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Molecular evolutionary and 3D protein structural analyses of Lactobacillus fermentum elongation factor Tu, a novel brain health promoting factor

Ong JS, Liu YW, Liong MT, Choi SB, Tsai YC, Low WY

Genomics, 2020 11;112(6):3915-3924.
PMID: 32629096 DOI: 10.1016/j.ygeno.2020.06.052

The role of microbiota in gut-brain communication has led to the development of probiotics promoting brain health. Here we report a genomic study of a Lactobacillus fermentum PS150 and its patented bioactive protein, elongation factor Tu (EF-Tu), which is associated with cognitive improvement in rats. The L. fermentum PS150 circular chromosome is 2,238,401 bp and it consists of 2281 genes. Chromosome comparisons with other L. fermentum strains highlighted a cluster of glycosyltransferases as potential candidate probiotic factors besides EF-Tu. Molecular evolutionary analyses on EF-Tu genes (tuf) in 235 bacteria species revealed one to three copies of the gene per genome. Seven tuf pseudogenes were found and three species only possessed pseudogenes, which is an unprecedented finding. Protein variability analysis of EF-Tu showed five highly variable residues (40 K, 41G, 42 L, 44 K, and 46E) on the protein surface, which warrant further investigation regarding their potential roles as binding sites.

Matched MeSH terms: Protein Conformation
Fulltext Anti-Obesity Attributes; UHPLC-QTOF-MS/MS-Based Metabolite Profiling and Molecular Docking Insights of Taraxacum officinale

Aabideen ZU, Mumtaz MW, Akhtar MT, Mukhtar H, Raza SA, Touqeer T, et al.

Molecules, 2020 Oct 26;25(21).
PMID: 33114490 DOI: 10.3390/molecules25214935

The naturopathic treatment of obesity is a matter of keen interest to develop efficient natural pharmacological routes for disease management with low or negligible toxicity and side effects. For this purpose, optimized ultrasonicated hydroethanolic extracts of Taraxacum officinale were evaluated for antiobesity attributes. The 2,2-diphenyl-1-picrylhydrazyl method was adopted to evaluate antioxidant potential. Porcine pancreatic lipase inhibitory assay was conducted to assess the in vitro antiobesity property. Ultra-high performance chromatography equipped with a mass spectrometer was utilized to profile the secondary metabolites in the most potent extract. The 60% ethanolic extract exhibited highest extract yield (25.05 ± 0.07%), total phenolic contents (123.42 ± 0.007 mg GAE/g DE), total flavonoid contents (55.81 ± 0.004 RE/g DE), DPPH-radical-scavenging activity (IC50 = 81.05 ± 0.96 µg/mL) and pancreatic lipase inhibitory properties (IC50 = 146.49 ± 4.24 µg/mL). The targeted metabolite fingerprinting highlighted the presence of high-value secondary metabolites. Molecular-binding energies computed by docking tool revealed the possible contribution towards pancreatic lipase inhibitory properties of secondary metabolites including myricetin, isomangiferin, icariside B4, kaempferol and luteolin derivatives when compared to the standard drug orlistat. In vivo investigations revealed a positive impact on the lipid profile and obesity biomarkers of obese mice. The study presents Taraxacum officinale as a potent source of functional bioactive ingredients to impart new insights into the existing pool of knowledge of naturopathic approaches towards obesity management.

Matched MeSH terms: Protein Conformation
Haloadaptation: insights from comparative modeling studies between halotolerant and non-halotolerant dehalogenases

Edbeib MF, Aksoy HM, Kaya Y, Wahab RA, Huyop F

J Biomol Struct Dyn, 2020 Aug;38(12):3452-3461.
PMID: 31422756 DOI: 10.1080/07391102.2019.1657498

Halophiles are extremophilic microorganisms that grow optimally at high salt concentrations by producing a myriad of equally halotolerant enzymes. Structural haloadaptation of these enzymes adept to thriving under high-salt environments, though are not fully understood. Herein, the study attempts an in silico investigation to identify and comprehend the evolutionary structural adaptation of a halotolerant dehalogenase, DehHX (GenBank accession number: KR297065) of the halotolerant Pseudomonas halophila, over its non-halotolerant counterpart, DehMX1 (GenBank accession number KY129692) produced by Pseudomonas aeruginosa. GC content of the halotolerant DehHX DNA sequence was distinctively higher (58.9%) than the non-halotolerant dehalogenases (55% average GC). Its acidic residues, Asp and Glu were 8.27% and 12.06%, respectively, compared to an average 5.5% Asp and 7% Glu, in the latter; but lower contents of basic and hydrophobic residues in the DehHX. The secondary structure of DehHX interestingly revealed a lower incidence of α-helix forming regions (29%) and a higher percentage of coils (57%), compared to 49% and 29% in the non-halotolerant homologues, respectively. Simulation models showed the DehHX is stable under a highly saline environment (25% w/v) by adopting a highly negative-charged surface with a concomitant weakly interacting hydrophobic core. The study thus, established that a halotolerant dehalogenase undergoes notable evolutionary structural changes related to GC content over its non-halotolerant counterpart, in order to adapt and thrive under highly saline environments.Communicated by Ramaswamy H. Sarma.

Matched MeSH terms: Protein Conformation, alpha-Helical
Fulltext Structural Basis of Specific Glucoimidazole and Mannoimidazole Binding by Os3BGlu7

Nutho B, Pengthaisong S, Tankrathok A, Lee VS, Ketudat Cairns JR, Rungrotmongkol T, et al.

Biomolecules, 2020 Jun 15;10(6).
PMID: 32549280 DOI: 10.3390/biom10060907

β-Glucosidases and β-mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 β-glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the B2,5 transition state (TS) conformation preferred for mannosides and glucosides via their preferred 4H3/4E TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the -1 subsite in a 4H3/4E conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the B2,5 conformation rapidly relaxed to a E3/4H3 conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the B2,5 conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant β-glycosidases.

Matched MeSH terms: Protein Conformation
Designing and evaluation of an antibody-targeted chimeric recombinant vaccine encoding Shigella flexneri outer membrane antigens

Kazi A, Hisyam Ismail CMK, Anthony AA, Chuah C, Leow CH, Lim BH, et al.

Infect Genet Evol, 2020 06;80:104176.
PMID: 31923724 DOI: 10.1016/j.meegid.2020.104176

Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection.

Matched MeSH terms: Protein Conformation
Comparative modelling studies of fruit bromelain using molecular dynamics simulation

Pang WC, Ramli ANM, Hamid AAA

J Mol Model, 2020 May 16;26(6):142.
PMID: 32417971 DOI: 10.1007/s00894-020-04398-1

Fruit bromelain is a cysteine protease accumulated in pineapple fruits. This proteolytic enzyme has received high demand for industrial and therapeutic applications. In this study, fruit bromelain sequences QIM61759, QIM61760 and QIM61761 were retrieved from the National Center for Biotechnology Information (NCBI) Genbank Database. The tertiary structure of fruit bromelain QIM61759, QIM61760 and QIM61761 was generated by using MODELLER. The result revealed that the local stereochemical quality of the generated models was improved by using multiple templates during modelling process. Moreover, by comparing with the available papain model, structural analysis provides an insight on how pro-peptide functions as a scaffold in fruit bromelain folding and contributing to inactivation of mature protein. The structural analysis also disclosed the similarities and differences between these models. Lastly, thermal stability of fruit bromelain was studied. Molecular dynamics simulation of fruit bromelain structures at several selected temperatures demonstrated how fruit bromelain responds to elevation of temperature.

Matched MeSH terms: Protein Conformation
Fulltext Nipah shell disorder, modes of infection, and virulence

Goh GK, Dunker AK, Foster JA, Uversky VN

Microb Pathog, 2020 Apr;141:103976.
PMID: 31940461 DOI: 10.1016/j.micpath.2020.103976

The Nipah Virus (NiV) was first isolated during a 1998-9 outbreak in Malaysia. The outbreak initially infected farm pigs and then moved to humans from pigs with a case-fatality rate (CFR) of about 40%. After 2001, regular outbreaks occurred with higher CFRs (~71%, 2001-5, ~93%, 2008-12). The spread arose from drinking virus-laden palm date sap and human-to-human transmission. Intrinsic disorder analysis revealed strong correlation between the percentage of disorder in the N protein and CFR (Regression: r2 = 0.93, p protein but, also, of M shell protein. The links among shell disorder, transmission modes, and virulence suggest mechanisms by which viruses are attenuated as they passed through different cell hosts from different animal species. These have implications for development of vaccines and epidemiological molecular analytical tools to contain outbreaks.

Matched MeSH terms: Protein Conformation
Fulltext Structure-Based Design of Antivirals against Envelope Glycoprotein of Dengue Virus

Anasir MI, Ramanathan B, Poh CL

Viruses, 2020 03 26;12(4).
PMID: 32225021 DOI: 10.3390/v12040367

Dengue virus (DENV) presents a significant threat to global public health with more than 500,000 hospitalizations and 25,000 deaths annually. Currently, there is no clinically approved antiviral drug to treat DENV infection. The envelope (E) glycoprotein of DENV is a promising target for drug discovery as the E protein is important for viral attachment and fusion. Understanding the structure and function of DENV E protein has led to the exploration of structure-based drug discovery of antiviral compounds and peptides against DENV infections. This review summarizes the structural information of the DENV E protein with regards to DENV attachment and fusion. The information enables the development of antiviral agents through structure-based approaches. In addition, this review compares the potency of antivirals targeting the E protein with the antivirals targeting DENV multifunctional enzymes, repurposed drugs and clinically approved antiviral drugs. None of the current DENV antiviral candidates possess potency similar to the approved antiviral drugs which indicates that more efforts and resources must be invested before an effective DENV drug materializes.

Matched MeSH terms: Protein Conformation
Fulltext Investigation of α-Glucosidase Inhibitory Metabolites from Tetracera scandens Leaves by GC-MS Metabolite Profiling and Docking Studies

Nokhala A, Siddiqui MJ, Ahmed QU, Ahamad Bustamam MS, Zakaria AZA

Biomolecules, 2020 02 12;10(2).
PMID: 32059529 DOI: 10.3390/biom10020287

Stone leaf (Tetracera scandens) is a Southeast Asian medicinal plant that has been traditionally used for the management of diabetes mellitus. The underlying mechanisms of the antidiabetic activity have not been fully explored yet. Hence, this study aimed to evaluate the α-glucosidase inhibitory potential of the hydromethanolic extracts of T. scandens leaves and to characterize the metabolites responsible for such activity through gas chromatography-mass spectrometry (GC-MS) metabolomics. Crude hydromethanolic extracts of different strengths were prepared and in vitro assayed for α-glucosidase inhibition. GC-MS analysis was further carried out and the mass spectral data were correlated to the corresponding α-glucosidase inhibitory IC50 values via an orthogonal partial least squares (OPLS) model. The 100%, 80%, 60% and 40% methanol extracts displayed potent α-glucosidase inhibitory potentials. Moreover, the established model identified 16 metabolites to be responsible for the α-glucosidase inhibitory activity of T. scandens. The putative α-glucosidase inhibitory metabolites showed moderate to high affinities (binding energies of -5.9 to -9.8 kcal/mol) upon docking into the active site of Saccharomyces cerevisiae isomaltase. To sum up, an OPLS model was developed as a rapid method to characterize the α-glucosidase inhibitory metabolites existing in the hydromethanolic extracts of T. scandens leaves based on GC-MS metabolite profiling.

Matched MeSH terms: Protein Conformation
Fulltext Pre-grafting histological studies of skin grafts cryopreserved in α helix antarctic yeast oriented antifreeze peptide (Afp1m)

Khan MS, Ibrahim SM, Adamu AA, Rahman MBA, Bakar MZA, Noordin MM, et al.

Cryobiology, 2020 02 01;92:26-33.
PMID: 31580830 DOI: 10.1016/j.cryobiol.2019.09.012

A number of living creatures in the Antarctic region have developed characteristic adaptation of cold weather by producing antifreeze proteins (AFP). Antifreeze peptide (Afp1m) fragment have been designed in the sequence of strings from native proteins. The objectives of this study were to assess the properties of Afp1m to cryopreserve skin graft at the temperature of -10 °C and -20 °C and to assess sub-zero injuries in Afp1m cryopreserved skin graft using light microscopic techniques. In the present study, a process was developed to cryopreserve Sprague-Dawley (SD) rat skin grafts with antifreeze peptide, Afp1m, α-helix peptide fragment derived from Glaciozyma antractica yeast. Its viability assessed by different microscopic techniques. This study also described the damages caused by subzero temperatures (-10 and -20 °C) on tissue cryopreserved in different concentrations of Afp1m (0.5, 1, 2, 5 and 10 mg/mL) for 72 h. Histological scores of epidermis, dermis and hypodermis of cryopreserved skin grafts showed highly significant difference (p 

Matched MeSH terms: Protein Conformation, alpha-Helical
Crystal structure of a neoagarobiose-producing GH16 family β-agarase from Persicobacter sp. CCB-QB2

Teh AH, Fazli NH, Furusawa G

Appl Microbiol Biotechnol, 2020 Jan;104(2):633-641.
PMID: 31784792 DOI: 10.1007/s00253-019-10237-y

PdAgaC from the marine bacterium Persicobacter sp. CCB-QB2 is a β-agarase belonging to the glycoside hydrolase family 16 (GH16). It is one of only a handful of endo-acting GH16 β-agarases able to degrade agar completely to produce neoagarobiose (NA2). The crystal structure of PdAgaC's catalytic domain, which has one of the highest Vmax value at 2.9 × 103 U/mg, was determined in order to understand its unique mechanism. The catalytic domain is made up of a typical β-jelly roll fold with two additional insertions, and a well-conserved but wider substrate-binding cleft with some minor changes. Among the unique differences, two unconserved residues, Asn226 and Arg286, may potentially contribute additional hydrogen bonds to subsites -1 and +2, respectively, while a third, His185 from one of the additional insertions, may further contribute another bond to subsite +2. These additional hydrogen bonds may probably have enhanced PdAgaC's affinity for short agaro-oligosaccharides such as neoagarotetraose (NA4), rendering it capable of binding NA4 strongly enough for rapid degradation into NA2.

Matched MeSH terms: Protein Conformation
Molecular characterization of Schistosoma mansoni tegument annexins and comparative analysis of antibody responses following parasite infection

Leow CY, Willis C, Leow CH, Hofmann A, Jones M

Mol Biochem Parasitol, 2019 12;234:111231.
PMID: 31628972 DOI: 10.1016/j.molbiopara.2019.111231

Schistosomes are parasitic blood flukes that infect approximately 250 million people worldwide. The disease known as schistosomiasis, is the second most significant tropical parasitic disease after malaria. Praziquantel is the only effective drug currently licensed for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Of these, annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the structural and immunobiochemical characterization of four homologous annexins namely annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni. Bioinformatics analysis showed that there was no signal peptide predicted for any annexin in this study. Further analysis showed that each of all four annexin protein possesses a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV), which contain five alpha-helices. The life cycle expression profile of each annexin was assessed using quantitative PCR. The results showed that the overall transcript levels of the each of four homologous annexins were relatively low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). Circular dichroism (CD) demonstrated that rAnnexin B30, rAnnexin B5a and rAnnexin 7a were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity was enhanced when rAnnexin B30, rAnnexin B5a and rAnnexin 7a was incubated in the presence of Ca2+. All annexin members evaluated in this study were immunolocalized to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. All four recombinant annexins were immunoreactive and they were recognized by the sera of mice infected with S. mansoni. In conclusion, the overall results present the molecular characterization of annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni in host-parasite interactions and strongly suggest that the molecules could be useful candidates for vaccine or diagnostic development.

Matched MeSH terms: Protein Conformation, alpha-Helical
Fulltext IntFOLD: an integrated web resource for high performance protein structure and function prediction

McGuffin LJ, Adiyaman R, Maghrabi AHA, Shuid AN, Brackenridge DA, Nealon JO, et al.

Nucleic Acids Res, 2019 07 02;47(W1):W408-W413.
PMID: 31045208 DOI: 10.1093/nar/gkz322

The IntFOLD server provides a unified resource for the automated prediction of: protein tertiary structures with built-in estimates of model accuracy (EMA), protein structural domain boundaries, natively unstructured or disordered regions in proteins, and protein-ligand interactions. The component methods have been independently evaluated via the successive blind CASP experiments and the continual CAMEO benchmarking project. The IntFOLD server has established its ranking as one of the best performing publicly available servers, based on independent official evaluation metrics. Here, we describe significant updates to the server back end, where we have focused on performance improvements in tertiary structure predictions, in terms of global 3D model quality and accuracy self-estimates (ASE), which we achieve using our newly improved ModFOLD7_rank algorithm. We also report on various upgrades to the front end including: a streamlined submission process, enhanced visualization of models, new confidence scores for ranking, and links for accessing all annotated model data. Furthermore, we now include an option for users to submit selected models for further refinement via convenient push buttons. The IntFOLD server is freely available at: http://www.reading.ac.uk/bioinf/IntFOLD/.

Matched MeSH terms: Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand
Fulltext Drug ReposER: a web server for predicting similar amino acid arrangements to known drug binding interfaces for potential drug repositioning

Ab Ghani NS, Ramlan EI, Firdaus-Raih M

Nucleic Acids Res, 2019 07 02;47(W1):W350-W356.
PMID: 31106379 DOI: 10.1093/nar/gkz391

A common drug repositioning strategy is the re-application of an existing drug to address alternative targets. A crucial aspect to enable such repurposing is that the drug's binding site on the original target is similar to that on the alternative target. Based on the assumption that proteins with similar binding sites may bind to similar drugs, the 3D substructure similarity data can be used to identify similar sites in other proteins that are not known targets. The Drug ReposER (DRug REPOSitioning Exploration Resource) web server is designed to identify potential targets for drug repurposing based on sub-structural similarity to the binding interfaces of known drug binding sites. The application has pre-computed amino acid arrangements from protein structures in the Protein Data Bank that are similar to the 3D arrangements of known drug binding sites thus allowing users to explore them as alternative targets. Users can annotate new structures for sites that are similarly arranged to the residues found in known drug binding interfaces. The search results are presented as mappings of matched sidechain superpositions. The results of the searches can be visualized using an integrated NGL viewer. The Drug ReposER server has no access restrictions and is available at http://mfrlab.org/drugreposer/.

Matched MeSH terms: Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand
Fulltext A neurotoxin that specifically targets Anopheles mosquitoes

Contreras E, Masuyer G, Qureshi N, Chawla S, Dhillon HS, Lee HL, et al.

Nat Commun, 2019 06 28;10(1):2869.
PMID: 31253776 DOI: 10.1038/s41467-019-10732-w

Clostridial neurotoxins, including tetanus and botulinum neurotoxins, generally target vertebrates. We show here that this family of toxins has a much broader host spectrum, by identifying PMP1, a clostridial-like neurotoxin that selectively targets anopheline mosquitoes. Isolation of PMP1 from Paraclostridium bifermentans strains collected in anopheline endemic areas on two continents indicates it is widely distributed. The toxin likely evolved from an ancestral form that targets the nervous system of similar organisms, using a common mechanism that disrupts SNARE-mediated exocytosis. It cleaves the mosquito syntaxin and employs a unique receptor recognition strategy. Our research has an important impact on the study of the evolution of clostridial neurotoxins and provides the basis for the use of P. bifermentans strains and PMP1 as innovative, environmentally friendly approaches to reduce malaria through anopheline control.

Matched MeSH terms: Protein Conformation
Evaluating the performance of MM/PBSA for binding affinity prediction using class A GPCR crystal structures

Yau MQ, Emtage AL, Chan NJY, Doughty SW, Loo JSE

J Comput Aided Mol Des, 2019 05;33(5):487-496.
PMID: 30989574 DOI: 10.1007/s10822-019-00201-3

The recent expansion of GPCR crystal structures provides the opportunity to assess the performance of structure-based drug design methods for the GPCR superfamily. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA)-based methods are commonly used for binding affinity prediction, as they provide an intermediate compromise of speed and accuracy between the empirical scoring functions used in docking and more robust free energy perturbation methods. In this study, we systematically assessed the performance of MM/PBSA in predicting experimental binding free energies using twenty Class A GPCR crystal structures and 934 known ligands. Correlations between predicted and experimental binding free energies varied significantly between individual targets, ranging from r = - 0.334 in the inactive-state CB1 cannabinoid receptor to r = 0.781 in the active-state CB1 cannabinoid receptor, while average correlation across all twenty targets was relatively poor (r = 0.183). MM/PBSA provided better predictions of binding free energies compared to docking scores in eight out of the twenty GPCR targets while performing worse for four targets. MM/PBSA binding affinity predictions calculated using a single, energy minimized structure provided comparable predictions to sampling from molecular dynamics simulations and may be more efficient when computational cost becomes restrictive. Additionally, we observed that restricting MM/PBSA calculations to ligands with a high degree of structural similarity to the crystal structure ligands improved performance in several cases. In conclusion, while MM/PBSA remains a valuable tool for GPCR structure-based drug design, its performance in predicting the binding free energies of GPCR ligands remains highly system-specific as demonstrated in a subset of twenty Class A GPCRs, and validation of MM/PBSA-based methods for each individual case is recommended before prospective use.

Matched MeSH terms: Protein Conformation
Optimisation of human VH domain antibodies specific to Mycobacterium tuberculosis heat shock protein (HSP16.3)

Soong JX, Chan SK, Lim TS, Choong YS

J Comput Aided Mol Des, 2019 03;33(3):375-385.
PMID: 30689080 DOI: 10.1007/s10822-019-00186-z

Mycobacterium tuberculosis (Mtb) 16.3 kDa heat shock protein 16.3 (HSP16.3) is a latency-associated antigen that can be targeted for latent tuberculosis (TB) diagnostic and therapeutic development. We have previously developed human VH domain antibodies (dAbs; clone E3 and F1) specific against HSP16.3. In this work, we applied computational methods to optimise and design the antibodies in order to improve the binding affinity with HSP16.3. The VH domain antibodies were first docked to the dimer form of HSP16.3 and further sampled using molecular dynamics simulation. The calculated binding free energy of the HSP16.3-dAb complexes showed non-polar interactions were responsible for the antigen-antibody association. Per-residue free energy decomposition and computational alanine scanning have identified one hotspot residue for E3 (Y391) and 4 hotspot residues for F1 (M394, Y396, R397 and M398). These hotspot residues were then mutated and evaluated by binding free energy calculations. Phage ELISA assay was carried out on the potential mutants (E3Y391W, F1M394E, F1R397N and F1M398Y). The experimental assay showed improved binding affinities of E3Y391W and F1M394E against HSP16.3 compared with the wild type E3 and F1. This case study has thus showed in silico methods are able to assist in optimisation or improvement of antibody-antigen binding.

Matched MeSH terms: Protein Conformation
Fulltext Structural and functional insights into TRiC chaperonin from a psychrophilic yeast, Glaciozyma antarctica

Yusof NA, Kamaruddin S, Abu Bakar FD, Mahadi NM, Abdul Murad AM

Cell Stress Chaperones, 2019 Mar;24(2):351-368.
PMID: 30649671 DOI: 10.1007/s12192-019-00969-1

Studies on TCP1-1 ring complex (TRiC) chaperonin have shown its indispensable role in folding cytosolic proteins in eukaryotes. In a psychrophilic organism, extreme cold temperature creates a low-energy environment that potentially causes protein denaturation with loss of activity. We hypothesized that TRiC may undergo evolution in terms of its structural molecular adaptation in order to facilitate protein folding in low-energy environment. To test this hypothesis, we isolated G. antarctica TRiC (GaTRiC) and found that the expression of GaTRiC mRNA in G. antarctica was consistently expressed at all temperatures indicating their importance in cell regulation. Moreover, we showed GaTRiC has the ability of a chaperonin whereby denatured luciferase can be folded to the functional stage in its presence. Structurally, three categories of residue substitutions were found in α, β, and δ subunits: (i) bulky/polar side chains to alanine or valine, (ii) charged residues to alanine, and (iii) isoleucine to valine that would be expected to increase intramolecular flexibility within the GaTRiC. The residue substitutions observed in the built structures possibly affect the hydrophobic, hydrogen bonds, and ionic and aromatic interactions which lead to an increase in structural flexibility. Our structural and functional analysis explains some possible structural features which may contribute to cold adaptation of the psychrophilic TRiC folding chamber.

Matched MeSH terms: Protein Conformation
PHA synthase (PhaC): interpreting the functions of bioplastic-producing enzyme from a structural perspective

Chek MF, Hiroe A, Hakoshima T, Sudesh K, Taguchi S

Appl Microbiol Biotechnol, 2019 Feb;103(3):1131-1141.
PMID: 30511262 DOI: 10.1007/s00253-018-9538-8

Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.

Matched MeSH terms: Protein Conformation, beta-Strand
Antitumor effects of berberine against EGFR, ERK1/2, P38 and AKT in MDA-MB231 and MCF-7 breast cancer cells using molecular modelling and in vitro study

Jabbarzadeh Kaboli P, Leong MP, Ismail P, Ling KH

Pharmacol Rep, 2019 Feb;71(1):13-23.
PMID: 30343043 DOI: 10.1016/j.pharep.2018.07.005

BACKGROUND: Berberine is an alkaloid plant-based DNA intercalator that affects gene regulation, particularly expression of oncogenic and tumor suppressor proteins. The effects of berberine on different signaling proteins remains to be elucidated. The present study aimed to identify the effects of berberine against key oncogenic proteins in breast cancer cells.
METHODS: Molecular docking and molecular dynamics simulations were used for EGFR, p38, ERK1/2, and AKT. The effects of berberine and lapatinib on MAPK and PI3K pathways in MDA-MB231 and MCF-7 cells were evaluated using immunoflorescence assays, and the amounts of phosphorylated kinases were compared to total kinases after treating with different concentrations of berberine.
RESULTS: Simulations showed berberine accurately interacted with EGFR, AKT, P38, and ERK1/2 active sites in silico (scores = -7.57 to -7.92 Kcal/mol) and decreased the levels of active forms of corresponding enzymes in both cell lines; however, berberine binding to p38 showed less stability. Cytotoxicity analysis indicated that MDA-MB231 cells were resistant to berberine compared to MCF-7 cells [72 h IC50 = 50 versus 15 μM, respectively). Also, lapatinib strongly activated AKT but suppressed EGFR in MDA-MB231 cells. The activity of EGFR, AKT, P38, and ERK1/2 were affected by berberine; however, berberine dramatically reduced EGFR and AKT phosphorylation.
CONCLUSION: By way of its multikinase inhibitory effects, berberine might be a useful replacement for lapatinib, an EGFR inhibitor which can cause acquired drug resistance in patients.

Matched MeSH terms: Protein Conformation

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