Tyr‐326 plays a critical role in controlling SecA–preprotein interaction

Kourtz, Lauralynn; Oliver, Donald B.

doi:10.1046/j.1365-2958.2000.02078.x

Cited by 44 publications

(39 citation statements)

References 60 publications

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“…Further kinetic studies on proOmpA translocation showed that the apparent K m for SecA is ϳ50 nM, which is ϳ3-fold higher as the K d value reported for the binding of SecA to the SecYEG complex (13 nM) (20). The apparent K m values for proOmpA is in the same range as reported for a chimeric protein containing the signal sequence of E. coli alkaline phosphatase and the mature portion of staphylococcal nuclease (570 Ϯ 150 nM) as assessed by the precursor protein-stimulated translocation ATPase activity of SecA (21). The SecYEG complex forms a protein conducting channel across the membrane, but the exact size of the pore is not known.…”

Section: Fluorescent Protein Translocation Assaymentioning

confidence: 50%

Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

Keyzer

Does

Driessen

2002

Journal of Biological Chemistry

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Section: Fluorescent Protein Translocation Assaymentioning

confidence: 50%

Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

Keyzer

Does

Driessen

2002

Journal of Biological Chemistry

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“…Of note, SecA2 Sg has the nine conserved motifs (Q, I, Ia, Ib, and II through VI) known to line the nucleotide binding cleft of SecA Ec and other helicases (35,44), although the IRA2 motifs IV and VI have nonconserved residues. The alignment also shows that SecA2 Sg has a tyrosine residue in the PPXD that is essential for preprotein binding and release by SecA Ec (27) and a tryptophan residue in IRA1 that coordinates preprotein binding with ATP hydrolysis (54). This suggests that SecA2 Sg is likely to coordinate the activities of preprotein binding and ATP hydrolysis in a manner similar to that of SecA Ec .…”

Section: Seca2mentioning

confidence: 87%

Characterization of Streptococcus gordonii SecA2 as a Paralogue of SecA

Bensing

Sullam

2009

J Bacteriol

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The accessory Sec system of Streptococcus gordonii is essential for transport of the glycoprotein GspB to the bacterial cell surface. A key component of this dedicated transport system is SecA2. The SecA2 proteins of streptococci and staphylococci are paralogues of SecA and are presumed to have an analogous role in protein transport, but they may be specifically adapted for the transport of large, serine-rich glycoproteins. We used a combination of genetic and biochemical methods to assess whether the S. gordonii SecA2 functions similarly to SecA. Although mutational analyses demonstrated that conserved amino acids are essential for the function of SecA2, replacing such residues in one of two nucleotide binding folds had only minor effects on SecA2 function. SecA2-mediated transport is highly sensitive to azide, as is SecA-mediated transport. Comparison of the S. gordonii SecA and SecA2 proteins in vitro revealed that SecA2 can hydrolyze ATP at a rate similar to that of SecA and is comparably sensitive to azide but that the biochemical properties of these enzymes are subtly different. That is, SecA2 has a lower solubility in aqueous solutions and requires higher Mg 2؉ concentrations for maximal activity. In spite of the high degree of similarity between the S. gordonii paralogues, analysis of SecA-SecA2 chimeras indicates that the domains are not readily interchangeable. This suggests that specific, unique contacts between SecA2 and other components of the accessory Sec system may preclude cross-functioning with the canonical Sec system.

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“…2, Table I) and protein translocation (Fig. 3C), is implicated in full-length preprotein cross-linking (31), and contains Tyr-326 that is important for preprotein interaction (41).…”

Section: Figmentioning

confidence: 99%

Allosteric Communication between Signal Peptides and the SecA Protein DEAD Motor ATPase Domain

Baud¹,

Karamanou²,

Sianidis³

et al. 2002

Journal of Biological Chemistry

107

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SecA, the preprotein translocase ATPase is built of an amino-terminal DEAD helicase motor domain bound to a regulatory C-domain. SecA recognizes mature and signal peptide preprotein regions. We now demonstrate that the amino-terminal 263 residues of the ATPase subdomain of the DEAD motor are necessary and sufficient for high affinity signal peptide binding. Binding is abrogated by deletion of residues 219 -244 that lie within SSD, a novel substrate specificity element of the ATPase subdomain. SSD is essential for protein translocation, is unique to SecA, and is absent from other DEAD proteins. Signal peptide binding to the DEAD motor is controlled in trans by the C-terminal intramolecular regulator of ATPase (IRA1) switch. IRA1 mutations that activate the DEAD motor ATPase also enhance signal peptide affinity. This mechanism coordinates signal peptide binding with ATPase activation. Signal peptide binding causes widespread conformational changes to the ATPase subdomain and inhibits the DEAD motor ATPase. This involves an allosteric mechanism, since binding occurs at sites that are distinct from the catalytic ATPase determinants. Our data reveal the physical determinants and sophisticated intramolecular regulation that allow signal peptides to act as allosteric effectors of the SecA motor.Several cellular polypeptides cross biological membranes prior to acquiring their native state. Such proteins are delivered by chaperone-like factors (e.g. signal recognition particle and SecB) (1, 2) to membranes and subsequently cross them through specialized pumps termed preprotein translocases or translocons (3-5). The bacterial Sec translocase comprises the membrane proteins SecYEGDFYajC and the peripheral ATPase SecA (4, 5). Several of these components are essential and conserved in the three domains of life. Secretory proteins bind to the SecA motor and activate its ATPase. This triggers SecA "insertion-deinsertion" cycles at SecYEG (6, 7), allowing processive translocase movement along the polymeric substrate (8) in defined steps (9, 10). Substrates are thought to transverse the bilayer through a putative "pore" formed by the essential SecYEA core (11-13). Proton motive force (14), SecG (15, 16), and SecDF (8, 16) regulate SecA cycling.Dimeric SecA is built of defined mechanical parts (17, 18). Each protomer (102 kDa) comprises a 68-kDa N-terminal domain (N68) homologous to ATPase motor domains of DEAD helicases (8,18,19) and a C-terminal (C34) dimerization domain (17,20). The SecA DEAD motor forms a proposed mononucleotide binding fold (18, 21) built from an amino-terminal region harboring a nucleotide binding subdomain (NBD) that contains Walker box A and B sequences (DEAD helicase motifs I and II; Fig. 2A) (22) and the intramolecular regulator of ATP hydrolysis (IRA2) subdomain (18). N68 displays a high, unregulated ATPase activity (17).Deletion and point mutants helped determine the basic features of SecA catalysis (17, 18). IRA1, an essentiaI molecular switch in C34, regulates the DEAD motor ATPase through C34/DEAD ...

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Tyr‐326 plays a critical role in controlling SecA–preprotein interaction

Cited by 44 publications

References 60 publications

Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

Characterization of Streptococcus gordonii SecA2 as a Paralogue of SecA

Allosteric Communication between Signal Peptides and the SecA Protein DEAD Motor ATPase Domain

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