The low-affinity ATP binding site of the Escherichia coli SecA dimer is localized at the subunit interface

The homodimeric SecA protein is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade. SecA contains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS2 that bind ATP with high and low affinity, respectively. The photoactivatable bifunctional cross-linking agent 3'-arylazido-8-azidoadenosine 5'-triphosphate (diN3ATP) was used to investigate the spatial arrangement of the nucleotide binding sites of SecA. DiN3ATP is an authentic ATP analogue as it supports SecA-dependent precursor protein translocation and translocation ATPase. UV-induced photo-cross-linking of the diN3ATP-bound SecA results in the formation of stable dimeric species of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1, was also photo-cross-linked by diN3ATP, whereas no cross-linking occurred with the NBS2 mutant R509K SecA. We concluded that the low-affinity NBS2, which is located in the carboxyl-terminal half of SecA, is the site of cross-linking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer.

The Sec system

Proteins designated to be secreted by Escherichia coli are synthesized with an amino-terminal signal peptide and associate as nascent chains with the export-specific chaperone SecB. Translocation occurs at a multisubunit membrane-bound enzyme termed translocase, which consists of a peripheral preprotein-binding site and an ATPase domain termed SecA, a core heterotrimeric integral membrane protein complex with SecY, SecE and SecG as subunits, and an accessory integral membrane protein complex containing SecD and SecE Major new insights have been gained into the cascade of preprotein targeting events and the enzymatic mechanism of preprotein translocation. It has become clear that preproteins are translocated in a stepwise fashion involving large nucleotide-induced conformational changes of the molecular motor SecA that propels the translocation reaction.

Thermodynamics of nucleotide binding to NBS-I of the Bacillus subtilis preprotein translocase subunit SecA

SecA is the dissociatable nucleotide and preprotein binding subunit of the bacterial translocase. The thermodynamics of nucleotide binding to soluble SecA at nucleotide binding site I were determined by isothermal titration calorimetry. Binding of ADP and non-hydrolyzable ATPγS is enthalpy-driven (ΔH0 of -14.44 and -5.56 kcal/mol, respectively), but is accompanied by opposite entropic contributions (ΔS0 of -18.25 and 9.55 cal/mol/K, respectively). ADP binding results in a large change in the heat capacity of SecA (ΔCp = -780 cal/mol/K). It is suggested that ADP binding promotes the interaction between the two thermodynamically discernible domains of SecA which is accompanied by a shielding of hydrophobic surface from solvent.

PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA–SecY interaction during the initiation of translocation

In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex

Identification of the Magnesium-binding Domain of the High-affinity ATP-binding Site of the Bacillus subtilis and Escherichia coli SecA Protein

The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD, that in combination with motif A is responsible for the Mg2+-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli secA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg2+-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg2+-ion when Mg2+-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.

Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA

In Escherichia coli, precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non-functional signal sequence. Δ8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB-SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.