Molecular dynamics and free energy studies on the carboxypeptidases complexed with peptide/small molecular inhibitor: Mechanism for drug resistance

Zhang, Hong; Yao, Yao; Yang, Huibin; Wang, Xia; Kang, Zhuo; Li, Yan; Li, Guohui; Wang, Yonghua

doi:10.1016/j.ibmb.2012.04.005

Cited by 10 publications

(9 citation statements)

References 47 publications

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“…A total of 400 snapshots from the trajectory were extracted every 20 ps, and the MM/PBSA and MM/GBSA calculations were performed on each snapshot. The binding free energies (ΔG) were computed according to the following equations [31]: where <…> indicates an average of an energy term along the MD simulation trajectory. ΔG gas and ΔG solv represent the vacuum and solvation binding free energies, respectively.…”

Section: Methodsmentioning

confidence: 99%

Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

et al. 2013

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New Delhi metallo-β-lactmase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactmases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1∼7 fold increases in kcat/Km values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.

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Section: Methodsmentioning

confidence: 99%

Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

et al. 2013

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“…To examine the change in the conformation and stability of the ligand bound complexes, studies demand MD simulations, as it provides valuable clues and details of the key interactions and orientation of the ligand in the final complex structure 15 . Long term MD simulations were performed for each system (wild-type + ATP, H44A + ATP, H47A + ATP, N69A + ATP, Q72A + ATP, K160A + ATP and Q164A + ATP) to study the dynamical behaviour of protein and ligand complex at atomics level.…”

Section: Resultsmentioning

confidence: 99%

Alanine mutation of the catalytic sites of Pantothenate Synthetase causes distinct conformational changes in the ATP binding region

Pandey

Grover

Goyal

et al. 2018

Sci Rep

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The enzyme Pantothenate synthetase (PS) represents a potential drug target in Mycobacterium tuberculosis. Its X-ray crystallographic structure has demonstrated the significance and importance of conserved active site residues including His44, His47, Asn69, Gln72, Lys160 and Gln164 in substrate binding and formation of pantoyl adenylate intermediate. In the current study, molecular mechanism of decreased affinity of the enzyme for ATP caused by alanine mutations was investigated using molecular dynamics (MD) simulations and free energy calculations. A total of seven systems including wildtype + ATP, H44A + ATP, H47A + ATP, N69A + ATP, Q72A + ATP, K160A + ATP and Q164A + ATP were subjected to 50 ns MD simulations. Docking score, MM-GBSA and interaction profile analysis showed weak interactions between ATP (substrate) and PS (enzyme) in H47A and H160A mutants as compared to wild-type, leading to reduced protein catalytic activity. However, principal component analysis (PCA) and free energy landscape (FEL) analysis revealed that ATP was strongly bound to the catalytic core of the wild-type, limiting its movement to form a stable complex as compared to mutants. The study will give insight about ATP binding to the PS at the atomic level and will facilitate in designing of nonreactive analogue of pantoyl adenylate which will act as a specific inhibitor for PS.The causative agent of tuberculosis (TB) is Mycobacterium tuberculosis (Mtb), a major infectious bacterium which spreads through droplets in the air. TB is second only to HIV/AIDS as the major killer across the globe 1 . Multi-drug resistant Mtb is becoming a regular health problem especially in immuno-compromised individuals with HIV 2 . This form of TB is more difficult to treat and as a result has higher mortality rate. Because of this, the discovery of drugs targeting novel pathways such as the synthesis of pantothenate has become increasingly important. The World Health statistics report in 2014 stated that 9.6 million people were diagnosed with TB of which 1.5 million people died 3 . PanC gene encodes Pantothenate Synthetase (PS; EC 6.3.2.1) responsible for producing pantothenate (vitamin B5), is a promising drug target owing to a few important reasons 4 . Firstly, it is present in all bacteria but absent in mammals, a key factor for the selective activity of drug molecule 5 . Secondly, Pantothenate is notable for its role in the synthesis of coenzyme A (CoA) and acyl carrier protein (ACP), essential components of fatty acid synthesis which maintain persistent growth and pathogenicity of the M. tuberculosis 6 . Lastly, Jacob et al., conducted research on a TB vaccine which compromised panC auxotrophs' growth and virulence rigorously supporting the theory of functional necessity of Pantothenate Synthetase pathway and enhancing its attractiveness as a potential antimicrobial target 7 . PS proceeds by Bi Uni Uni Bi Ping Pong kinetic reactions; it catalyzes the ATP dependent condensation of pantoate with beta-alanine via a pantoyl adenylate intermediate as follo...

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“…4 Previously, carboxypeptidases (CPs) have been validated to play many diverse biological roles by removing C-terminal amino acids from proteins and peptides. 5,6 Genomic disorders in CPs have been revealed to lead to numerous diseases. [7][8][9] In mammals, the M14 family of CPs, which comprises 25 family members, is the largest family of enzymes that mediates C-terminal residue cleavage.…”

Section: Introductionmentioning

confidence: 99%

<p>Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway</p>

Ye¹,

An²,

Wang³

et al. 2020

CMAR

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Background: Carboxypeptidase X, M14 family member 2 (CPXM2) has been reported to be involved with several human malignancies. However, the impact of CPXM2 on human hepatocellular carcinoma (HCC) tumorigenesis has not been studied. Materials and Methods: Using immunohistochemistry, the detailed CPXM2 expression patterns were examined in HCC cell lines and tissues. Additionally, a hepatic stellate cell line overexpressing CPXM2 and an HCC CPXM2-knockdown cell line were established by lipofection of an expression plasmid or short hairpin RNA, respectively. The transfection efficiencies were confirmed by reverse transcription-quantitative PCR, Western blotting and immunofluorescence. Moreover, Western blotting was conducted to determine the phosphorylation levels of the tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (Stat1) pathway. Furthermore, gp130-specific hairpin RNA was used to knockdown gp130 expression in hepatic stellate cells overexpressing CPXM2. The malignant phenotype of cultured HCC cells was assessed by a Cell Counting Kit-8 (CCK8) assay, plate cloning assay, Matrigel invasion assay and wound-healing assay in vitro. Results: It was demonstrated that CPXM2 was upregulated in HCC, and its upregulation predicted a poor prognosis. Besides, the upregulation of CPXM2 markedly enhanced the metastatic potential of HCC via the gp130/JAK2/Stat1 signaling pathway in vitro. Conclusion: In summary, this evidence suggests a positive role for CPXM2 in HCC progression via modulation of the gp130/JAK2/Stat1 signaling pathway in HCC.

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Molecular dynamics and free energy studies on the carboxypeptidases complexed with peptide/small molecular inhibitor: Mechanism for drug resistance

Cited by 10 publications

References 47 publications

Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

Alanine mutation of the catalytic sites of Pantothenate Synthetase causes distinct conformational changes in the ATP binding region

<p>Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway</p>

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