that target the proteins to the TAT complex includes the consensus sequence Ser/Thr-Arg-Arg-(X)-Phe-Leu-Lys (with X being any polar amino acid)[86]. When the proteins reach the TAT complex, a H+ _{proton gradient}

Chapter 1: Structure and function of outer-membrane proteins

The SecYEG translocon is a multi-protein complex composed of the three proteins SecY, SecE and SecG [88]. A homologue of the bacteria SEC translocon, the translocon Sec61αβγ, has been identified in Eukaryotes [89]. Although the mechanism of translocation through the SEC complex has not been fully understood, it is clear that SecA plays a major role [90]. During translocation, in fact, SecA partially insert itself through the translocon pore and through several cycle of binding and hydrolysis of ATP gives the energy necessary for the pre- OMPs to translocate [91][90].

In the SEC pathway, once the protein has crossed the IM, a signal peptidase cleaves the signal peptide and releases the pre-OMPs in the periplasm. The pre- OMPs are recognizes by different chaperons (SurA, Skp, FkpA and Spy) whose role is to keep the pre-OMPs unfolded, avoiding their aggregation [92][93]. In case of misfolded proteins, a particular chaperone with protease activity, DegP, binds and hence degrades the pre-OMPs [94]. Once the chaperons bind the pre- OMPs, they are delivered to the BAM complex.

In E. coli the BAM machinery is a multi-protein complex composed of BamA, an outer-membrane protein, and four lipoproteins, BamBCDE, linked to the inner leaflet by a N-terminus modification [95], [96]. BamA is a 16-β-stranded barrel with an extended periplasmic domain comprising of five repetitions of a POTRA (polypeptide-translocation-associated) domain (Figure 1.11) [97],[98]. The POTRAs present a β-α-α-β-β architecture and although structurally identical they share low sequence identity. The POTRAs domain mediates BamA and BamB

Chapter 1: Structure and function of outer-membrane proteins

interaction. Deletion of the last three POTRAs (P3-5) is lethal for E. coli, but not for Neisseria gonorrhea [99], which implies that the importance of the single POTRAs on the bacterial viability is species-specific.

Figure 1.11: Crystal structures of BamA and BamB. (A) BamA structure is colored in

yellow and POTRAs domains in blue. (B) The lipoprotein BamB in complex with P3-5 POTRAs domains of BamA (PDB codes: 4K3B and4XGA, respectively).

BamA interacts to BamC and BamE through BamD. While deletion of BamB, BamC and BamE is tolerable, deletion of BamA and BamD severely impairs the OMPs assembly leading to the bacteria death. BamA and BamD the only essential

Chapter 1: Structure and function of outer-membrane proteins

Although the BAM complex has been structurally characterized, its mechanism of action has yet to be fully understood. Several in vitro studies, have demonstrated that most of the OMPs can spontaneously (but slowly) fold and insert themself into an artificial lipid bilayer [100] [101]. These results have brought to the idea that, in vivo, the BAM complex might increase the kinetic of the OMPs insertion in the OM, making the process more favorable [81]. A BAM-assisted model has therefore been proposed to explain the OMPs assembly. According to this model, the BAM complex plays a dual role: (1) to direct the OMPs to the OM and (2) to destabilize the OM and consequently facilitate the OMP insertion [102]. Nevertheless, another model, known as “BAM budding model”, has also been introduced to explain the BamA-mediated insertion [103]. According to such model, the unfolded OMP would use the first β strand (β1) of BamA as a template to start the folding process. The growing OMP would therefore insert inside the BamA β-barrel, forming a BamA-preOMP intermediate complex. Eventually a lateral opening of BamA would lead the fully folded OMP to “bud” and insert into the outer-membrane [104]. Although more studies need to be conducted to elucidate this convoluted mechanism, it is likely that both mechanisms play an important role in the OMPs biogenesis and insertion [105].

Chapter 1: Structure and function of outer-membrane proteins