Characterization of the C-terminal binding domain from bacterial Enzyme I

Modulation of enzyme structure and flexibility by substrate/ligand binding provides an important source of enzyme function regulation. Unfortunately, our understanding of the fundamental mechanisms coupling protein dynamics to biological function is still largely incomplete, therefore limiting our ability to harness protein conformational dynamics in order to regulate enzymatic activity. Here we couple variable temperature (VT) NMR, particularly relaxation dispersion experiments, X-ray crystallography, computer simulations, protein engineering, and enzyme kinetic assays to explore the role of structural heterogeneity and conformational disorder in regulation of the C-terminal substrate binding domain (EIC) of bacterial Enzyme I (EI). In particular, we investigate the relationship between structure, conformational dynamics, and biological function of four EIC constructs: the wild type mesophilic enzyme (eEIC), a thermophilic homologue (tEIC), and two hybrid constructs engineered by incorporating the active site loops of the mesophilic enzyme into the scaffold of the thermophilic enzyme (etEIC), and vice versa (teEIC). Through this characterization we provide evidence that the four EIC constructs are structurally similar and that holo EIC undergoes an exchange between a disordered expanded inactive state and a more ordered compact active state. Furthermore, we report that the population of the active state dictates the effective turnover number and that this functional regulation is achieved by tuning the thermodynamic balance between the active and inactive states providing rational for thermal adaption (i.e. why thermophilic homologues exhibit lower activity than their mesophilic counterpart at low temperatures but increased activity comparable to its mesophilic homologue at higher temperatures). We demonstrate that altering thermal stability, conformational flexibility, and enzymatic activity through the hybridization of mesophilic/thermophilic enzyme pairs is a promising strategy for protein engineering in the field of biotechnology

Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra

Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6–10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days

Characterization of the C-terminal binding domain from bacterial Enzyme I

Modulation of enzyme structure and flexibility by substrate/ligand binding provides an important source of enzyme function regulation. Unfortunately, our understanding of the fundamental mechanisms coupling protein dynamics to biological function is still largely incomplete, therefore limiting our ability to harness protein conformational dynamics in order to regulate enzymatic activity. Here we couple variable temperature (VT) NMR, particularly relaxation dispersion experiments, X-ray crystallography, computer simulations, protein engineering, and enzyme kinetic assays to explore the role of structural heterogeneity and conformational disorder in regulation of the C-terminal substrate binding domain (EIC) of bacterial Enzyme I (EI). In particular, we investigate the relationship between structure, conformational dynamics, and biological function of four EIC constructs: the wild type mesophilic enzyme (eEIC), a thermophilic homologue (tEIC), and two hybrid constructs engineered by incorporating the active site loops of the mesophilic enzyme into the scaffold of the thermophilic enzyme (etEIC), and vice versa (teEIC). Through this characterization we provide evidence that the four EIC constructs are structurally similar and that holo EIC undergoes an exchange between a disordered expanded inactive state and a more ordered compact active state. Furthermore, we report that the population of the active state dictates the effective turnover number and that this functional regulation is achieved by tuning the thermodynamic balance between the active and inactive states providing rational for thermal adaption (i.e. why thermophilic homologues exhibit lower activity than their mesophilic counterpart at low temperatures but increased activity comparable to its mesophilic homologue at higher temperatures). We demonstrate that altering thermal stability, conformational flexibility, and enzymatic activity through the hybridization of mesophilic/thermophilic enzyme pairs is a promising strategy for protein engineering in the field of biotechnology.</p

Hybrid thermophilic/mesophilic enzymes reveal a role for conformational disorder in regulation of bacterial Enzyme I

Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.This is a manuscript of an article published as Dotas, Rochelle R., Trang T. Nguyen, Charles E. Stewart Jr, Rodolfo Ghirlando, Davit A. Potoyan, and Vincenzo Venditti. "Hybrid thermophilic/mesophilic enzymes reveal a role for conformational disorder in regulation of bacterial Enzyme I." Journal of Molecular Biology (2020). DOI: 10.1016/j.jmb.2020.05.024.</p

A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.This article is published as Purslow JA, Thimmesch JN, Sivo V, Nguyen TT, Khatiwada B, Dotas RR and Venditti V (2021) A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis. Front. Mol. Biosci. 8:699203. DOI: 10.3389/fmolb.2021.699203. Copyright 2021 Purslow, Thimmesch, Sivo, Nguyen, Khatiwada, Dotas and Venditti. Attribution 4.0 International (CC BY 4.0). Posted with permission

Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra

Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6–10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.This article is published as Evangelidis, Thomas, Santrupti Nerli, Jiří Nováček, Andrew E. Brereton, P. Andrew Karplus, Rochelle R. Dotas, Vincenzo Venditti, Nikolaos G. Sgourakis, and Konstantinos Tripsianes. "Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra." Nature Communications 9, no. 1 (2018): 384. DOI: 10.1038/s41467-017-02592-z. Posted with permission.</p