SheikIsmail_Amino Acid Insertions in the Hinge Region of HIV Protease Affect Enzyme Kinetics, Conformational Stability and Dynamics
Ontology highlight
ABSTRACT:
SUBMITTER: 
Zaahida Sheik Ismail
PROVIDER: MODEL2207110001 | BioModels | 2025-10-24
REPOSITORIES: BioModels
Publications
HIV Protease Hinge Region Insertions at Codon 38 Affect Enzyme Kinetics, Conformational Stability and Dynamics.
Sheik Ismail Zaahida Z Worth Roland R Mosebi Salerwe S Sayed Yasien Y
The protein journal 20230708 5
HIV-1 protease is essential for the production of mature, infectious virions and is a major target in antiretroviral therapy. We successfully purified a HIV-1 subtype C variant, L38↑N↑L<sup>- 4</sup>, containing an insertion of asparagine and leucine at position 38 without the four background mutations - K20R, E35D, R57K, V82I using a modified purification protocol. Isothermal titration calorimetry indicated that 50% of the variant protease sample was in the active conformation compared to 62% o ...[more]
Similar Datasets
Project description:HIV-1 protease is essential for the production of mature, infectious virions and is a major target in antiretroviral therapy. We successfully purified a HIV-1 subtype C variant, L38↑N↑L- 4, containing an insertion of asparagine and leucine at position 38 without the four background mutations - K20R, E35D, R57K, V82I using a modified purification protocol. Isothermal titration calorimetry indicated that 50% of the variant protease sample was in the active conformation compared to 62% of the wild type protease. The secondary structure composition of the variant protease was unaffected by the double insertion. The specific activity and kcat values of the variant protease were approximately 50% lower than the wild type protease values. The variant protease also exhibited a 1.6-fold increase in kcat/KM when compared to the wild type protease. Differential scanning calorimetry showed a 5 °C increase in Tm of the variant protease, indicating the variant was more stable than the wild type. Molecular dynamics simulations indicated the variant was more stable and compact than the wild type protease. A 3-4% increase in the flexibility of the hinge regions of the variant protease was observed. In addition, increased flexibility of the flaps, cantilever and fulcrum regions of the variant protease B chain was observed. The variant protease sampled only the closed flap conformation indicating a potential mechanism for drug resistance. The present study highlights the direct impact of a double amino acid insertion in hinge region on enzyme kinetics, conformational stability and dynamics of an HIV-1 subtype C variant protease.
| S-EPMC10480237 | biostudies-literature
Project description:Targeted degradation of proteins by chimeric chemical inducers of degradation (CIDEs) has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a CIDE remain poorly understood, and potent target engagement by a CIDE does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an EGFR mutant-selective degrader that potently binds to wildtype and mutant EGFR but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life is the primary determinant driving mutant-selective degradation. We present a cryo-EM structure of a CRL2VHL:CIDE:EGFR complex and show that potent target degradation can be achieved in the absence of CIDE-induced protein-protein interactions. These results highlight the importance of considering target conformation during CIDE development as well as leveraging ligand binding kinetics to achieve robust target degradation.
2022-03-31 | MSV000089175 | MassIVE
Project description:Cytotoxic T lymphocytes (CTLs) have been suggested to play an important role in controlling human immunodeficiency virus (HIV-1 or simply HIV) infection. HIV, due to its high mutation rate, can evade recognition of T cell responses by generating escape variants that cannot be recognized by HIV-specific CTLs. Although HIV escape from CTL responses has been well documented, factors contributing to the timing and the rate of viral escape from T cells have not been fully elucidated. Fitness costs associated with escape and magnitude of the epitope-specific T cell response are generally considered to be the key in determining timing of HIV escape. Several previous analyses generally ignored the kinetics of T cell responses in predicting viral escape by either considering constant or maximal T cell response; several studies also considered escape from different T cell responses to be independent. Here, we focus our analysis on data from two patients from a recent study with relatively frequent measurements of both virus sequences and HIV-specific T cell response to determine impact of CTL kinetics on viral escape. In contrast with our expectation, we found that including temporal dynamics of epitope-specific T cell response did not improve the quality of fit of different models to escape data. We also found that for well-sampled escape data, the estimates of the model parameters including T cell killing efficacy did not strongly depend on the underlying model for escapes: models assuming independent, sequential, or concurrent escapes from multiple CTL responses gave similar estimates for CTL killing efficacy. Interestingly, the model assuming sequential escapes (i.e., escapes occurring along a defined pathway) was unable to accurately describe data on escapes occurring rapidly within a short-time window, suggesting that some of model assumptions must be violated for such escapes. Our results thus suggest that the current sparse measurements of temporal CTL dynamics in blood bear little quantitative information to improve predictions of HIV escape kinetics. More frequent measurements using more sensitive techniques and sampling in secondary lymphoid tissues may allow to better understand whether and how CTL kinetics impacts viral escape.
| S-EPMC5810297 | biostudies-literature
Project description:Investigation of whole genome expression level changes in Bacillus anthracis Sterne deltaClpX mutant compared to the wild-type strain after growth in nutrient rich media. The deltaClpX mutant used in this study is described in McGillivray et al. 2009. ClpX Protease Contributes to Antimicrobial Peptide Resistance and Virulence Phenotypes of Bacillus anthracis. Journal of Innate Immunity 1(5): 494-506.
2018-02-14 | GSE94398 | GEO
Project description:We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions. These experimental constraints eliminated many classes of potential interactions, and narrowed the model to a single interaction scheme with two non-equivalent interfaces acting to form Gags into a hexamer, and a third interface acting to link hexamers together. This model was able to form into a hexameric structure with correct lattice spacing and reproduced biologically relevant growth rates. We explored the effect of genomic RNA seeding punctum growth, finding that RNA may be a factor in locally concentrating Gags to initiate assembly. The simulation results infer that completion of assembly cannot be governed simply by Gag binding kinetics. However the addition of membrane curvature suggests that budding of the virion from the plasma membrane could factor into slowing incorporation of Gag at an assembly site resulting in virions of the same size and number of Gag molecules independent of Gag concentration or the time taken to complete assembly. To corroborate the results of our simulation model, we developed an analytic model for Gag assembly finding good agreement with the simulation results.
| S-EPMC5909904 | biostudies-literature
Project description:Protease activity dependent proteome change of COS7 cells expressing CAPN10a or CAPN10c was analyzed using iTRAQ reagents. iTRAQ114 and 116 for WT, iTRAQ115 and 117 for C73S mutant.
2022-01-01 | PXD026552 | JPOST Repository
Project description:Multiple mutations are typically required to significantly improve protein stability or aggregation kinetics. However, when several substitutions are made in a single protein, the mutations can potentially interact in a nonadditive manner, resulting in epistatic effects, which can hamper protein-engineering strategies to improve thermostability or aggregation kinetics. Here, we have examined the role of protein dynamics in mediating epistasis between pairs of mutations. With Escherichia coli transketolase (TK) as a model, we explored the epistatic interactions between two single variants H192P and A282P, and also between the double-mutant H192P/A282P and two single variants, I365L or G506A. Epistasis was determined for several measures of protein stability, including the following: the free-energy barrier to kinetic inactivation, ∆∆G ‡; thermal transition midpoint temperatures, T m; and aggregation onset temperatures, T agg Nonadditive epistasis was observed between neighboring mutations as expected, but also for distant mutations located in the surface and core regions of different domains. Surprisingly, the epistatic behaviors for each measure of stability were often different for any given pairwise recombination, highlighting that kinetic and thermodynamic stabilities do not always depend on the same structural features. Molecular-dynamics simulations and a pairwise cross-correlation analysis revealed that mutations influence the dynamics of their local environment, but also in some cases the dynamics of regions distant in the structure. This effect was found to mediate epistatic interactions between distant mutations and could therefore be exploited in future protein-engineering strategies.
| S-EPMC6255212 | biostudies-literature
Project description:Sequencing of mononucleosomal DNA from asynchronous cells of Schizosaccharomyces pombe WT and S2A mutant
2015-06-22 | GSE59768 | GEO
Project description:Sequencing of mononucleosomal DNA from asynchronous cells of Schizosaccharomyces pombe WT and S2A mutant Mononucleosomal DNA from asynchronous S. pombe WT (1 sample) and S2A mutant cells (2 samples) were sequenced (Illumina HiSeq 2000) using the single-end read protocol
2015-06-22 | E-GEOD-59768 | biostudies-arrayexpress
Project description:Although the crystal structures of the HIV-1 gp120 core bound and pre-bound by CD4 are known, the details of dynamics involved in conformational equilibrium and transition in relation to gp120 function have remained elusive. The homology models of gp120 comprising the N- and C-termini and loops V3 and V4 in the CD4-bound and CD4-unbound states were built and subjected to molecular dynamics (MD) simulations to investigate the differences in dynamic properties and molecular motions between them. The results indicate that the CD4-bound gp120 adopted a more compact and stable conformation than the unbound form during simulations. For both the unbound and bound gp120, the large concerted motions derived from essential dynamics (ED) analyses can influence the size/shape of the ligand-binding channel/cavity of gp120 and, therefore, were related to its functional properties. The differences in motion direction between certain structural components of these two forms of gp120 were related to the conformational interconversion between them. The free energy calculations based on the metadynamics simulations reveal a more rugged and complex free energy landscape (FEL) for the unbound than for the bound gp120, implying that gp120 has a richer conformational diversity in the unbound form. The estimated free energy difference of ∼-6.0 kJ/mol between the global minimum free energy states of the unbound and bound gp120 indicates that gp120 can transform spontaneously from the unbound to bound states, revealing that the bound state represents a high-probability "ground state" for gp120 and explaining why the unbound state resists crystallization. Our results provide insight into the dynamics-and-function relationship of gp120, and facilitate understandings of the thermodynamics, kinetics and conformational control mechanism of HIV-1 gp120.
| S-EPMC4126740 | biostudies-literature