crc press - E-Lib FK UWKS

Signal Peptides 299
that binds denatured mature portions of proteins. 17,26,27 If so, how does SecB discriminate
preproteins from others?
To reconcile the paradox, a kinetic partitioning model, which indicates that
retardation of protein folding by signal peptides is the major factor for SecB to
discriminate its substrates, has been proposed by Hardy and Randall. 28,29 However,
the binding rate of SecB is much faster than rates of protein synthesis and its folding 21
and the substrate specificity of SecB does not seem to explain its in vivo preference
with preproteins. 27 Mutations on signal peptides or on early mature regions can
change apparent SecB dependency; thus the ability to interact with the transport
machinery of a protein may be the key factor to determining its apparent SecB
dependency. 30 Recently, the crystal structure of SecB from Haemophilus influenzae
was determined, 31 which shed some light on this paradox, although the structure
binding its substrate would be desirable for detailed discussion. 18,19
SecB is a highly acidic homotetramer. A mutational study implies that the
tetramer is a dimer of dimers, 32 which was confirmed by the x-ray structure. In its
quaternary structure, SecB seems to have two long grooves that are proposed to be
the unfolded polypeptide binding sites. 31 The groove consists roughly of two subsites:
one lined with conserved aromatic residues, the other a shallow floor rich in hydrophobic
residues. Possibly, proteins in their translocation-competent state wrap
around SecB. The SecB tetramer also has two potential SecA-binding sites, which
are solvent-exposed flat surfaces rich in acidic residues. In addition, four exposed
arms of SecB may embrace the SecA protein. Thus, subsequent binding among
SecB, SecA, and preprotein could be the determinant of specific signal recognition.
14.2.1.2 SecA
In Escherichia coli, SecA is a large protein (901 residues) that works as a
homodimer. 33,34 SecA is a part of the bacterial protein translocase complex; it binds
to SecY through its N-terminal domain but also loosely binds to anionic phospholipids
possibly through its C-terminal domain. 35-37 The exact role of SecA in preprotein
translocation has not been fully understood; its RNA helicase activity is not
required in vivo for efficient protein translocation. 38 SecA also has the ATPase
activity: its binding to SecB 39 and to anionic phospholipids 40,41 induces its conformational
change, thus stimulating its ATPase activity, especially in the presence of
SecYEG.
Synthetic signal peptides were originally reported as inhibitors of ATPase activity
without their mature portion, 40,42 although some recent experiments show that they
stimulate ATPase activity in the presence of lipids. 43,44 However, a soluble N-terminal
fragment of SecA was shown to bind signal peptides more tightly and this binding
inhibits its ATPase activity. 45 It has been regarded that SecA is an ATP-driven motor
protein and pushes preproteins into the membrane. 46 According to the membrane
insertion hypothesis, SecA cycles insertion and deinsertion processes with the preprotein
by utilizing the driving force of ATP hydrolysis, 47,48 but the detailed mechanism
has yet to be elucidated. 19 As another driving force, the proton motive force
(pmf) also seems to contribute significantly to the (late stage of) translocation
process. 49