The crystal structure of <i>Mycobacterium tuberculosis</i> high-temperature requirement A protein reveals an autoregulatory mechanism

Gupta, Arvind Kumar; Behera, Debashree; Gopal, B.

doi:10.1107/s2053230x18016217

Cited by 1 publication

(9 citation statements)

References 30 publications

(31 reference statements)

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“…tuberculosis PepD structure (PDB ID: 2Z9I ) and monomeric apo form of M. tuberculosis HtrA (PDB ID: 6IEO ). Prior to the simulations, the protein structures are first energy-minimized using the conjugate gradient method to remove high-energy contacts.…”

Section: Methodsmentioning

confidence: 99%

“…Crystallization trails were performed using the conditions reported earlier . Briefly, mutants of freshly purified M.…”

Section: Methodsmentioning

confidence: 99%

“…In apo HtrA, the residue H270 of the catalytic triad (H270, D306, and S387) was seen to be flipped away, leading to an inactive (tense) active site topology. 23 In two of the three MD simulations of the apo form of HtrA, the catalytic triad was unable to sample the relaxed conformation at the μs timescale. The replica exchange simulations also further corroborated the findings from the unbiased simulations.…”

Section: Active Site Topology Is Altered By Effectormentioning

confidence: 99%

“…Using the simulation data of DegS and PepD, we calculated differences in the electrostatic energy map between the apo and holo forms of these proteins leading us to identify residues that undergo substantial changes in the electrostatic interaction energy when the effector ligand is bound to the PDZ domain. 18,23,24 We hypothesized that residues with substantial changes in electrostatic energy upon effector binding would be critical to the allosteric mechanism in DegS and PepD. 11,12,25,26 Combining information from these electrostatic interaction maps with Shannon information entropy calculations allowed us to identify other residues involved in allosteric regulation.…”

Section: Introductionmentioning

confidence: 99%

“…tuberculosis PepD, and used that information to identify residues that play a critical role in allosteric communication. Using the simulation data of DegS and PepD, we calculated differences in the electrostatic energy map between the apo and holo forms of these proteins leading us to identify residues that undergo substantial changes in the electrostatic interaction energy when the effector ligand is bound to the PDZ domain. ,, We hypothesized that residues with substantial changes in electrostatic energy upon effector binding would be critical to the allosteric mechanism in DegS and PepD. ,,, Combining information from these electrostatic interaction maps with Shannon information entropy calculations allowed us to identify other residues involved in allosteric regulation. , We further hypothesized that these residues, critical to allosteric regulation in HtrA, would be located at structurally similar locations in HtrA homologues if the allosteric network is conserved in this enzyme family. To validate these hypotheses and predictions from simulation data, and to probe the allosteric mechanism, we engineered mutations in M.…”

Section: Introductionmentioning

confidence: 99%

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Allosteric Determinants in High Temperature Requirement A Enzymes Are Conserved and Regulate the Population of Active Conformations

et al. 2023

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High temperature requirement A (HtrA) are allosterically regulated enzymes wherein effector binding to the PDZ domain triggers proteolytic activity. Yet, it remains unclear if the inter-residue network governing allostery is conserved across HtrA enzymes. Here, we investigated and identified the interresidue interaction networks by molecular dynamics simulations on representative HtrA proteases, Escherichia coli DegS and Mycobacterium tuberculosis PepD, in effector-bound and free forms. This information was used to engineer mutations that could potentially perturb allostery and conformational sampling in a different homologue, M. tuberculosis HtrA. Mutations in HtrA perturbed allosteric regulation�a finding consistent with the hypothesis that the inter-residue interaction network is conserved across HtrA enzymes. Electron density from data collected on cryo-protected HtrA crystals revealed that mutations altered the topology of the active site. Ensemble models fitted into electron density calculated from room-temperature diffraction data showed that only a fraction of these models had a catalytically competent active site conformation alongside a functional oxyanion hole thus providing experimental evidence that these mutations influenced conformational sampling. Mutations at analogous positions in the catalytic domain of DegS perturbed the coupling between effector binding and proteolytic activity, thus confirming the role of these residues in the allosteric response. The finding that a perturbation in the conserved inter-residue network alters conformational sampling and the allosteric response suggests that an ensemble allosteric model best describes regulated proteolysis in HtrA enzymes.

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Section: Methodsmentioning

confidence: 99%

“…Crystallization trails were performed using the conditions reported earlier . Briefly, mutants of freshly purified M.…”

Section: Methodsmentioning

confidence: 99%

Section: Active Site Topology Is Altered By Effectormentioning

confidence: 99%