Gatzeva-topalova, P. Z., Warner, L. R., Pardi, A. & Sousa, M. C. Structure and Flexibility of the Complete Periplasmic Domain of BamA : The Protein Insertion Machine of the Outer Membrane. Struct. Des. 18, 1492–1501 (2010).
The outer membrane of gram-negative bacteria are characterized as one that has outer membrane proteins (OMPs). The correct folding and insertion of these OMPs allow for the beta-barrel structure to be embedded into the outer membrane; however, the mechanism mediating this process is still unclear.
Figure 1: Outer Membrane Protein Biogenesis Overview- The outer membrane precursors is originally synthesized in the cytosol before being processed and translocated across the inner membrane and into the periplasm. Chaperone proteins will then bind to and shuttle the protein to the BAM complex where it will be directionally inserted into the outer membrane.
There are several key players that are involved in OMP biosynthesis. The OMP precursor is synthesized in the cytosol and is tagged with a signal sequence. This for it to be post-translationally targeted to the SecYEG translocase. Following translocation into the periplasm and cleavage of the signal sequence, chaperone proteins will bind to the nascent OMP and shuttle them to the BAM complex. This multiprotein complex allows for directional insertion of the OMPs into the outer membrane.
The Beta-barrel Assembly Machine (BAM) is anchored by the beta-barrel BamA plus four lipoproteins (Bam B/C/D/E). BamA is found in all gram-negative bacteria and consists of a C-terminal beta barrel and five N-terminal polypeptide transport associated (PORTA) repeats. The structure of the first four PORTA’s have been reported, but PORTA five has not.
The goal of this paper is to determine the structure of PORTA 5 with respect with the other PORTA domains of BamA. The authors solved a crystal structure of PORTA 4-5 from E. coli BamA at 2.7 Å. PORTA5 displays the characteristic PORTA structure of two alpha helices packaged against a mixed three-strand beta sheet. The interface of PORTA4 and 5 is bridged by a 3 amino acid linker (G344 N345 R346) which forms an L-shaped conformation in the crystal between the two domains (Fig. 2A). There are several interactions that are conserved within the interface of the domains (Fig. 2B).
Figure 2- E. coli BamA PORTA4-5 crystal structure. A) L-shaped conformation in the crystal. Superposition of the two molecules in the asymmetric unit (Chain A and B). PORTA4 chain A is in salmon; PORTA5 chain A is green; grey is chain B. B) Interface of PORTA 4 and 5. Guanidinium group of R314 makes a salt bridge with D383 and a hydrogen bond with S379. Hydrogen bonds between main chain atoms are shown with red dotted lines. Interactions of R346 in the linker stabilize interface by HB to Y315 in 4 and forming a cation pi- interaction with W376 in 5. All conserved interactions in chain B except for R314.
The authors validated their crystal structure through solution NMR and SAXS experiments as independent methods for determining the orientation of the two PORTA domains. By looking at a pool of 100 structures with randomized orientations – varying the torsion angles between the three amino acid linker- the lowest energy structures were found to be in agreement with the solved crystal structure.
The crystal structure of BamA POTRA1-4 has been solved in two conformations. The extended and bent models differ in bending at linker between POTRA 2 and 3. When POTRA4 from BamA POTRA4-5 was superimposed with the same domain in POTRA1-4, this made a spliced model of the entire periplasmic domain of BamA. Subsequent analysis of the structure suggested two rigid arms- PORTA 1-2 and PORTA3-4 with a hinge point between 2 and 3. This was also validated by SAXS.