Structural analysis of thermostabilizing mutations of cocaine esterase.


Abstract

Cocaine is considered to be the most addictive of all substances of abuse and mediates its effects by inhibiting monoamine transporters, primarily the dopamine transporters. There are currently no small molecules that can be used to combat its toxic and addictive properties, in part because of the difficulty of developing compounds that inhibit cocaine binding without having intrinsic effects on dopamine transport. Most of the effective cocaine inhibitors also display addictive properties. We have recently reported the use of cocaine esterase (CocE) to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality. However, wild-type CocE is relatively unstable at physiological temperatures (tau(1/2) approximately 13 min at 37 degrees C), presenting challenges for its development as a viable therapeutic agent. We applied computational approaches to predict mutations to stabilize CocE and showed that several of these have increased stability both in vitro and in vivo, with the most efficacious mutant (T172R/G173Q) extending half-life up to 370 min. Here we present novel X-ray crystallographic data on these mutants that provide a plausible model for the observed enhanced stability. We also more extensively characterize the previously reported variants and report on a new stabilizing mutant, L169K. The improved stability of these engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans. Study holds ProTherm entries: 25711, 25712, 25713, 25714, 25715 Extra Details: cocaine esterase; computational; drug abuse; thermostable

Submission Details

ID: EgYJ8JuN

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:56 p.m.

Version: 1

Publication Details
Narasimhan D;Nance MR;Gao D;Ko MC;Macdonald J;Tamburi P;Yoon D;Landry DM;Woods JH;Zhan CG;Tesmer JJ;Sunahara RK,Protein Eng. Des. Sel. (2010) Structural analysis of thermostabilizing mutations of cocaine esterase. PMID:20436035
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3I2K 2010-06-16 1.51 Cocaine Esterase, wild type, bound to a DTT adduct
3PUI 2011-09-21 1.53 Cocaine Esterase with mutations G4C, S10C
1JU3 2001-12-21 1.58 BACTERIAL COCAINE ESTERASE COMPLEX WITH TRANSITION STATE ANALOG
3IDA 2010-03-02 1.6 Thermostable Cocaine Esterase with mutations L169K and G173Q, bound to DTT adduct
1JU4 2001-12-21 1.63 BACTERIAL COCAINE ESTERASE COMPLEX WITH PRODUCT
1L7R 2003-02-11 1.64 Tyr44Phe Mutant of Bacterial Cocaine Esterase cocE
3I2H 2010-06-16 1.65 Cocaine Esterase with mutation L169K, bound to DTT adduct
1L7Q 2003-02-11 1.76 Ser117Ala Mutant of Bacterial Cocaine Esterase cocE
3I2J 2010-06-16 2.01 Cocaine Esterase, wild type, without a ligand
3I2I 2010-06-16 2.14 Cocaine Esterase with mutation T172R, bound to DTT adduct
3PUH 2011-09-21 2.3 Cocaine Esterase, wild-type biologically active dimer
4P08 2014-07-16 2.34 Engineered thermostable dimeric cocaine esterase
3I2G 2010-06-16 2.5 Cocaine Esterase with mutation G173Q, bound to DTT adduct
3I2F 2010-06-16 2.5 Cocaine Esterase with mutations T172R / G173Q, bound to DTT adduct

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cocaine esterase Q9L9D7 COCE_RHOSM