| Abstract |
About 1/3 of the proteins produced in the cytoplasm of bacteria need to be
translocated to extracytoplasmic locations. The cytoplasmic membrane is an
impenetrable barrier that holds life’s macromolecules compartmentalized. Protein
crossing of the membrane hydrophobic bilayer is thermodynamically unfavorable and
does not occur spontaneously. To tackle this challenge, cells have evolved at least 16
systems to translocate proteins into and across the cytoplasmic membrane. One of
these, the Sec system is the only ubiquitous and essential for viability.
In bacteria the Sec system consists of the SecY/SecE/SecG (SecYEG hereafter)
membrane-embedded heterotrimeric complex, which forms the channel through
which proteins are threaded, and the cytoplasmic motor SecA.
Proteins destined to be translocated outside the cytoplasmic membrane are produced
as preproteins, bearing short and conserved aminoterminal extensions called signal
peptides and mature domains, that are not conserved in their length or
physicochemical properties. Signal peptides are cleaved once the translocation of the
preprotein is almost completed. SecA interacts with preproteins in the cytoplasm or
after it has formed a complex with SecYEG at the membrane. Next SecA consumes
energy in the form of ATP, to produce mechanical work that somehow pushes and
threads preproteins through the SecYEG pore. After a few cycles of ATP hydrolysis
(depending on the size of the preproteins) the signal peptide is cleaved and the mature
domain is released in the periplasmic space for further processing.
The aim of this study was to determine the region on the translocase holoenzyme
(SecA-SecYEG) where preproteins dock for the first time. We also wanted to
determine which are the molecular/structural events that lead to the activation of the
translocase in response to preprotein binding.
Our results show that preproteins interact with the flat cytoplasmic surface of SecA,
when in complex with SecYEG. Signal peptides and mature domains utilize
independent binding sites on SecA; mature domains bind on the flat surface formed
by all 4 domains of SecA(NBD, Nucleotide Binding Domain, IRA2, Intramolecular
Regulator of ATP-hydrolysis 2, PBD, Preprotein Binding Domain and the C-domain);
and signal peptides bind on a shallow groove along PBD. Mature domains might interact first with SecA due to their sheer size (in relation to the signal peptide)
making their stochastic binding more probable. PBD rotates to adjust the spacing
between the two independent binding sites in order to ensure optimal synergistic
preprotein binding (signal peptide and mature domain binding).
The initial interaction of preproteins with the SecA-SecYEG complex induces the
transmition of an activation signal that leads to the production of preprotein
translocation mechanical work. The transmition of the activation signal occurs through
structural alterations that occur throughout SecA. Amino acids on the surface of PBD
participate in the transduction of the activation signal through interactions with
SecYEG and other proteinaceous moieties. Furthermore, PBD position seems to affect
the activation of SecA. However, PBD swiveling is not the mechanical event that
pushes preproteins through SecYEG.
An additional mechanism SecA uses to control its activation is its oligomerization
during preprotein secretion. SecA interacts with SecYEG and preproteins and gets
activated as a dimer. However, SecA must monomerize to proceed to the later steps of
the reactions and complete the threading of the preprotein through the SecYEG pore.
|
Ο νανοκινητήρας της έκκρισης πρωτεϊνών : μοριακός μηχανισμός
