pK(a) coupling at the intein active site : implications for the coordination mechanism of protein splicing with a conserved aspartate
Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pK(a) of 6.1, two units above the normal pK(a) of aspartate. The elevated pK(a) of D422 is coupled to the depressed pK(a) of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pK(a) to normal, while a D422G mutation increases the C1 pK(a) from 7.5 to 8.5. The pK(a) coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N-S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2011 |
|---|---|
| Erschienen: |
2011 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:133 |
|---|---|
| Enthalten in: |
Journal of the American Chemical Society - 133(2011), 26 vom: 06. Juli, Seite 10275-82 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Du, Zhenming [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
30KYC7MIAI |
|---|
| Anmerkungen: |
Date Completed 20.08.2012 Date Revised 20.10.2021 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1021/ja203209f |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM208547282 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM208547282 | ||
| 003 | DE-627 | ||
| 005 | 20231224004202.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231224s2011 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1021/ja203209f |2 doi | |
| 028 | 5 | 2 | |a pubmed24n0695.xml |
| 035 | |a (DE-627)NLM208547282 | ||
| 035 | |a (NLM)21604815 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Du, Zhenming |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a pK(a) coupling at the intein active site |b implications for the coordination mechanism of protein splicing with a conserved aspartate |
| 264 | 1 | |c 2011 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 20.08.2012 | ||
| 500 | |a Date Revised 20.10.2021 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pK(a) of 6.1, two units above the normal pK(a) of aspartate. The elevated pK(a) of D422 is coupled to the depressed pK(a) of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pK(a) to normal, while a D422G mutation increases the C1 pK(a) from 7.5 to 8.5. The pK(a) coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N-S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a Protons |2 NLM | |
| 650 | 7 | |a Solutions |2 NLM | |
| 650 | 7 | |a Aspartic Acid |2 NLM | |
| 650 | 7 | |a 30KYC7MIAI |2 NLM | |
| 650 | 7 | |a Rec A Recombinases |2 NLM | |
| 650 | 7 | |a EC 2.7.7.- |2 NLM | |
| 700 | 1 | |a Zheng, Yuchuan |e verfasserin |4 aut | |
| 700 | 1 | |a Patterson, Melissa |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Yangzhong |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Chunyu |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Journal of the American Chemical Society |d 1945 |g 133(2011), 26 vom: 06. Juli, Seite 10275-82 |w (DE-627)NLM00000569X |x 1520-5126 |7 nnns |
| 773 | 1 | 8 | |g volume:133 |g year:2011 |g number:26 |g day:06 |g month:07 |g pages:10275-82 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1021/ja203209f |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 133 |j 2011 |e 26 |b 06 |c 07 |h 10275-82 | ||