Defining the Escherichia coli SecA Dimer Interface Residues through In Vivo Site-Specific Photo-Cross-Linking

Abstract

The motor protein SecA is a core component of the bacterial general secretory (Sec) pathway and is essential for cell viability. Despite evidence showing that SecA exists in a dynamic monomer-dimer equilibrium favoring the dimeric form in solution and in the cytoplasm, there is considerable debate as to the quaternary structural organization of the SecA dimer. Here, a site-directed photo-cross-linking technique was utilized to identify residues on the Escherichia coli SecA (ecSecA) dimer interface in the cytosol of intact cells. The feasibility of this method was demonstrated with residue Leu6, which is essential for ecSecA dimerization based on our analytical ultracentrifugation studies of SecA L6A and shown to form the cross-linked SecA dimer in vivo with p-benzoyl-phenylalanine (pBpa) substituted at position 6. Subsequently, the amino terminus (residues 2 to 11) in the nucleotide binding domain (NBD), Phe263 in the preprotein binding domain (PBD), and Tyr794 and Arg805 in the intramolecular regulator of the ATPase 1 domain (IRA1) were identified to be involved in ecSecA dimerization. Furthermore, the incorporation of pBpa at position 805 did not form a cross-linked dimer in the SecA ⌬2-11 context, indicating the possibility that the amino terminus may directly contact Arg805 or that the deletion of residues 2 to 11 alters the topology of the naturally occurring ecSecA dimer.

The Sec System: Protein Export in Escherichia coli

“…6). A separate, extensive in vivo photocrosslinking study identified crosslinks that were not consistent with any one dimer (171). This may mean that in vivo minor populations of several dimeric forms exist and were trapped.…”

Human Eukaryotic Initiation Factor 4G (eIF4G) Protein Binds to eIF3c, -d, and -e to Promote mRNA Recruitment to the Ribosome

“…To address this, we employed an alternate method to confirm which of the eIF3c, -d, -l, and -e subunits interact with eIF4G. The unnatural amino acid p-benzoylphenylalanine (Bpa) has been used recently to explore multi-factor interactions in vivo and in vitro in bacteria, yeast, and mammalian systems (31)(32)(33)(34)(35)(36). Bpa is photoactivatable at 350 -360 nm, preferentially reacts with C-H bonds versus water, and can be activated reversibly.…”

SecA functions in vivo as a discrete anti‐parallel dimer to promote protein transport

“…The low efficiency of this technique in comparison to disulfide crosslinking assures that it is minimally perturbing of any natural protein interaction equilibrium. This approach has been utilized previously to map both the SecA-SecY and SecA dimer interfaces (Mori and Ito 2006, Das and Oliver 2011, Yu, Wowor et al 2013), although only cytosolic SecA was examined in the latter study with somewhat equivocal results.…”