frster-resonance-energy-transfer-mapping-new-methodology-to-elucidate

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

Wesleyan University

To identify cell envelope proteins of Borrelia burgdorferi, the causative agent of Lyme disease, we constructed a library of B. burgdorferi genes fused to the Escherichia coli phoA gene, which expresses enzymatically active alkaline phosphatase. One such gene, oppA-1, encodes a predicted polypeptide with significant similarities to various peptide-binding proteins of ABC-type transporters. Immediately downstream of oppA-1 are two genes, oppA-2 and oppA-3, whose predicted polypeptide products show strong similarities in their amino acid sequences to OppA-1, including a sequence that resembles the most highly conserved region in peptide-binding proteins. By labeling with [3H]palmitate, OppA-1, OppA-2, and OppA-3 were shown to be lipoproteins. DNA hybridization analysis showed that the oppA-1 oppA-2 oppA-3 region is located on the linear chromosome of B. burgdorferi, and the genes are conserved among different Borrelia species that cause Lyme disease (B. burgdorferi, B. garinii, and B. afzelli), suggesting that all three homologous genes are important to the maintenance of Lyme disease spirochetes in one or more of their hosts.

Lyme disease-causing Borrelia species encode multiple lipoproteins homologous to peptide-binding proteins of ABC-type transporters.

secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell. This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling. Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation. Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.

Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an intact gene X signal sequence and an active translocon.

The conserved cp32 plasmid family of Borrelia burgdorferi was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308-7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. An Escherichia coli mutant containing the blyAB locus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction of sheA by blyAB expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with E. coli and B. burgdorferi indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage in B. burgdorferi dramatically stimulated blyAB expression. Our results provide the first evidence of a prophage-encoded holin within Borrelia.

Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system.

SecA is an ATPase and motor protein that drives protein translocation across the bacterial plasma membrane. In Escherichia coli SecA levels are regulated by the secretion needs of the cell utilizing secM, which encodes a secreted protein. Previous studies demonstrated that this regulation requires a translational pause within secM, whose duration regulates the accessibility of the secA Shine-Dalgarno sequence on secM secA mRNA. Here we provide evidence that translocon "pulling" of nascent SecM is what regulates the duration of the secM translational pause, and thus secA expression levels, thereby providing direct support for this model.

Translocon "pulling" of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation.

&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC;&#x8D5B;&#x5EB7;&#x4F7F;&#x7528; ATP &#x7EC6;&#x80DE;&#x819C;&#x8DE8;&#x9A71;&#x52A8;&#x5668;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x8FD0;&#x8F93;&#x3002;&#x8C37;&#x6C28;&#x9178; 210 &#x8D5B;&#x5EB7; ATP &#x7ED3;&#x5408;&#x7F51;&#x7AD9;&quot;DEVD&quot;&#x6C83;&#x514B; B &#x6BCD;&#x9898;&#x4E2D;&#x88AB;&#x63D0;&#x51FA;&#x6765;&#x4F5C;&#x4E3A;&#x50AC;&#x5316;&#x57FA;&#x7840; ATP &#x6C34;&#x89E3; (&#x4EA8;&#x7279;&#x3001; J.F.&#x3001; Weinkauf S.&#xFF0C;&#x4EA8;&#x5229;&#xFF0C;&#x9EA6;&#x514B;&#x5C3C;&#x53E4;&#x62C9;&#x65AF;&#xFF0C;J.J.Fak&#x3001; L.&#x4F53;&#x80B2;&#x3001; &#x5965;&#x5229;&#x5F17;&#x3001; D.B.&#x548C; Deisenhofer&#xFF0C;J.&#xFF08;2002 &#x5E74;&#xFF09; &#x79D1;&#x5B66; 297&#xFF0C;2018&#x5E74; 2026&#x5E74;)&#x3002;&#x7B26;&#x5408;&#x8FD9;&#x4E00;&#x5047;&#x8BBE;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x8BE5;&#x7A81;&#x53D8;&#x7684;&#x8C37;&#x6C28;&#x9178; 210 &#x5929;&#x51AC;&#x6C28;&#x9178;&#x7ED3;&#x679C;&#x4E2D;&#x7684;&#x4E09;&#x78F7;&#x9178;&#x817A;&#x82F7;&#x6C34;&#x89E3;&#x7387;&#x51CF;&#x5C11;&#x4E86; 90-fold &#x5206;&#x522B;&#x4E0E;&#x91CE;&#x751F;&#x578B;&#x8D5B;&#x5EB7;&#xFF0C;0.3 27 s(-1)&#xFF0C;&#x4E0E; s(-1) &#x76F8;&#x6BD4;&#x3002;&#x8D5B;&#x5EB7; E210D &#x4E5F;&#x4F1A;&#x91CA;&#x653E; ADP &#x4E0E;&#x91CE;&#x751F;&#x578B;&#x8D5B;&#x5EB7;&#xFF0C;&#x6697;&#x793A;&#x9664;&#x4E86;&#x4F5C;&#x4E3A;&#x50AC;&#x5316;&#x7684;&#x57FA;&#x5730;&#xFF0C;&#x8C37;&#x6C28;&#x9178; 210 &#x53EF;&#x80FD;&#x63F4;&#x52A9;&#x4EE5;&#x53CA;&#x6210;&#x4EA4;&#x91CF;&#x76F8;&#x6BD4;&#x901F;&#x5EA6;&#x6162;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x76F8;&#x77DB;&#x76FE;&#x65E9;&#x4E9B;&#x65F6;&#x5019;&#x7684;&#x62A5;&#x544A;&#xFF0C;&#x5EFA;&#x8BAE; 133 &#x5929;&#x51AC;&#x6C28;&#x9178;&#x4F5C;&#x4E3A;&#x50AC;&#x5316;&#x57FA;&#x5730; (&#x4F50;&#x85E4;&#xFF0C;K.&#xFF0C;&#x68EE;&#x559C;&#x6717;&#xFF0C;H.&#x3001; &#x5409;&#x7530;&#xFF0C;M.&#x548C;&#x6C34;&#x5C9B;&#xFF0C;&#x6761; (1996 &#x5E74;) J.Biol.&#x5316;&#x5B66; 271&#xFF0C;17439 17444)&#x3002;&#x8D5B;&#x5EB7; D133N &#x7A81;&#x53D8;&#x4F53;&#x7684;&#x7814;&#x7A76;&#x4E2D;&#x4F7F;&#x7528;&#x7684;&#x91CD;&#x65B0;&#x8BC4;&#x4F30;&#x786E;&#x8BA4;&#x5176;&#x635F;&#x5931;&#x7684; ATPase &#x548C;&#x819C;&#x8F6C;&#x8FD0;&#x6D3B;&#x52A8;&#xFF0C;&#x4F46;&#x5947;&#x602A;&#x7684;&#x662F;&#xFF0C;&#x7C7B;&#x4F3C;&#x7684;&#x8D5B;&#x5EB7; D133A &#x7A81;&#x53D8;&#x4FDD;&#x7559;&#x5145;&#x5206;&#x7684;&#x6D3B;&#x52A8;&#xFF0C;&#x63ED;&#x793A;&#x8FD9;&#x79CD;&#x6B8B;&#x4F59;&#x4E0D;&#x5728;&#x50AC;&#x5316;&#x53CD;&#x5E94;&#x4E2D;&#x53D1;&#x6325;&#x5173;&#x952E;&#x4F5C;&#x7528;&#x3002;

&#x5B88;&#x6052;&#x7684;&#x8C37;&#x6C28;&#x9178;&#x6B8B;&#x7559;&#x5728;&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC;&#x8D5B;&#x5EB7; ATPase &#x673A;&#x5236;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#x3002;

Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the "DEVD" Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s(-1) versus 27 s(-1), respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439-17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis.

Role of a conserved glutamate residue in the Escherichia coli SecA ATPase mechanism.

Escherichia coli SecA utilise l&#x26;#39;ATP pour entra&#xEE;ner le transport de prot&#xE9;ines dans les membranes des cellules. Glutamate 210 dans le &#x26;quot;DEVD&#xAB; motif Walker B de la SECA ATP-binding site a &#xE9;t&#xE9; propos&#xE9; que la base catalytique pour l&#x26;#39;hydrolyse d&#x26;#39;ATP (Hunt, JF, Weinkauf, S., Henry, L., Fak, JJ, McNicholas, P. , Oliver, DB, et Deisenhofer, J. (2002) Sciences 297, 2018-2026). Conform&#xE9;ment &#xE0; cette hypoth&#xE8;se, nous trouvons que la mutation du glutamate 210 &#xE0; des r&#xE9;sultats aspartate dans une r&#xE9;duction de 90 fois du taux de l&#x26;#39;hydrolyse d&#x26;#39;ATP par rapport avec le type sauvage Seca, 0,3 s (-1) contre 27 s (-1), respectivement. SECA-E210D ADP lib&#xE8;re &#xE9;galement &#xE0; un rythme plus lent comparativement &#xE0; SecA de type sauvage, ce qui sugg&#xE8;re que, en plus de servir comme base catalytique, le glutamate 210 pourrait aider le chiffre d&#x26;#39;affaires aussi bien. Nos r&#xE9;sultats contredisent un rapport plus t&#xF4;t que propos&#xE9; aspartate 133, la base catalytique (Sato, K., Mori, H., Yoshida, M., et Mizushima, S. (1996) J. Biol. Chem. 271, 17439-17444) . R&#xE9;&#xE9;valuation du mutant SecA-D133N utilis&#xE9; dans cette &#xE9;tude confirme la perte de l&#x26;#39;ATPase et les activit&#xE9;s de translocation membranaire, mais &#xE9;tonnamment, l&#x26;#39;analogue SecA-D133A mutant conserve pleine activit&#xE9;, r&#xE9;v&#xE9;lant que ce r&#xE9;sidu ne joue pas un r&#xF4;le cl&#xE9; dans la catalyse.

R&#xF4;le d&#x26;#39;un r&#xE9;sidu de glutamate conserv&#xE9;e dans le Escherichia Coli SecA M&#xE9;canisme ATPase

Rolle eines konservierten Glutamat-Rest in dem Escherichia coli SecA ATPase-Mechanismus.

Escherichia coli SecA utilizza ATP per guidare il trasporto delle proteine attraverso le membrane cellulari. Glutammato 210 il motivo &quot;Marta&quot; Walker B del sito SecA ATP-associazione &egrave; stata proposta come base catalitica per idrolisi dell&#39;ATP (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, B. D. e Deisenhofer, J. (2002) scienza 297, 2018-2026). Coerente con questa ipotesi, troviamo quella mutazione del glutammato 210 risultati aspartato in una 90-fold riduzione del tasso di idrolisi di ATP rispetto al wild type SecA, 0,3 s(-1) versus s(-1) 27, rispettivamente. SecA-E210D rilascia anche ADP a un ritmo pi&ugrave; lento rispetto al wild type SecA, suggerendo che oltre a servire come base catalitica, glutammato 210 potrebbe aiuto fatturato pure. I nostri risultati contraddicono un report precedente che ha proposto come base catalitica aspartato 133 (Sato, K., Mori, H., Yoshida, M. e Mizushima, s. (1996) j Biol Chem. 271, 17439-17444). Rivalutazione del mutante SecA-D133N utilizzato in questo studio conferma la sua perdita di attivit&agrave; di traslocazione ATPasi e membrana, ma sorprendentemente, il mutante analogo SecA-D133A conserva la piena attivit&agrave;, rivelando che questo residuo non gioca un ruolo chiave nella catalisi.

Ruolo di un residuo di glutammato conservata nel meccanismo SecA atpasi di Escherichia coli.

&#x5927;&#x8178;&#x83CC;&#x30BB;&#x30AB;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x819C;&#x3092;&#x6A2A;&#x5207;&#x3063;&#x3066;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x8F38;&#x9001;&#x3092;&#x99C6;&#x52D5;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;ATP&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30BB;&#x30AB;ATP-&#x7D50;&#x5408;&#x90E8;&#x4F4D;&#x306E; &#x26;quot;DEVD&#x26;quot;&#x30A6;&#x30A9;&#x30FC;&#x30AB;&#x30FC;B&#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x306B;210&#x3092;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x306F;&#x3001;ATP&#x52A0;&#x6C34;&#x5206;&#x89E3;&#x306E;&#x89E6;&#x5A92;&#x5869;&#x57FA;&#xFF08;&#x30CF;&#x30F3;&#x30C8;&#x3001;JF&#x3001;Weinkauf&#x3001;S.&#x3001;&#x30D8;&#x30F3;&#x30EA;&#x30FC;&#x3001;L.&#x3001;FAK&#x3001;JJ&#x3001;McNicholas&#x3001;P.&#x3068;&#x3057;&#x3066;&#x63D0;&#x6848;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;&#x30AA;&#x30EA;&#x30D0;&#x30FC;&#x3001;DB&#x3001;&#x304A;&#x3088;&#x3073;Deisenhofer&#x3001;J.&#xFF08;2002&#xFF09;&#x79D1;&#x5B66;297&#x3001;2018&#x5E74;&#x304B;&#x3089;2026&#x5E74;&#xFF09;&#x3002;&#x3053;&#x306E;&#x4EEE;&#x8AAC;&#x3068;&#x4E00;&#x81F4;&#x3057;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x305D;&#x308C;&#x305E;&#x308C;&#x91CE;&#x751F;&#x578B;&#x30BB;&#x30AB;&#x3001;0.3&#x79D2;&#xFF08;-1&#xFF09;&#x5BFE;27&#x306E;&#xFF08;-1&#xFF09;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;ATP&#x306E;&#x52A0;&#x6C34;&#x5206;&#x89E3;&#x901F;&#x5EA6;&#x306E;90&#x500D;&#x306E;&#x6E1B;&#x5C11;&#x3067;&#x30A2;&#x30B9;&#x30D1;&#x30E9;&#x30AE;&#x30F3;&#x9178;&#x306E;&#x7D50;&#x679C;&#x306B;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x306E;&#x5909;&#x7570;&#x306F;210&#x3092;&#x898B;&#x3064;&#x3051;&#x308B;&#x3002;&#x30BB;&#x30AB;-E210D&#x306F;&#x307E;&#x305F;&#x3001;&#x89E6;&#x5A92;&#x5869;&#x57FA;&#x3068;&#x3057;&#x3066;&#x50CD;&#x304F;&#x306B;&#x52A0;&#x3048;&#x3066;&#x3001;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;210&#x306F;&#x3001;&#x540C;&#x69D8;&#x306B;&#x58F2;&#x4E0A;&#x9AD8;&#x3092;&#x52A9;&#x3051;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3001;&#x91CE;&#x751F;&#x578B;&#x30BB;&#x30AB;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x9045;&#x3044;&#x901F;&#x5EA6;&#x3067;ADP&#x3092;&#x89E3;&#x653E;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x89E6;&#x5A92;&#x5869;&#x57FA;&#x3068;&#x3057;&#x3066;&#x30A2;&#x30B9;&#x30D1;&#x30E9;&#x30AE;&#x30F3;&#x9178;133&#x3092;&#x63D0;&#x6848;&#x3057;&#x305F;&#x4EE5;&#x524D;&#x306E;&#x5831;&#x544A;&#x3068;&#x77DB;&#x76FE;&#x3059;&#x308B;&#xFF08;&#x4F50;&#x85E4;&#x3001;K.&#x3001;&#x68EE;&#x3001;H.&#x3001;&#x5409;&#x7530;&#x3001;M.&#x3001;&#x304A;&#x3088;&#x3073;&#x6C34;&#x5CF6;&#x3001;S.&#xFF08;1996&#xFF09;J.&#x8BB8;&#x6587;&#x732E;&#x3002;271&#x3001;17439&#x304B;&#x3089;17444&#xFF09;&#x304C;&#x3002;&#x305D;&#x306E;&#x7814;&#x7A76;&#x3067;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x30BB;&#x30AB;-D133N&#x5909;&#x7570;&#x4F53;&#x306E;&#x518D;&#x8A55;&#x4FA1;&#x306F;&#x3001;&#x305D;&#x306E;ATPase&#x6D3B;&#x6027;&#x306E;&#x640D;&#x5931;&#x3068;&#x819C;&#x8F38;&#x9001;&#x6D3B;&#x52D5;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x305F;&#x304C;&#x3001;&#x610F;&#x5916;&#x306B;&#x3082;&#x3001;&#x985E;&#x4F3C;&#x3057;&#x305F;&#x30BB;&#x30AB;-D133A&#x5909;&#x7570;&#x4F53;&#x306F;&#x3001;&#x3053;&#x306E;&#x6B8B;&#x57FA;&#x304C;&#x89E6;&#x5A92;&#x4F5C;&#x7528;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x306A;&#x3044;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x3001;&#x5B8C;&#x5168;&#x306A;&#x6D3B;&#x6027;&#x3092;&#x4FDD;&#x6301;&#x3057;&#x307E;&#x3059;&#x3002;

&#x5927;&#x8178;&#x83CC;&#x30BB;&#x30AB;ATPase&#x306E;&#x6A5F;&#x69CB;&#x3067;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x6B8B;&#x57FA;&#x306E;&#x5F79;&#x5272;

&#xB300;&#xC7A5;&#xADE0; SecA ATP&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC138;&#xD3EC; &#xB9C9;&#xC5D0;&#xC11C; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC218;&#xC1A1;&#xC744; &#xB4DC;&#xB77C;&#xC774;&#xBE0C;. SecA ATP &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8;&#xC758; &quot;DEVD&quot; &#xC6CC;&#xCEE4; B &#xBAA8;&#xD2F0;&#xBE0C;&#xC5D0; &#xC870;&#xBBF8;&#xB8CC; 210 ATP &#xAC00;&#xC218;&#xBD84;&#xD574;&#xC5D0; &#xB300; &#xD55C; &#xCD09;&#xB9E4; &#xAE30;&#xBC18;&#xC73C;&#xB85C; &#xC81C;&#xC548; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; (&#xC0AC;&#xB0E5;, J. F., Weinkauf, S., &#xD5E8;&#xB9AC;, L., &#xD30D;, &#xC81C;&#xC774; &#xC81C;&#xC774;, McNicholas, P., &#xC62C;&#xB9AC;&#xBC84;, D. B.&#xC640; &#xB85C;&#xBCA0;&#xB974;&#xD2B8;, &#xC81C;&#xC774; (2002) &#xACFC;&#xD559; 297, 2018-2026). &#xC774; &#xAC00;&#xC124;&#xACFC; &#xC77C;&#xCE58;, &#xC6B0;&#xB9AC;&#xB294; &#xC870;&#xBBF8;&#xB8CC; 210 90-fold ATP &#xAC00;&#xC218;&#xBD84;&#xD574; &#xC18D;&#xB3C4; &#xAC10;&#xC18C;&#xC5D0; aspartate &#xACB0;&#xACFC;&#xB97C; &#xAC01;&#xAC01; &#xC57C;&#xC0DD; &#xD0C0;&#xC785; SecA, 0.3 s(-1) 27 s(-1) &#xB300;&#xC5D0; &#xBE44;&#xD574; &#xADF8; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xCC3E;&#xC73C;&#xC2ED;&#xC2DC;&#xC624;. SecA E210D &#xB610;&#xD55C; &#xC57C;&#xC0DD; &#xD0C0;&#xC785; SecA, &#xCD09;&#xB9E4; &#xAE30;&#xBC18;&#xC73C;&#xB85C; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xAC83; &#xC678;&#xC5D0;&#xB3C4; &#xC870;&#xBBF8;&#xB8CC; 210 &#xC218;&#xB3C4; &#xD68C;&#xC804;&#xC728; &#xB610;&#xD55C; &#xC751;&#xAE09; &#xC81C;&#xC548;&#xC5D0; &#xBE44;&#xD574; &#xB290;&#xB9B0; &#xC18D;&#xB3C4;&#xB85C; ADP&#xB97C; &#xD574;&#xC81C; &#xD569;&#xB2C8;&#xB2E4;. &#xCD09;&#xB9E4; &#xC810;&#xC73C;&#xB85C; aspartate 133&#xC744; &#xC81C;&#xC548; &#xC774;&#xC804; &#xBCF4;&#xACE0;&#xC11C;&#xB97C; &#xBD80;&#xC815; &#xD558;&#xB294; &#xACB0;&#xACFC; (&#xC0AC;&#xD1A0; &#xCF00;&#xC774;, &#xBAA8;&#xB9AC; &#xD5E4;, &#xC694;&#xC2DC;&#xB2E4;, M., &#xBC0F; S&#xC758; (1996) J. Biol. &#xD654;&#xD559; 271, 17439 17444 &#xBBF8;&#xC988;&#xC2DC;&#xB9C8;). &#xADF8; &#xC5F0;&#xAD6C;&#xC5D0; &#xC0AC;&#xC6A9; &#xB41C; SecA D133N &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xC7AC;&#xD3C9;&#xAC00; ATPase &#xB9C9; &#xC804; &#xD65C;&#xB3D9;&#xC758; &#xC190;&#xC2E4;&#xC744; &#xD655;&#xC778; &#xD558;&#xC9C0;&#xB9CC; &#xC720;&#xC0AC;&#xD55C; SecA D133A &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB180;&#xB78D;&#xAC8C;&#xB3C4;, &#xC804;&#xCCB4; &#xD65C;&#xB3D9; &#xC720;&#xC9C0; &#xB429;&#xB2C8;&#xB2E4; &#xACF5;&#xAC1C;&#xC774; &#xC794;&#xB958;&#xBB3C; &#xCD09;&#xB9E4;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC7AC;&#xC0DD; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xBCD1;&#xC6D0; &#xC131; &#xB300;&#xC7A5;&#xADE0;&#xC758; SecA ATPase &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0;&#xC11C; &#xBCF4;&#xC874;&#xB41C; &#xC870;&#xBBF8;&#xB8CC; &#xC794;&#xB958;&#xBB3C;&#xC758; &#xC5ED;&#xD560;.

Escherichia coli SecA utiliza ATP para conducir el transporte de prote&#xED;nas a trav&#xE9;s de las membranas celulares. Glutamato 210 en el &#x26;quot;DEVD&#x26;quot; motivo Walker B del SecA ATP-sitio de uni&#xF3;n ha sido propuesto como la base catal&#xED;tica para la hidr&#xF3;lisis de ATP (Hunt, JF, Weinkauf, S., Henry, L., Fak, JJ, McNicholas, P. , Oliver, DB, y Deisenhofer, J. (2002) Ciencia 297, 2018-2026). De acuerdo con esta hip&#xF3;tesis, encontramos que la mutaci&#xF3;n de glutamato 210 a resultados aspartato en una reducci&#xF3;n de 90 veces de la tasa de hidr&#xF3;lisis de ATP en comparaci&#xF3;n con el tipo salvaje Seca, 0,3 s (-1) frente a 27 s (-1), respectivamente. Seca-E210D tambi&#xE9;n libera ADP a un ritmo m&#xE1;s lento en comparaci&#xF3;n con SecA tipo silvestre, lo que sugiere que adem&#xE1;s de servir como la base catal&#xED;tica, el glutamato 210 podr&#xED;a ayudar el volumen de negocios tambi&#xE9;n. Nuestros resultados contradicen un informe anterior que propon&#xED;a aspartato 133 como la base catal&#xED;tica (Sato, K., Mori, H., Yoshida, M., y Mizushima, S. (1996) J. Biol.. Chem. 271 de 17.439 a 17.444) . Re-evaluaci&#xF3;n de la mutante D133N-Seca utilizados en ese estudio confirma su p&#xE9;rdida de la ATPasa de la membrana y las actividades de la translocaci&#xF3;n, pero sorprendentemente, el an&#xE1;logo Seca-D133A mutante conserva plena actividad, revelando que este residuo no juega un papel clave en la cat&#xE1;lisis.

El papel de un residuo de glutamato conservadas en el Escherichia Coli Mecanismo SecA ATPasa

The Sec-dependent protein translocation pathway promotes the transport of proteins into or across the bacterial plasma membrane. SecA ATPase has been shown to be a nanomotor that associates with its protein cargo as well as the SecYEG channel complex and to undergo ATP-driven cycles of membrane insertion and retraction that promote stepwise protein translocation. Previous studies have shown that both the 65-kDa N-domain and 30-kDa C-domain of SecA appear to undergo such membrane cycling. In the present study we performed in vivo sulfhydryl labeling of an extensive collection of monocysteine secA mutants under topologically specific conditions to identify regions of SecA that are accessible to the trans side of the membrane in its membrane-integrated state. Our results show that distinct regions of five of six SecA domains were labeled under these conditions, and such labeling clusters to a single face of the SecA structure. Our results demarcate an extensive face of SecA that interacts with SecYEG and is in fluid contact with the protein-conducting channel. The observed domain-specific labeling patterns should also provide important constraints on model building efforts in this dynamic system.

In vivo membrane topology of Escherichia coli SecA ATPase reveals extensive periplasmic exposure of multiple functionally important domains clustering on one face of SecA.

Identification of the signal peptide-binding domain within SecA ATPase is an important goal for understanding the molecular basis of SecA preprotein recognition as well as elucidating the chemo-mechanical cycle of this nanomotor during protein translocation. In this study, Forster resonance energy transfer methodology was employed to map the location of the SecA signal peptide-binding domain using a collection of functional monocysteine SecA mutants and alkaline phosphatase signal peptides labeled with appropriate donor-acceptor fluorophores. Fluorescence anisotropy measurements yielded an equilibrium binding constant of 1.4 or 10.7 muM for the alkaline phosphatase signal peptide labeled at residue 22 or 2, respectively, with SecA, and a binding stoichiometry of one signal peptide bound per SecA monomer. Binding affinity measurements performed with a monomer-biased mutant indicate that the signal peptide binds equally well to SecA monomer or dimer. Distance measurements determined for 13 SecA mutants show that the SecA signal peptide-binding domain encompasses a portion of the preprotein cross-linking domain but also includes regions of nucleotide-binding domain 1 and particularly the helical scaffold domain. The identified region lies at a multidomain interface within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel. Our FRET-mapped binding domain, in contrast to the domain identified by NMR spectroscopy, includes the two-helix finger that has been shown to interact with the preprotein during translocation and lies at the entrance to the protein-conducting channel in the recently determined SecA-SecYEG structure.

Mapping of the signal peptide-binding domain of Escherichia coli SecA using Förster resonance energy transfer.

The two major components of the Eubacteria Sec-dependent protein translocation system are the heterotrimeric channel-forming component SecYEG and its binding partner, the SecA ATPase nanomotor. Once bound to SecYEG, the preprotein substrate, and ATP, SecA undergoes ATP-hydrolytic cycles that drive the stepwise translocation of proteins. Although a previous site-directed in vivo photocross-linking study (Mori, H., and Ito, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16159-16164) elucidated residues of SecY needed for interaction with SecA, no reciprocal study for SecA protein has been reported to date. In the present study we mapped residues of SecA that interact with SecY or SecG utilizing this approach. Our results show that distinct domains of SecA on two halves of the molecule interact with two corresponding SecY partners as well as with the central cytoplasmic domain of SecG. Our data support the in vivo relevance of the Thermotoga maritima SecA·SecYEG crystal structure that visualized SecYEG interaction for only one-half of SecA as well as previous studies indicating that SecA normally binds two molecules of SecYEG.

Mapping of the SecA·SecY and SecA·SecG interfaces by site-directed in vivo photocross-linking.

SecA signal peptide interaction is critical for initiating protein translocation in the bacterial Sec-dependent pathway. Here, we have utilized the recent nuclear magnetic resonance (NMR) and Förster resonance energy transfer studies that mapped the location of the SecA signal peptide-binding site to design and isolate signal peptide-binding-defective secA mutants. Biochemical characterization of the mutant SecA proteins showed that Ser226, Val310, Ile789, Glu806, and Phe808 are important for signal peptide binding. A genetic system utilizing alkaline phosphatase secretion driven by different signal peptides was employed to demonstrate that both the PhoA and LamB signal peptides appear to recognize a common set of residues at the SecA signal peptide-binding site. A similar system containing either SecA-dependent or signal recognition particle (SRP)-dependent signal peptides along with the prlA suppressor mutation that is defective in signal peptide proofreading activity were employed to distinguish between SecA residues that are utilized more exclusively for signal peptide recognition or those that also participate in the proofreading and translocation functions of SecA. Collectively, our data allowed us to propose a model for the location of the SecA signal peptide-binding site that is more consistent with recent structural insights into this protein translocation system.

Characterization of the Escherichia coli SecA signal peptide-binding site.

Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotide-binding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secAΔ519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi(r)) and suppression of signal sequence defects (PrlD). The SecAΔ(519-547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.

The variable subdomain of Escherichia coli SecA functions to regulate SecA ATPase activity and ADP release.

The Sec machinery constitutes the major pathway for protein translocation in bacteria. SecA is thought to act as a molecular motor driving translocation of the preprotein across the membrane by repeated ATP-driven cycles of insertion and retraction at the translocon channel. SecA is predominately a dimer under physiological conditions; however, its oligomeric state during active protein translocation is still unresolved. Five SecA crystal structures have been determined, each displaying a different dimer interface, suggesting that SecA may adopt different dimer configurations. In this study, a Förster resonance energy transfer approach was utilized with nine functional monocysteine SecA mutants labeled with appropriate dyes to determine the predominant solution state dimer. Three different dye pairs allowed interprotomer distances ranging from 20 to 140 Å to be investigated. Comparison of 15 experimentally determined distances with those predicted from X-ray structures showed the greatest agreement with the Bacillus subtilis SecA antiparallel dimer structure [Hunt, J., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhfer, J. (2002) Science 297, 2018-2026]. The binding of two signal peptides to SecA was also examined to determine their effect on SecA dimer structure. We found that the SecA dimer is maintained upon peptide binding; however, the preprotein cross-linking domain (PPXD) and helical wing domain regions experience significant conformational changes, and the PPXD movement is greatly enhanced by binding of an extended signal peptide containing 19 additional residues. Modeling of an "open" antiparallel dimer structure suggests that binding of preprotein to SecA induces an activated open conformation suitable for binding to SecYEG.

Defining the solution state dimer structure of Escherichia coli SecA using Förster resonance energy transfer.

SecYEG protein of bacteria or Sec61αβγ of eukaryotes is a universally conserved heterotrimeric protein channel complex that accommodates the partitioning of membrane proteins into the lipid bilayer as well as the secretion of proteins to the trans side of the plasma or endoplasmic reticular membrane, respectively. SecYEG function is facilitated by cytosolic partners, mainly a nascent chain-ribosome complex or the SecA ATPase motor protein. Extensive efforts utilizing both biochemical and biophysical approaches have been made to determine whether SecYEG functions as a monomer or a dimer, but such approaches have often generated conflicting results. Here we have employed site-specific in vivo photo-cross-linking or cysteine cross-linking, along with co-immunoprecipitation or SecA footprinting techniques to readdress this issue. Our findings show that the SecY dimer to monomer ratio is relatively constant regardless of whether translocons are actively engaged with protein substrate or not. Under the former conditions the SecY dimer can be captured associated with a translocon-jammed substrate, indicative of SecY dimer function. Furthermore, SecA ATPase can be cross-linked to two copies of SecY when the complex contains a translocation intermediate. Collectively, our results suggest that SecYEG dimers are functional units of the translocon. 

Determination of the Oligomeric State of SecYEG Protein Secretion Channel Complex Using in Vivo Photo- and Disulfide Cross-linking.

The bacterial Sec-dependent system is the major protein-biogenic pathway for protein secretion across the cytoplasmic membrane or insertion of integral membrane proteins into the phospholipid bilayer. The mechanism of SecA-driven protein transport across the SecYEG channel complex has remained controversial with conflicting claims from biochemical and structural studies regarding the depth and extent of SecA insertion into SecYEG during ongoing protein transport. Here we utilized site-specific  photo-crosslinking to thoroughly map SecY regions that are in contact with SecA during its insertion cycle. An arabinose-inducible, rapidly folding OmpA-GFP chimera was utilized to jam the SecYEG channels with an arrested substrate protein to "freeze" them in their SecA-inserted state. Examination of 117 sites distributed throughout SecY indicated that SecA not only interacts extensively with the cytosolic regions of SecY as shown previously, but it also interacts with most of the transmembrane helices and periplasmic regions of SecY, with a clustering of interaction sights around the lateral gate and pore ring regions. Our observations support previous reports of SecA membrane insertion during  protein transport as well as those documenting the membrane penetration properties of this protein. They suggest that one or more SecA regions transiently integrate into the heart of the SecY channel complex to span the membrane to promote the protein transport cycle. These findings indicate that high-resolution structural information about the membrane-inserted state of SecA is still lacking and will be critical for elucidating the bacterial protein transport mechanism.

The SecA protein deeply penetrates into the SecYEG channel during insertion, contacting most channel transmembrane helices and periplasmic regions.

Characterization of Sec-dependent bacterial protein transport has often relied on an  protein translocation system comprised in part of  inverted inner membrane vesicles or, more recently, purified SecYEG translocons reconstituted into liposomes using mostly a single substrate (proOmpA). A paper published in this issue (P. Bariya and L. Randall, J Bacteriol 201:e00493-18, 2019, https://doi.org/10.1128/JB.00493-18) finds that inclusion of SecA protein during SecYEG proteoliposome reconstitution dramatically improves the number of active translocons. This experimentally useful and intriguing result that may arise from SecA membrane integration properties is discussed here. Furthermore, determination of the rate-limiting transport step for nine different substrates implicates the mature region distal to the signal peptide in the observed rate constant differences, indicating that more nuanced transport models that respond to differences in protein sequence and structure are needed.

Article	Year
Lyme disease-causing Borrelia species encode multiple lipoproteins homologous to peptide-binding proteins of ABC-type transporters. Infection and immunity\| PubMed ID: 9712756	1998
Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an intact gene X signal sequence and an active translocon. Journal of bacteriology\| PubMed ID: 9748461	1998
Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system. Journal of bacteriology\| PubMed ID: 11073925	2000
Translocon "pulling" of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation. Journal of bacteriology\| PubMed ID: 14594848	2003
Role of a conserved glutamate residue in the Escherichia coli SecA ATPase mechanism. The Journal of biological chemistry\| PubMed ID: 15710614	2005
In vivo membrane topology of Escherichia coli SecA ATPase reveals extensive periplasmic exposure of multiple functionally important domains clustering on one face of SecA. The Journal of biological chemistry\| PubMed ID: 17166834	2007
Mapping of the signal peptide-binding domain of Escherichia coli SecA using Förster resonance energy transfer. Biochemistry\| PubMed ID: 20025247	2010
Mapping of the SecA·SecY and SecA·SecG interfaces by site-directed in vivo photocross-linking. The Journal of biological chemistry\| PubMed ID: 21317284	2011
Characterization of the Escherichia coli SecA signal peptide-binding site. Journal of bacteriology\| PubMed ID: 22056930	2012
The variable subdomain of Escherichia coli SecA functions to regulate SecA ATPase activity and ADP release. Journal of bacteriology\| PubMed ID: 22389482	2012
Defining the solution state dimer structure of Escherichia coli SecA using Förster resonance energy transfer. Biochemistry\| PubMed ID: 23484952	2013
Determination of the Oligomeric State of SecYEG Protein Secretion Channel Complex Using in Vivo Photo- and Disulfide Cross-linking. The Journal of biological chemistry\| PubMed ID: 26747607	2016
The SecA protein deeply penetrates into the SecYEG channel during insertion, contacting most channel transmembrane helices and periplasmic regions. The Journal of biological chemistry\| PubMed ID: 28986446	2017
Substrate Proteins Take Shape at an Improved Bacterial Translocon. Journal of bacteriology\| PubMed ID: 30322856	2019

Donald B. Oliver

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