Structure Report for: 5IKX

Mutations : M364L, I419F, A472T, I505T, K530E, D554G authors Jackson, C.J., Liu,J-W., Carr, P.D., Younis, F., Pandey, G., Coppin, C., Meirelles, T., Ollis, D.L., Tawfik, D.S., Weik, M., Oakeshott, J.G.

Title: Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability
Fraser NJ, Liu JW, Mabbitt PD, Correy GJ, Coppin CW, Lethier M, Perugini MA, Murphy JM, Oakeshott JG and Jackson CJ <1 more author(s)> Fraser NJ, Liu JW, Mabbitt PD, Correy GJ, Coppin CW, Lethier M, Perugini MA, Murphy JM, Oakeshott JG, Weik M, Jackson CJ (- 1)
Ref: Journal of Molecular Biology, 428:2359, 2016 : PubMed
Abstract


ESTHER: Fraser_2016_J.Mol.Biol_428_2359
PubMedSearch: Fraser 2016 J.Mol.Biol 428 2359
PubMedID: 27016206
Gene_locus related to this paper: luccu-E3aest7
Inhibitor(s) related to this paper: DEUP

Abstract

Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the alphaE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcalphaE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcalphaE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization.



        

Title: Structure and function of an insect alpha-carboxylesterase (alphaEsterase7) associated with insecticide resistance
Jackson CJ, Liu JW, Carr PD, Younus F, Coppin C, Meirelles T, Lethier M, Pandey G, Ollis DL and Oakeshott JG <2 more author(s)> Jackson CJ, Liu JW, Carr PD, Younus F, Coppin C, Meirelles T, Lethier M, Pandey G, Ollis DL, Russell RJ, Weik M, Oakeshott JG (- 2)
Ref: Proc Natl Acad Sci U S A, 110:10177, 2013 : PubMed
Abstract


ESTHER: Jackson_2013_Proc.Natl.Acad.Sci.U.S.A_110_10177
PubMedSearch: Jackson 2013 Proc.Natl.Acad.Sci.U.S.A 110 10177
PubMedID: 23733941
Gene_locus related to this paper: luccu-E3aest7

Abstract

Insect carboxylesterases from the alphaEsterase gene cluster, such as alphaE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcalphaE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of alpha-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcalphaE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical alpha/beta-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal alpha-helix that serves as a membrane anchor. Soaking of LcalphaE7 crystals in OPs led to the capture of a crystallographic snapshot of LcalphaE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcalphaE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants ( approximately 10(6) M(-1) s(-1)) indicative of a natural substrate.