2L8L

Structure of an engineered splicing intein mutant based on Mycobacterium tuberculosis RecA

Summary for 2L8L

• Entry DOI: 10.2210/pdb2l8l/pdb
• NMR Information: BMRB: 17414
• Descriptor: Endonuclease PI-MtuI (1 entity in total)
• Functional Keywords: hydrolase
• Biological source: Mycobacterium tuberculosis
• Cellular location: Cytoplasm (By similarity): P0A5U4
• Total number of polymer chains: 1
• Total formula weight: 15423.51

Authors

Du, Z.,Wang, C. (deposition date: 2011-01-19, release date: 2012-01-04, Last modification date: 2024-05-15)

Primary citation

Du, Z.,Zheng, Y.,Patterson, M.,Liu, Y.,Wang, C.
pK(a) coupling at the intein active site: implications for the coordination mechanism of protein splicing with a conserved aspartate.
J.Am.Chem.Soc., 133:10275-10282, 2011

PubMed Abstract: 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.

PubMed: 21604815
DOI: 10.1021/ja203209f

Experimental method

SOLUTION NMR