Preprotein Substrate Research Articles

Sequence-specific Binding of prePhoD to Soluble TatAd Indicates Protein-mediated Targeting of the Tat Export in Bacillus subtilis

The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.

The Role of the Membrane-spanning Domain of Type I Signal Peptidases in Substrate Cleavage Site Selection

Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane. This limits the enzyme-substrate interactions such that cleavage occurs at only one site. In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2). With purified full-length as well as truncated constructs of both B. subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.

Bacterial preprotein translocase: mechanism and conformational dynamics of a processive enzyme.

Preprotein translocase, the membrane transporter for secretory proteins, is a processive enzyme. It comprises the membrane proteins SecYEG(DFYajC) and the peripheral ATPase SecA, which acts as a motor subunit. Translocase subunits form dynamic complexes in the lipid bilayer and build an aqueous conduit through which preprotein substrates are transported at the expense of energy. Preproteins bind to translocase and trigger cycles of ATP binding and hydrolysis that drive a transition of SecA between two distinct conformational states. These changes are transmitted to SecG and lead to inversion of its membrane topology. SecA conformational changes promote directed migration of the polymeric substrate through the translocase, in steps of 20-30 aminoacyl residues. Translocase dissociates from the substrate only after the whole preprotein chain length has been transported to the trans side of the membrane, where it is fully released.

The N-domain of the signal recognition particle 54-kDa subunit promotes efficient signal sequence binding.

The signal recognition particle 54-kDa subunit (SRP54) binds to the signal sequences of nascent presecretory and transmembrane proteins. Previous studies have shown that signal sequences bind to the C-terminal methionine-rich domain of the protein (M-domain), but have raised the possibility that either the N-terminal domain (N-domain) or the central guanosine triphosphatase module (GTPase-domain) also contribute to signal-sequence-binding activity. We have generated a series of N-domain and GTPase-domain mutants to investigate this issue further. Mutations in a conserved N-domain motif (ALLEADV) produced significant defects in signal sequence binding that correlate with the severity of the mutation. The magnitude of the defect was independent of the preprotein substrate, which suggested that the mutations do not alter the specificity of signal sequence recognition. The N-domain mutants also showed defects in promoting the translocation of presecretory proteins across the membrane of microsomal vesicles, but these defects appeared to be a direct consequence of the reduction in signal-sequence-binding activity and not separate effects of the mutations. By contrast, mutations in the guanosine triphosphatase consensus sequence had no effect on signal sequence binding, but instead severely impaired protein translocation activity. These results indicate that a principal function of the SRP54 N-domain is to promote efficient signal sequence binding. These data also suggest that the SRP54 GTPase regulates the cycle of signal sequence binding and release, perhaps by modulating the relative orientation of the N- and M-domains.

Use of Site-directed Chemical Modification to Study an Essential Lysine in Escherichia coli Leader Peptidase

Escherichia coli leader peptidase, which catalyzes the cleavage of signal peptides from pre-proteins, is an essential, integral membrane serine peptidase that has its active site residing in the periplasmic space. It contains a conserved lysine residue that has been proposed to act as the general base, abstracting the proton from the side chain hydroxyl group of the nucleophilic serine 90. To help elucidate the role of the essential lysine 145 in the activity of E. coli leader peptidase, we have combined site-directed mutagenesis and chemical modification methods to introduce unnatural amino acid side chains at the 145-position. We show that partial activity can be restored to an inactive K145C leader peptidase mutant by reacting it with 2-bromoethylamine.HBr to produce a lysine analog (gamma-thia-lysine) at the 145-position. Modification with the reagents 3-bromopropylamine.HBr and 2-mercaptoethylamine also allowed for partial restoration of activity showing that there is some flexibility in the length requirements of this essential residue. Modification with (2-bromoethyl)trimethylammonium.Br to form a positively charged, nontitratable side chain at the 145-position failed to restore activity to the inactive K145C leader peptidase mutant. This result, along with an inactive K145R mutant result, supports the claim that the lysine side chain at the 145-position is essential due to its ability to form a hydrogen bond(s) or to act as a general base rather than because of an ability to form a critical salt bridge. We find that leader peptidase processes the pre-protein substrate, pro-OmpA nuclease A, with maximum efficiency at pH 9.0, and apparent pKa values for titratable groups at approximately 8.7 and 9.3 are revealed. We show that the lysine modifier maleic anhydride inhibits leader peptidase by reacting with lysine 145. The results of this study are consistent with the hypothesis that the lysine at the 145-position of leader peptidase functions as the active site general base. A model of the active site region of leader peptidase is presented based on the structure of the E. coli UmuD', and a mechanism for bacterial leader peptidase is proposed.

21] Bacterial leader peptidase 1

Publisher Summary This chapter highlights bacterial leader peptidase 1. It describes the purification of leader peptidase, its substrate specificity, sequence homology of the leader peptidase 1 genes, and techniques to probe the structure and function of this protein. Proteins that are secreted out of the cell or translocated across lipid bilayers are usually made with N-terminal extension peptides, termed “leader sequences.” In bacteria, two separate leader peptidases process proteins. Leader peptidase 1 cleaves the majority of the preproteins destined to the cell surface, whereas leader peptidase 2 only processes preproteins that have been first modified with a lipid molecule at the +1 position. The Escherichia coli leader peptidase 1 is an integral membrane protein of 323 amino acid residues. Its physiological role is to release exported proteins from the membrane by removing the leader sequence. Leader peptidase can be assayed using a radiolabeled preprotein substrate or a peptide substrate. It is purified by membrane isolation, Triton X-100 extraction, diethylaminoethyl (DEAE) chromatography, and chromatofocusing.