Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity

Ozkan

2018

IJMS

β-lactamases are enzymes produced by bacteria to hydrolyze β-lactam antibiotics as a common mechanism of resistance. Evolution in such enzymes has been rendering a wide variety of antibiotics impotent, therefore posing a major threat. Clinical and in vitro studies of evolution in TEM-1 β-lactamase have revealed a large number of single point mutations that are responsible for driving resistance to antibiotics and/or inhibitors. The distal locations of these mutations from the active sites suggest that these allosterically modulate the antibiotic resistance. We investigated the effects of resistance driver mutations on the conformational dynamics of the enzyme to provide insights about the mechanism of their long-distance interactions. Through all-atom molecular dynamics (MD) simulations, we obtained the dynamic flexibility profiles of the variants and compared those with that of the wild type TEM-1. While the mutational sites in the variants did not have any direct van der Waals interactions with the active site position S70 and E166, we observed a change in the flexibility of these sites, which play a very critical role in hydrolysis. Such long distance dynamic interactions were further confirmed by dynamic coupling index (DCI) analysis as the sites involved in resistance driving mutations exhibited high dynamic coupling with the active sites. A more exhaustive dynamic analysis, using a selection pressure for ampicillin and cefotaxime resistance on all possible types of substitutions in the amino acid sequence of TEM-1, further demonstrated the observed mechanism. Mutational positions that play a crucial role for the emergence of resistance to new antibiotics exhibited high dynamic coupling with the active site irrespective of their locations. These dynamically coupled positions were neither particularly rigid nor particularly flexible, making them more evolvable positions. Nature utilizes these sites to modulate the dynamics of the catalytic sites instead of mutating the highly rigid positions around the catalytic site.

Section: Resultsmentioning

confidence: 99%

Mutations Utilize Dynamic Allostery to Confer Resistance in TEM-1 β-lactamase

Ozkan

2018

IJMS

“…Particularly, in the human branch, there is an approximately six-fold decrease in the kinetic rates between its first ancestor, AECA, and the modern-day Human THRX. Our earlier work on protein evolution shows that nature uses distal sites that are dynamically coupled with the active sites to control the active site's dynamics [49][50][51]. Thus, we analysed how the DCI (see Material and methods for the definition) of the active site changed during THRX evolution (figure 3a).…”

Section: (B) Dynamic Coupling Of the Active Site Alters Throughout Evmentioning

confidence: 99%

Ancient thioredoxins evolved to modern-day stability–function requirement by altering native state ensemble

Huihui

Ghosh

et al. 2018

Phil. Trans. R. Soc. B

Self Cite

Thioredoxins (THRXs)-small globular proteins that reduce other proteins-are ubiquitous in all forms of life, from Archaea to mammals. Although ancestral thioredoxins share sequential and structural similarity with the modern-day (extant) homologues, they exhibit significantly different functional activity and stability. We investigate this puzzle by comparative studies of their (ancient and modern-day THRXs') native state ensemble, as quantified by the dynamic flexibility index (DFI), a metric for the relative resilience of an amino acid to perturbations in the rest of the protein. Clustering proteins using DFI profiles strongly resemble an alternative classification scheme based on their activity and stability. The DFI profiles of the extant proteins are substantially different around the α3, α4 helices and catalytic regions. Likewise, allosteric coupling of the active site with the rest of the protein is different between ancient and extant THRXs, possibly explaining the decreased catalytic activity at low pH with evolution. At a global level, we note that the population of low-flexibility (called hinges) and high-flexibility sites increases with evolution. The heterogeneity (quantified by the variance) in DFI distribution increases with the decrease in the melting temperature typically associated with the evolution of ancient proteins to their modern-day counterparts.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

“…Particularly, in the human branch, there is an approximately sixfold decrease in the kinetic rates between its first ancestor, AECA, and the modern-day Human Thrx. Our earlier work on protein evolution shows that nature utilizes distal sites that are dynamically coupled with the active sites to control the active site's dynamics [49][50][51]. Thus, we analyzed how the Dynamic Coupling Index (DCI, see methods section for the definition) of the active site changed during Thrx evolution ( Figure 3A).…”

Section: Dynamic Coupling Of the Active Site Alters Throughout The Evmentioning

confidence: 99%

“…Recently, we have extended this method to identify dynamic coupling between any given residue and functionally important residues by introducing a new metric called the dynamic coupling index (DCI). DCI metric can identify sites that are distal to functional sites but impact active site dynamics through dynamic allosteric coupling [49,50,76]. This type of allosteric coupling is important; sites with strong dynamic allosteric coupling to functionally critical residues (Dynamic Allosteric Residue Coupling (DARC) spots), regardless of separation distance, contribute to the function as well.…”

Section: Dynamic Flexibility Index (Dfi)mentioning

confidence: 99%

Ancient Thioredoxins Evolved to Modern Day Stability-Function Requirement by Altering Native State Ensemble

Huihui

Ghosh

et al. 2018

Preprint

Thioredoxins (Thrxs) -small globular proteins that reduce other proteins -are ubiquitous in all forms of life, from archaea to mammals. Although ancestral Thioredoxins share sequential and structural similarity with the modern day (extant) homologs, they exhibit significantly different functional activity and stability. We investigate this puzzle by comparative studies of their (ancient and modern day Thrxs') native state ensemble, as quantified by the Dynamic Flexibility Index (DFI), a metric for the relative resilience of an amino acid to perturbations in the rest of the protein.Clustering proteins using DFI profiles strongly resembles an alternate classification scheme based on their activity and stability. The DFI profiles of the extant proteins are substantially different around the α3, α4 helices and catalytic regions. Likewise, allosteric coupling of the active site with the rest of the protein is different between ancient and extant Thrxs, possibly explaining the decreased catalytic activity at low pH with evolution. At a global level, we note that the population of low flexibility (called hinges) and high flexibility sites increases with evolution. The heterogeneity (quantified by the variance) in DFI distribution increases with the decrease in the melting temperature typically associated with the evolution of ancient proteins to their modernday counterparts. Introduction:Modern proteins have evolved through small changes from ancient times. Much of this information is encoded in protein classes from different species in the three kingdoms of life (bacteria, archaea, and eukarya). With the advances in phylogenetics and DNA-synthesis techniques, the various ancient genes, including those from the last common ancestors of bacteria, bilaterian animals, and vertebrates, have been resurrected in the laboratories. These studies have provided crucial insights about the environmental adaptations and the evolution of functions [1-8]: (i) ancestral proteins are more robust showing high thermal and chemical stability [9][10][11][12][13], and more interestingly, (ii) protein structures are conserved more than protein sequences throughout the molecular evolution [12][13][14][15]. Thus, the current challenge in molecular evolution is to understand the molecular mechanism of how nature alters the function and biophysical properties through amino acid substitutions, while conserving the 3-D structure.