A molecular dynamics (MD) simulation study of the enzymatic portion of cholera toxin; cholera toxin A-1 polypeptide (CTA1) was performed at 283, 310 and 323 K. From total energy analysis it was ...observed that this toxin is stable thermodynamically and these outcomes were likewise confirmed by root mean square deviations (RMSD) investigations. The Cα root mean square fluctuation (RMSF) examinations revealed that there are a number of residues inside CTA1, which can be used as target for designing and synthesizing inhibitory drugs, in order to inactivate cholera toxin inside the human body. The fluctuations in the radius of gyration and hydrogen bonding in CTA1 proved that protein unfolding and refolding were normal routine phenomena in its structure at all temperatures. Solvent accessible surface area study identified the hydrophilic nature of the CTA1, and due to this property it can be a potential biological weapon. The structural identification (STRIDE) algorithm for proteins was successfully used to determine the partially disordered secondary structure of CTA1. On account of this partially disordered secondary structure, it can easily deceive the proteolytic enzymes of the endoplasmic reticulum of host cells.
Thermal stability is of great importance for industrial enzymes. Here we explored the thermal-stable mechanism of thermophilic nitrile hydratases (NHases) utilizing a molecular dynamic simulation. At ...a nanosecond timescale, profiles of root mean square fluctuation (RMSF) of two thermophilic NHases, 1UGQ and 1V29, under enhancing thermal stress were carried out at 300
K, 320
K, 350
K and 370
K, respectively. Results showed that the region A1 (211–231
aa) and A2 (305–316
aa) in 1UGQ, region B1 (186–192
aa) in 1V29, and most of terminal ends in both enzymes are hyper-sensitive. Salt-bridge analyses revealed that in one hand, salt-bridges contributed to maintaining the rigid structure and stable performance of the thermophilic 1UGQ and 1V29; in the other hand, salt-bridges involved in thermal sensitive regions are relatively weak and prone to be broken at elevated temperature, thereby cannot hold the stable conformation of the spatial neighborhood. In 1V29, region A1 was stabilized by a well-organized hook–hook like cluster with multiple salt-bridge interactions, region A2 was stabilized by two strong salt-bridge interactions of GLU52-ARG332 and GLU334-ARG332. In 1UGQ, the absence of a charged residue decreased its thermal sensitivity of region B1, and the formation of a small β-sheet containing a stable salt-bridge in C-β-terminal significantly enhanced its thermal stability. By radius of gyration calculation containing or eliminating the thermal sensitive regions, we quantified the contribution of thermal sensitive regions for thermal sensitivity of 1UGQ and 1V29. Consequently, we presented strategies to improve thermal stability of the industrialized mesophilic NHase by introducing stable salt-bridge interactions into its thermal sensitive regions.
In biomedical and drug delivery treatments, protein Ca2+-ATPase in the lipid bilayer (plasma) membrane plays a key role by reducing multidrug resistance of the cancerous cells. The lipid bilayer ...membrane and the protein Ca2+-ATPase were simulated by utilising the Gromacs software and by applying the all-atom/united atom and coarse-grained models. The initial structure of Ca2+-ATPase was derived from X-ray diffraction and electron microscopy patterns and was placed in a simulated bilayer membrane of dipalmitoylphosphatidylcholine. The conformational changes were investigated by evaluating the root mean square deviation, root mean square fluctuation, order parameter, diffusion coefficients, partial density, thickness and area per lipid.
Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of a specific amino acid. There are two classes of aminoacyl-tRNA synthetases. Class I usually exists as a monomeric or dimeric form and ...has two highly conserved sequence motifs. Functionally, it aminoacylates at the 2′-OH of an adenosine nucleotide. While, class II normally exists as a dimeric or tetrameric form and consists of three highly conserved sequence motifs. It aminoacylates at the 3′-OH of the same adenosine. Aspartyl-tRNA synthetase (AspRS) belongs to class II aaRSs, is not only important to sustain the mechanism of protein fidelity by specifically recognizing its cognate amino acid; but also equally significant in the aminoacylation of tRNA
Asp. Several crystal structures of AspRS have been reported yet but no structural information is available for mammalian AspRS. In this study, we have applied template-based modeling/structure prediction to elucidate structural details of two mammalian AspRS from
Homo sapiens and
Mus musculus. The resultant models showed excellent stereochemistry similar to the crystal structure of yeast. A 5
ns molecular dynamics (MD) simulation was also performed to study the conformational changes occur in the flipping loop region (279–285). The root mean square fluctuation (RMSF) graph shows movements mostly in the catalytic site and in the flipping loop region while the main secondary structure maintained fairly stable conformations.
Stabilization of a protein using cavity‐filling strategy has hardly been successful because of unfavorable van der Waals contacts. We succeeded in stabilizing lysozymes by cavity‐filling mutations. ...The mutations were checked by a simple energy minimization in advance. It was shown clearly that the sum of free energy change caused by the hydrophobicity and the cavity size was correlated very well with protein stability. We also considered the aromatic–aromatic interaction. It is reconfirmed that the cavity‐filling mutation in a hydrophobic core is a very useful method to stabilize a protein when the mutation candidate is selected carefully.
The arylamine
N-acetyltransferases (NAT; EC 2.3.1.5) are xenobiotic-metabolizing enzymes (XME) that catalyze the transfer of an acetyl group from acetylCoA (Ac-CoA) to arylamine, hydrazines and their
...N-hydroxylated metabolites. Eukaryotes may have up to three NAT isoforms, but
Mesorhizobium loti is the only prokaryote with two functional NAT isoforms (MLNAT1 and MLNAT2). The three-dimensional structure of MLNAT1 has been determined (Holton, S.J., Dairou, J., Sandy, J., Rodrigues-Lima, F., Dupret, J.M., Noble, M.E.M. and Sim, E. (2005) Structure of
Mesorhizobium loti arylamine
N-acetyltransferase 1. Acta Cryst, F61, 14–16). No MLNAT2 crystals have yet been produced, despite the production of sufficient quantities of pure protein. Using purified recombinant MLNAT1 and MLNAT2, we showed here that MLNAT1 was intrinsically more stable than MLNAT2. To test whether different structural features could explain these differences in intrinsic stability, we constructed a high-quality homology model for MLNAT2 based on far UV-CD data. Despite low levels of sequence identity with other prokaryotic NAT enzymes (≈28% identity), this model suggests that MLNAT2 adopts the characteristic three-domain NAT fold. More importantly, molecular dynamics simulations on the structures of MLNAT1 and MLNAT2 suggested that MLNAT2 was less stable than MLNAT1 due to differences in amino-acid sequence/structure features in the α/β lid domain.
The bacterium
Agrobacterium tumefaciens mediates quorum sensing via TraR, a
N-3-oxo-octanoyl-
l-homoserine lactone (OOHL) signaling transcription factor. TraR consists of an N-terminal Per-Arnt-Sim ...domain (PAS-domain) containing OOHL and a C-terminal helix-turn-helix motif (HTF-motif) binding DNA, thus comprising a complete signaling pathway. Using Molecular Dynamics, we investigate the influence of OOHL on the dynamics of the protein. The OOHL binding pocket is in contact with water, leading to dynamic changes in OOHL–protein interactions. These rearrangements increase fluctuations in the PAS-domain, and, interestingly, also enhance fluctuations in the HTF-motif. Our results imply OOHL in inducing fluctuations in TraR that may facilitate DNA binding.
Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin α‐KTx12.1 interacts with various K+ channels, suggesting its capacity to match diverse channel ...pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied α‐KTx12.1 using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an α‐helix and a triple‐stranded β‐sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pKa of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin's coherent motions. In conclusion, His28, with its low pKa value, provides α‐KTx12.1 with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins' functionality.
Glutamate synthase (GltS) is a complex iron–sulfur flavoprotein that catalyzes the reductive transfer of L‐glutamine amide group to the C2 carbon of 2‐oxoglutarate yielding two molecules of ...L‐glutamate. Molecular dynamics calculations in explicit solvent were carried out to gain insight into the conformational flexibility of GltS and into the role played by the enzyme substrates in regulating the catalytic cycle. We have modelled the free (unliganded) form of Azospirillum brasilense GltS α subunit and the structure of the reduced enzyme in complex with the L‐glutamine and 2‐oxoglutarate substrates starting from the crystallographically determined coordinates of the GltS α subunit in complex with L‐methionine sulphone and 2‐oxoglutarate. The present 4‐ns molecular dynamics calculations reveal that the GltS glutaminase site may exist in a catalytically inactive conformation unable to bind glutamine, and in a catalytically competent conformation, which is stabilized by the glutamine substrate. Substrates binding also induce (1) closure of the loop formed by residues 263–271 with partial shielding of the glutaminase site from solvent, and (2) widening of the ammonia tunnel entrance at the glutaminase end to allow for ammonia diffusion toward the synthase site. The Q‐loop of glutamate synthase, which acts as an active site lid in other amidotransferases, seems to maintain an open conformation. Finally, binding of L‐methionine sulfone, a glutamine analog that mimics the tetrahedral transient species occurring during its hydrolysis, causes a coordinated rigid‐body motion of segments of the glutaminase domain that results in the inactive conformation observed in the crystal structure of GltS α subunit.
The replication of HIV-1 is strongly enhanced by a small membrane protein called virus protein U (Vpu). Vpu achieves its task by (a) interacting with CD4, the HIV-1 receptor, and (b) by amplifying ...particle release at the site of the plasma membrane. While the first role is due to interactions of the cytoplasmic site of Vpu with CD4, the second role may be due to ion channel activity caused by the self-assembly of the protein. Recently, a blocker has been proposed which abolishes channel activity. In this chapter, the mechanism of blocking is described using computational methods, including a brief overview of other viral ion channel blockers.
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