Displaying publications 21 - 40 of 53 in total

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  1. 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 Domains
  2. Fulltext The Role of Surface Exposed Lysine in Conformational Stability and Functional Properties of Lipase from Staphylococcus Family
    Ahmad NN, Ahmad Kamarudin NH, Leow ATC, Rahman RNZRA
    Molecules, 2020 Aug 25;25(17).
    PMID: 32854267 DOI: 10.3390/molecules25173858
    Surface charge residues have been recognized as one of the stability determinants in protein. In this study, we sought to compare and analyse the stability and conformational dynamics of staphylococcal lipase mutants with surface lysine mutation using computational and experimental methods. Three highly mutable and exposed lysine residues (Lys91, Lys177, Lys325) were targeted to generate six mutant lipases in silico. The model structures were simulated in water environment at 25 °C. Our simulations showed that the stability was compromised when Lys177 was substituted while mutation at position 91 and 325 improved the stability. To illustrate the putative alterations of enzyme stability in the stabilising mutants, we characterized single mutant K325G and double mutant K91A/K325G. Both mutants showed a 5 °C change in optimal temperature compared to their wild type. Single mutant K325G rendered a longer half-life at 25 °C (T1/2 = 21 h) while double mutant K91A/K325G retained only 40% of relative activity after 12 h incubation. The optimal pH for mutant K325G was shifted from 8 to 9 and similar substrate preference was observed for the wild type and two mutants. Our findings indicate that surface lysine mutation alters the enzymatic behaviour and, thus, rationalizes the functional effects of surface exposed lysine in conformational stability and activity of this lipase.
    Matched MeSH terms: Protein Domains
  3. C-terminal splice variants of P/Q-type Ca2+ channel CaV2.1 α1 subunits are differentially regulated by Rab3-interacting molecule proteins
    Hirano M, Takada Y, Wong CF, Yamaguchi K, Kotani H, Kurokawa T, et al.
    J Biol Chem, 2017 06 02;292(22):9365-9381.
    PMID: 28377503 DOI: 10.1074/jbc.M117.778829
    Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.
    Matched MeSH terms: Protein Domains
  4. Site-directed mutagenesis: role of lid region for T1 lipase specificity
    Mohamed RA, Salleh AB, Leow TC, Yahaya NM, Abdul Rahman MB
    Protein Eng. Des. Sel., 2018 06 01;31(6):221-229.
    PMID: 30239965 DOI: 10.1093/protein/gzy023
    A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region. BSLA lipase was chosen as the reference protein for T1 lipase modification since it contains no lid. Initially, structures of both enzymes were compared via protein-protein superimposition in 3D-space and the location of the lid region of T1 lipase was highlighted. A total of three variants of T1 lipase without lid were successfully designed by referring to BSLA lipase (a lipase without lid). The ability of T1 lipase without lid variants in degrading P(3HB) was investigated quantitatively. All the variants showed activity towards the substrate which confirmed that T1 lipase without lid is indeed able to degrade P(3HB). In addition, D2 was recorded to have the highest activity amongst other variants. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation but also P(3HB) by simply removing the lid region.
    Matched MeSH terms: Protein Domains
  5. Expression and characterization of functional domains of FK506-binding protein 35 from Plasmodium knowlesi
    Goh CKW, Silvester J, Wan Mahadi WNS, Chin LP, Ying LT, Leow TC, et al.
    Protein Eng. Des. Sel., 2018 12 01;31(12):489-498.
    PMID: 31120120 DOI: 10.1093/protein/gzz008
    The FK506-binding protein of Plasmodium knowlesi (Pk-FKBP35) is considerably a viable antimalarial drug target, which belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) protein family member. Structurally, this protein consists of an N-terminal FK506-binding domain (FKBD) and a C-terminal tetratricopeptide repeat domain (TPRD). This study aims to decipher functional properties of these domains as a platform for development of novel antimalarial drugs. Accordingly, full-length Pk-FKBP35 as well as its isolated domains, Pk-FKBD and Pk-TPRD were overexpressed, purified, and characterized. The results showed that catalytic PPIase activity was confined to the full-length Pk-FKBP35 and Pk-FKBD, suggesting that the catalytic activity is structurally regulated by the FKBD. Meanwhile, oligomerization analysis revealed that Pk-TPRD is essential for dimerization. Asp55, Arg60, Trp77 and Phe117 in the Pk-FKBD were considerably important for catalysis as underlined by significant reduction of PPIase activity upon mutations at these residues. Further, inhibition activity of Pk-FKBP35 towards calcineurin phosphatase activity revealed that the presence of FKBD is essential for the inhibitory property, while TPRD may be important for efficient binding to calcineurin. We then discussed possible roles of FKBP35 in Plasmodium cells and proposed mechanisms by which the immunosuppressive drug, FK506, interacts with the protein.
    Matched MeSH terms: Protein Domains
  6. Current strategies in extending half-lives of therapeutic proteins
    Zaman R, Islam RA, Ibnat N, Othman I, Zaini A, Lee CY, et al.
    J Control Release, 2019 05 10;301:176-189.
    PMID: 30849445 DOI: 10.1016/j.jconrel.2019.02.016
    Macromolecular protein and peptide therapeutics have been proven to be effective in treating critical human diseases precisely. Thanks to biotechnological advancement, a huge number of proteins and peptide therapeutics were made their way to pharmaceutical market in past few decades. However, one of the biggest challenges to be addressed for protein therapeutics during clinical application is their fast degradation in serum and quick elimination owing to enzymatic degradation, renal clearance, liver metabolism and immunogenicity, attributing to the short half-lives. Size and hydrophobicity of protein molecules make them prone to kidney filtration and liver metabolism. On the other hand, proteasomes responsible for protein destruction possess the capability of specifically recognizing almost all kinds of foreign proteins while avoiding any unwanted destruction of cellular components. At present almost all protein-based drug formulations available in market are administered intravenously (IV) or subcutaneously (SC) with high dosing at frequent interval, eventually creating dose-fluctuation-related complications and reducing patient compliance vastly. Therefore, artificially increasing the therapeutic half-life of a protein by attaching to it a molecule that increases the overall size (eg, PEG) or helps with receptor mediated recycling (eg, albumin), or manipulating amino acid chain in a way that makes it more prone towards aggregate formation, are some of the revolutionary approaches to avoid the fast degradation in vivo. Half-life extension technologies that are capable of dramatically enhancing half-lives of proteins in circulation (2-100 folds) and thus improving their overall pharmacokinetic (PK) parameters have been successfully applied on a wide range of protein therapeutics from hormones and enzymes, growth factor, clotting factor to interferon. The focus of the review is to assess the technological advancements made so far in enhancing circulatory half-lives and improving therapeutic potency of proteins.
    Matched MeSH terms: Protein Domains
  7. Fulltext Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes
    Polat OK, Uno M, Maruyama T, Tran HN, Imamura K, Wong CF, et al.
    J Am Soc Nephrol, 2019 09;30(9):1587-1603.
    PMID: 31266820 DOI: 10.1681/ASN.2018070756
    BACKGROUND: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved.

    METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.

    RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton.

    CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.

    Matched MeSH terms: Protein Domains
  8. A molecular dynamics approach to explore the structural characterization of cataract causing mutation R58H on human γD crystallin
    Karunakaran R, Srikumar PS
    Mol Cell Biochem, 2018 Dec;449(1-2):55-62.
    PMID: 29532225 DOI: 10.1007/s11010-018-3342-8
    The crystallins are a family of monomeric proteins present in the mammalian lens and mutations in these proteins cause various forms of cataracts. The aim of our current study is to emphasize the structural characterization of aggregation propensity of mutation R58H on γD crystallin using molecular dynamics (MD) approach. MD result revealed that difference in the sequence level display a wide variation in the backbone atomic position, and thus exhibits rigid conformational dynamics. Changes in the flexibility of residues favoured to increase the number of intra-molecular hydrogen bonds in mutant R58H. Moreover, notable changes in the hydrogen bonding interaction resulted to cause the misfolding of mutant R58H by introducing α-helix. Principal component analysis (PCA) result suggested that mutant R58H showed unusual conformational dynamics along the two principal components when compared to the wild-type (WT)-γD crystallin. In a nutshell, the increased surface hydrophobicity could be the cause of self-aggregation of mutant R58H leading to aculeiform cataract.
    Matched MeSH terms: Protein Domains
  9. Identification of regions affecting enzyme activity, substrate binding, dimer stabilization and polyhydroxyalkanoate (PHA) granule morphology in the PHA synthase of Aquitalea sp. USM4
    Lim H, Chuah JA, Chek MF, Tan HT, Hakoshima T, Sudesh K
    Int J Biol Macromol, 2021 Sep 01;186:414-423.
    PMID: 34246679 DOI: 10.1016/j.ijbiomac.2021.07.041
    Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized by microorganisms as intracellular energy reservoirs under stressful environmental conditions. PHA synthase (PhaC) is the key enzyme responsible for PHA biosynthesis, but the importance of its N- and C-terminal ends still remains elusive. Six plasmid constructs expressing truncation variants of Aquitalea sp. USM4 PhaC (PhaC1As) were generated and heterologously expressed in Cupriavidus necator PHB-4. Removal of the first six residues at the N-terminus enabled the modulation of PHA composition without altering the PHA content in cells. Meanwhile, deletion of 13 amino acids from the C-terminus greatly affected the catalytic activity of PhaC1As, retaining only 1.1-7.4% of the total activity. Truncation(s) at the N- and/or C-terminus of PhaC1As gradually diminished the incorporation of comonomer units, and revealed that the N-terminal region is essential for PhaC1As dimerization whereas the C-terminal region is required for stabilization. Notably, transmission electron microscopy analysis showed that PhaC modification affected the morphology of intracellular PHA granules, which until now is only known to be regulated by phasins. This study provided substantial evidence and highlighted the significance of both the N- and C-termini of PhaC1As in regulating intracellular granule morphology, activity, substrate specificity, dimerization and stability of the synthase.
    Matched MeSH terms: Protein Domains
  10. Fulltext JMJD8 is a novel endoplasmic reticulum protein with a JmjC domain
    Yeo KS, Tan MC, Lim YY, Ea CK
    Sci Rep, 2017 11 13;7(1):15407.
    PMID: 29133832 DOI: 10.1038/s41598-017-15676-z
    Jumonji C (JmjC) domain-containing proteins have been shown to regulate cellular processes by hydroxylating or demethylating histone and non-histone targets. JMJD8 belongs to the JmjC domain-only family that was recently shown to be involved in angiogenesis and TNF-induced NF-κB signaling. Here, we employed bioinformatic analysis and immunofluorescence microscopy to examine the physiological properties of JMJD8. We demonstrated that JMJD8 localizes to the lumen of endoplasmic reticulum and that JMJD8 forms dimers or oligomers in vivo. Furthermore, we identified potential JMJD8-interacting proteins that are known to regulate protein complex assembly and protein folding. Taken together, this work demonstrates that JMJD8 is the first JmjC domain-containing protein found in the lumen of the endoplasmic reticulum that may function in protein complex assembly and protein folding.
    Matched MeSH terms: Protein Domains
  11. Fulltext Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability
    Veno J, Ahmad Kamarudin NH, Mohamad Ali MS, Masomian M, Raja Abd Rahman RNZ
    Int J Mol Sci, 2017 Nov 04;18(11).
    PMID: 29113034 DOI: 10.3390/ijms18112202
    In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386) was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C) demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C) and displayed a more prolonged half-life in the range of 40-60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.
    Matched MeSH terms: Protein Domains
  12. Selective binding of pyrene in subdomain IB of human serum albumin: Combining energy transfer spectroscopy and molecular modelling to understand protein binding flexibility
    Ling I, Taha M, Al-Sharji NA, Abou-Zied OK
    Spectrochim Acta A Mol Biomol Spectrosc, 2018 Apr 05;194:36-44.
    PMID: 29316482 DOI: 10.1016/j.saa.2018.01.005
    The ability of human serum albumin (HSA) to bind medium-sized hydrophobic molecules is important for the distribution, metabolism, and efficacy of many drugs. Herein, the interaction between pyrene, a hydrophobic fluorescent probe, and HSA was thoroughly investigated using steady-state and time-resolved fluorescence techniques, ligand docking, and molecular dynamics (MD) simulations. A slight quenching of the fluorescence signal from Trp214 (the sole tryptophan residue in the protein) in the presence of pyrene was used to determine the ligand binding site in the protein, using Förster's resonance energy transfer (FRET) theory. The estimated FRET apparent distance between pyrene and Trp214 was 27Å, which was closely reproduced by the docking analysis (29Å) and MD simulation (32Å). The highest affinity site for pyrene was found to be in subdomain IB from the docking results. The calculated equilibrium structure of the complex using MD simulation shows that the ligand is largely stabilized by hydrophobic interaction with Phe165, Phe127, and the nonpolar moieties of Tyr138 and Tyr161. The fluorescence vibronic peak ratio I1/I3 of bound pyrene inside HSA indicates the presence of polar effect in the local environment of pyrene which is less than that of free pyrene in buffer. This was clarified by the MD simulation results in which an average of 5.7 water molecules were found within 0.5nm of pyrene in the binding site. Comparing the fluorescence signals and lifetimes of pyrene inside HSA to that free in buffer, the high tendency of pyrene to form dimer was almost completely suppressed inside HSA, indicating a high selectivity of the binding pocket toward pyrene monomer. The current results emphasize the ability of HSA, as a major carrier of several drugs and ligands in blood, to bind hydrophobic molecules in cavities other than subdomain IIA which is known to bind most hydrophobic drugs. This ability stems from the nature of the amino acids forming the binding sites of the protein that can easily adapt their shape to accommodate a variety of molecular structures.
    Matched MeSH terms: Protein Domains
  13. Fulltext N-terminal domain of SARS CoV-2 spike protein mutation associated reduction in effectivity of neutralizing antibody with vaccinated individuals
    Singh Y, Fuloria NK, Fuloria S, Subramaniyan V, Meenakshi DU, Chakravarthi S, et al.
    J Med Virol, 2021 Oct;93(10):5726-5728.
    PMID: 34232521 DOI: 10.1002/jmv.27181
    Matched MeSH terms: Protein Domains
  14. Fulltext FKBP22 from the psychrophilic bacterium Shewanella sp. SIB1 selectively binds to the reduced state of insulin to prevent its aggregation
    Budiman C, Goh CKW, Arief II, Yusuf M
    Cell Stress Chaperones, 2021 Mar;26(2):377-386.
    PMID: 33247372 DOI: 10.1007/s12192-020-01183-0
    FKBP22 of a psychrophilic bacterium, Shewanella sp. SIB1 (SIB1 FKBP22), is a member of peptidyl-prolyl cis-trans isomerase (PPIase) and consists of N- and C-domains responsible for chaperone-like and PPIase catalytic activities, respectively. The chaperone-like activity of SIB1 FKBP22 was previously evidenced by its ability to prevent dithiothreitol (DTT)-induced insulin aggregation. Nevertheless, the mechanism by which this protein inhibits the aggregation remains unclear. To address this, the binding affinity of SIB1 FKBP22 to the native or reduced states of insulin was examined using surface plasmon resonance (SPR). The native and reduced states refer to insulin in the absence or DTT presence, respectively. The SPR sensorgram showed that SIB1 FKBP22 binds specifically to the reduced state of insulin, with a KD value of 37.31 ± 3.20 μM. This binding was facilitated by the N-domain, as indicated by the comparable KD values of the N-domain and SIB1 FKBP22. Meanwhile, the reduced state of insulin was found to have no affinity towards the C-domain. The KD value of SIB1 FKBP22 was slightly decreased by NaCl but was not severely affected by FK506, a specific FKBP inhibitor. Similarly, the prevention of DTT-induced aggregation by SIB1 FKBP22 was also modulated by the N-domain and was not affected by FK506. Further, the reduced and native states of insulin had no effect on the catalytic efficiency (kcat/KM) of SIB1 FKBP22 towards a peptide substrate. Nevertheless, the reduced state of insulin slightly reduced the catalytic efficiency towards refolding RNase T1, at up to 1.5-fold lower than in the absence of insulin. These results suggested that the binding event was mainly facilitated by hydrophobic interaction and was independent from its PPIase activity. Altogether, a possible mechanism by which SIB1 FKBP22 prevents DTT-induced insulin aggregation was proposed.
    Matched MeSH terms: Protein Domains
  15. Fulltext The Role of the Z-DNA Binding Domain in Innate Immunity and Stress Granules
    Chiang C, Li Y, Ng SK
    Front Immunol, 2020;11:625504.
    PMID: 33613567 DOI: 10.3389/fimmu.2020.625504
    Both DNA and RNA can maintain left-handed double helical Z-conformation under physiological condition, but only when stabilized by Z-DNA binding domain (ZDBD). After initial discovery in RNA editing enzyme ADAR1, ZDBD has also been described in pathogen-sensing proteins ZBP1 and PKZ in host, as well as virulence proteins E3L and ORF112 in viruses. The host-virus antagonism immediately highlights the importance of ZDBD in antiviral innate immunity. Furthermore, Z-RNA binding has been shown to be responsible for the localization of these ZDBD-containing proteins to cytoplasmic stress granules that play central role in coordinating cellular response to stresses. This review sought to consolidate current understanding of Z-RNA sensing in innate immunity and implore possible roles of Z-RNA binding within cytoplasmic stress granules.
    Matched MeSH terms: Protein Domains
  16. Fulltext Structural studies of Chikungunya virus maturation
    Yap ML, Klose T, Urakami A, Hasan SS, Akahata W, Rossmann MG
    Proc Natl Acad Sci U S A, 2017 12 26;114(52):13703-13707.
    PMID: 29203665 DOI: 10.1073/pnas.1713166114
    Cleavage of the alphavirus precursor glycoprotein p62 into the E2 and E3 glycoproteins before assembly with the nucleocapsid is the key to producing fusion-competent mature spikes on alphaviruses. Here we present a cryo-EM, 6.8-Å resolution structure of an "immature" Chikungunya virus in which the cleavage site has been mutated to inhibit proteolysis. The spikes in the immature virus have a larger radius and are less compact than in the mature virus. Furthermore, domains B on the E2 glycoproteins have less freedom of movement in the immature virus, keeping the fusion loops protected under domain B. In addition, the nucleocapsid of the immature virus is more compact than in the mature virus, protecting a conserved ribosome-binding site in the capsid protein from exposure. These differences suggest that the posttranslational processing of the spikes and nucleocapsid is necessary to produce infectious virus.
    Matched MeSH terms: Protein Domains
  17. Identification of the cell binding domain in Nipah virus G glycoprotein using a phage display system
    Lam CW, AbuBakar S, Chang LY
    J Virol Methods, 2017 05;243:1-9.
    PMID: 28082163 DOI: 10.1016/j.jviromet.2017.01.004
    Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus with unusual broad host tropism and is designated as a Category C pathogen by the U.S. National Institute of Allergy and Infectious Diseases. NiV infection is initiated after binding of the viral G glycoprotein to the host cell receptor. The aim of this study was to map the NiV G glycoprotein cell binding domain using a phage display system. The NiV G extracellular domain was truncated and displayed as attachment proteins on M13 phage g3p minor coat protein. The binding efficiency of recombinant phages displaying different regions of NiV G to mammalian cells was evaluated. Results showed that regions of NiV G consisting of amino acids 396-602 (recombinant phage G4) and 498-602 (recombinant phage G5) demonstrated the highest binding to both Vero (5.5×103 cfu/ml and 5.6×103 cfu/ml) and THP-1 cells (3.5×103 cfu/ml and 2.9×103 cfu/ml). However, the binding of both of these recombinant phages to THP-1 cells was significantly lower than to Vero cells, and this could be due to the lack of primary host cell receptor expression on THP-1 cells. Furthermore, the binding between these two recombinant phages was competitive suggesting that there was a common host cell attachment site. This study employed an approach that is suitable for use in a biosafety level 2 containment laboratory without the need to use live virus to show that NiV G amino acids 498-602 play an important role for attachment to host cells.
    Matched MeSH terms: Protein Domains
  18. Fulltext Reductive evolution in outer membrane protein biogenesis has not compromised cell surface complexity in Helicobacter pylori
    Webb CT, Chandrapala D, Oslan SN, Bamert RS, Grinter RD, Dunstan RA, et al.
    Microbiologyopen, 2017 12;6(6).
    PMID: 29055967 DOI: 10.1002/mbo3.513
    Helicobacter pylori is a gram-negative bacterial pathogen that chronically inhabits the human stomach. To survive and maintain advantage, it has evolved unique host-pathogen interactions mediated by Helicobacter-specific proteins in the bacterial outer membrane. These outer membrane proteins (OMPs) are anchored to the cell surface via a C-terminal β-barrel domain, which requires their assembly by the β-barrel assembly machinery (BAM). Here we have assessed the complexity of the OMP C-terminal β-barrel domains employed by H. pylori, and characterized the H. pyloriBAM complex. Around 50 Helicobacter-specific OMPs were assessed with predictive structural algorithms. The data suggest that H. pylori utilizes a unique β-barrel architecture that might constitute H. pylori-specific Type V secretions system. The structural and functional diversity in these proteins is encompassed by their extramembrane domains. Bioinformatic and biochemical characterization suggests that the low β-barrel-complexity requires only minimalist assembly machinery. The H. pylori proteins BamA and BamD associate to form a BAM complex, with features of BamA enabling an oligomerization that might represent a mechanism by which a minimalist BAM complex forms a larger, sophisticated machinery capable of servicing the outer membrane proteome of H. pylori.
    Matched MeSH terms: Protein Domains
  19. TCR-like domain antibody against Mycobacterium tuberculosis (Mtb) heat shock protein antigen presented by HLA-A*11 and HLA-A*24
    Dass SA, Norazmi MN, Acosta A, Sarmiento ME, Tye GJ
    Int J Biol Macromol, 2020 Jul 15;155:305-314.
    PMID: 32240734 DOI: 10.1016/j.ijbiomac.2020.03.229
    T cell receptor (TCR)-like antibodies, obtained with the use of phage display technology, sandwich the best of the both arms of the adaptive immune system. In this study, in vitro selections against the latency associated Mycobacterium tuberculosis (Mtb) heat shock protein 16 kDa antigen (16 kDa) presented by HLA-A*011 and HLA-A*24 were carried out with the use of a human domain phage antibody library. TCR-like domain antibodies (A11Ab and A24Ab) were successfully generated recognizing 16 kDa epitopes presented by HLA-A*011 and HLA-A*24 molecules respectively. Both antibodies were found to be functional in soluble form and exhibited strong binding capacity against its targets. The results obtained support the future evaluation of these ligands for the development of diagnostic and therapeutic tools for tuberculosis infection.
    Matched MeSH terms: Protein Domains
  20. Fulltext A novel de novo NLRC4 mutation reinforces the likely pathogenicity of specific LRR domain mutation
    Chear CT, Nallusamy R, Canna SW, Chan KC, Baharin MF, Hishamshah M, et al.
    Clin Immunol, 2020 02;211:108328.
    PMID: 31870725 DOI: 10.1016/j.clim.2019.108328
    Autoinflammatory disorders are characterized by dysregulated innate immune response, resulting in recurrent uncontrolled systemic inflammation and fever. Gain-of-function mutations in NLRC4 have been described to cause a range of autoinflammatory disorders. We report a twelve-year-old Malay girl with recurrent fever, skin erythema, and inflammatory arthritis. Whole exome sequencing and subsequent bidirectional Sanger sequencing identified a heterozygous missense mutation in NLRC4 (NM_001199138: c.1970A > T). This variant was predicted to be damaging in silico, was absent in public and local databases and occurred in a highly conserved residue in the leucine-rich repeat (LRR) domain. Cytokine analysis showed extremely high serum IL-18 and IL-18/CXCL9 ratio, consistent with other NLRC4-MAS patients. In summary, we identified the first patient with a novel de novo heterozygous NLRC4 gene mutation contributing to autoinflammatory disease in Malaysia. Our findings reinforce the likely pathogenicity of specific LRR domain mutations in NLRC4 and expand the clinical spectrum of NLRC4 mutations.
    Matched MeSH terms: Protein Domains
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