Current Research in Structural Biology最新文献

Tobacco etch virus Protease (TEVp), a cysteine protease, is renowned for its remarkable specific proteolysis, making it an invaluable tool for removing fusion tags from recombinant proteins. However, TEV protease's inherent insolubility limits its broad application. Fusion constructs like an N-terminal MBP fusion, known for its improved solubility, have been employed for TEVp production to address this issue. In this study, we fused the TEVp with the N-terminal domain of the spider silk protein, specifically utilizing a charge-reversed mutant (D40K/K65D) of the N-terminal domain of major ampullate spidroin-1 protein from Euprosthenops australis, referred to as NT*. This fusion construct contains a TEVp cleavage site, enabling intracellular self-processing and the release of a His₇-tagged protease. The significant increase in soluble protein expression allowed us to purify approximately 90–100 mg of TEVp from a 1-L E. coli culture, surpassing previous findings by a considerable margin. The enzyme remained stable and catalytically active even after several months of storage in a deep freezer (−80 °C).

Myotubularin or MTM1 is a lipid phosphatase that regulates vesicular trafficking in the cell. The MTM1 gene is mutated in a severe form of muscular disease, X-linked myotubular myopathy or XLMTM, affecting 1 in 50,000 newborn males worldwide. There have been several studies on the disease pathology of XLMTM, but the structural effects of missense mutations of MTM1 are underexplored due to the unavailability of a crystal structure. MTM1 consists of three domains-a lipid-binding N-terminal GRAM domain, the phosphatase domain and a coiled-coil domain which aids dimerisation of Myotubularin homologs. While most mutations reported to date map to the phosphatase domain of MTM1, the other two domains on the sequence are also frequently mutated in XLMTM. To understand the overall structural and functional effects of missense mutations on MTM1, we curated several missense mutations and performed in silico and in vitro studies. Apart from significantly impaired binding to substrate, abrogation of phosphatase activity was observed for a few mutants. Possible long-range effects of mutations from non-catalytic domains on phosphatase activity were observed as well. Coiled-coil domain mutants have been characterised here for the first time in XLMTM literature.

A. baumannii is a ubiquitously found gram-negative, multi-drug resistant bacterial species from the ESKAPE family of pathogens known to be the causative agent for hospital-acquired infections such as pneumonia, meningitis, endocarditis, septicaemia and urinary tract infections. A. baumannii is implicated as a contributor to bloodstream infections in approximately 2% of all worldwide infections. Hence, exploring novel therapeutic agents against the bacterium is essential. LpxA or UDP-N-acetylglucosamine acetyltransferase is an essential enzyme important in Lipid A biosynthesis which catalyses the reversible transfer of an acetyl group on the glucosamine 3-OH of the UDP-GlcNAc which is a crucial step in the biosynthesis of the protective Lipopolysaccharides (LPS) layer of the bacteria which upon disruption can lead to the elimination of the bacterium which delineates LpxA as an appreciable drug target from A. baumannii. The present study performs high throughput virtual screening of LpxA against the enamine-HTSC-large-molecule library and performs toxicity and ADME screening to identify the three promising lead molecules subjected to molecular dynamics simulations. Global and essential dynamics analysis of LpxA and its complexes along with FEL and MM/PBSA based binding free energy delineate Z367461724 and Z219244584 as potential inhibitors against LpxA from A. baumannii.

Human serum albumin (HSA) is a multi-domain macromolecule with diverse ligand binding capability because of its ability to allow allosteric modulation despite being a monomeric protein. Physiologically, HSA act as the primary carrier for various exogenous and endogenous compounds and fatty acids, and alter the pharmacokinetic properties of several drugs. It has antioxidant properties and is utilized therapeutically to improve the drug delivery of pharmacological agents for the treatment of several disorders. The flexibility of albumin in holding various types of drugs coupled with a variety of modifications makes this protein a versatile drug carrier with incalculable potential in therapeutics. This review provides a brief outline of the different structural properties of HSA, and its various binding sites, moreover, an overview of the genetic, biomedical, and allosteric modulation of drugs and drug delivery aspects of HSA is also included, which may be helpful in guiding advanced clinical applications and further research on the therapeutic potential of this extraordinary protein.

CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III–B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), causative agent of the coronavirus disease-2019 (COVID-19) has resulted in several deaths and severe economic losses throughout the world. The spike protein in the virus binds to the human ACE-2 receptor in order to mediate virus-host interactions required for the viral transmission. Since first report of the SARS-CoV-2 sequence during December 2019 from patient infected with the virus in Wuhan, China, the virus has undergone rapid changes leading to mutations comprising substitutions, deletions and insertions in the sequence resulting in several variants of the virus that were more virulent and transmissible or less virulent but highly transmissible. The timely intervention with COVID-19 vaccines proved to be effective in controlling the number of infections. However, rapid mutations in the virus led to the lowering of vaccine efficacies being administered to people. In May 2023, the World Health Organization declared COVID-19 was not a public health emergency of international concern anymore. In order to take stock of mutations in the virus from early days to nearly end of COVID-19 pandemic, sequence analyses of the SARS-CoV-2 spike proteins available in the NCBI Virus database was carried out. The mutations and invariant residues in the SARS-CoV-2 spike protein sequences relative to the reference sequence were analysed. The location of the invariant residues and residues at interface of the protein chains in the spike protein trimer complex structure were examined. A total of 111,298 non-redundant SARS-CoV-2 spike protein sequences representing 2,345,585 spike proteins in the NCBI Virus database showed mutations at 1252 of the 1273 positions in the amino acid sequence. The mutations represented 6129 different mutation types in the sequences analysed. Besides, some sequences also contained insertion mutations. The SARS-CoV-2 spike protein sequences represented 1435 lineages. In addition, several spike protein sequences with mutations whose lineages were either ‘not classified’ or were ‘unclassifiable’ indicated the virus could still be evolving.

Atherosclerosis is a chronic inflammatory disease characterized by plaque build-up in the arteries, leading to the obstruction of blood flow. Macrophages are the primary immune cells found in the atherosclerotic lesions and are directly involved in atherosclerosis progression. Macrophages are derived from extravasating blood monocytes. The monocytic CD40 receptor is important for monocyte recruitment on the endothelium expressing the CD40 ligand (CD40L). Thus, targeting monocyte/macrophage interaction with the endothelium by inhibiting CD40-CD40L interaction may be a promising strategy for attenuating atherosclerosis. Monoclonal antibodies have been used against this target but shows various complications. We used an array of computer-aided drug discovery tools and molecular docking approaches to design a therapeutic inhibitory peptide that could efficiently bind to the critical residues (82Y, 84D, and 86N) on the CD40 receptor essential for the receptor's binding to CD40L. The initial screen identified a parent peptide with a high binding affinity to CD40, but the peptide exhibited a positive hepatotoxicity score. We then designed several novel peptidomimetic derivatives with higher binding affinities to CD40, good physicochemical properties, and negative hepatotoxicity as compared to the parent peptide. Furthermore, we conducted molecular dynamics simulations for both the apo and complexed forms of the receptor with ligand, and screened peptides to evaluate their stability. The designed peptidomimetic derivatives are promising therapeutics targeting the CD40-CD40L interaction and may potentially be used to attenuate atherosclerosis.

The overall diffraction precision index (DPI) of a biological macromolecule crystal structure was first described by Cruickshank in 1999. This topical review proceeds from this point and describes the subsequent elaboration of the index to individual atom coordinates. Additional developments were introduced by the availability of a webserver, which provides a transformed PDB entry with individual atom coordinate errors derived from applying the DPI method using the parameters provided by the authors and then subsequently added to the PDB file. This webserver has been extensively used and harnessed in describing non-covalent distance error estimates as well as assessing the significance, or otherwise, of atom movements in a variety of studies. The standard uncertainties on a biological macromolecule's atomic displacement parameters (the ‘B factors’) has been an entirely different challenge but is obviously important since the crystallographic community has developed the habit of quoting B factors to a false precision in papers. This can convey a false certainty in the dynamics of a structure. A method involving parallelisation of workflows for diffraction image data processing does however offer estimates of the precision of B factors.

Ddi1 is a multidomain protein that belongs to the ubiquitin receptor family of proteins. The Ddi1 proteins contain a highly conserved retroviral protease (RVP)-like domain along with other domains. The severity of opportunistic infections, caused by parasitic protozoa in AIDS patients, was found to decline when HIV protease inhibitors were used in antiretroviral therapy. Parasite growth was shown to be suppressed by a few of the inhibitors targeting Ddi1 present in these parasites. In this study, the binding of HIV protease inhibitors to the RVP domain of Ddi1 from Toxoplasma gondii and Cryptosporidium hominis; and the binding of ubiquitin to the ubiquitin-associated domain of Ddi1 from these two parasites were established using Biolayer Interferometry. The crystal structures of the RVP domains of Ddi1 from T. gondii and C. hominis were determined; they form homodimers similar to those observed in HIV protease and the reported structures of the same domain from Saccharomyces cerevisiae, Leishmania major and humans. The native form of the domain showed an open dimeric structure and a normal mode analysis revealed that it can take up a closed conformation resulting from relative movements of the subunits. Based on the crystal structure of the RVP domain of Ddi1 from L. major, a seven residue peptide inhibitor was designed and it was shown to bind to the RVP domain of Ddi1 from L. major by Biolayer Interferometry. This peptide was modified using computational methods and was shown to have a better affinity than the initial peptide.

Plasmoredoxin is a 22 ​kDa thiol–disulfide oxidoreductase involved in cellular redox regulatory processes and antioxidant defense. The 1.6 ​Å structure of the protein, solved via X-ray crystallography, adopts a modified thioredoxin fold. The structure reveals that plasmoredoxin, unique for malarial parasites, forms a new subgroup of thioredoxin-like proteins together with tryparedoxin, unique for kinetoplastids. Unlike most members of this superfamily, Plrx does not have a proline residue within the CxxC redox motif. In addition, the Plrx structure has a distinct C-terminal domain. Similar to human thioredoxin, plasmoredoxin forms monomers and dimers, which are also structurally similar to the human thioredoxin dimer, and, as in humans, plasmoredoxin is inactive as a dimer. Monomer–dimer equilibrium depends on the surrounding redox conditions, which could support the parasite in reacting to oxidative challenges. Based on structural considerations, the residues of the dimer interface are likely to interact with target proteins. In contrast to human and Plasmodium falciparum thioredoxin, however, there is a cluster of positively charged residues at the dimer interface of plasmoredoxin. These intersubunit (lysine) residues might allow binding of the protein to cellular membranes or to plasminogen. Malaria parasites lack catalase and glutathione peroxidase and therefore depend on their other glutathione and thioredoxin-dependent redox relays. Plasmoredoxin could be part of a so far unknown electron transfer system that only occurs in these parasites. Since the surface charge of plasmoredoxin differs significantly from other members of the thioredoxin superfamily, its three-dimensional structure can provide a model for designing selective redox-modulatory inhibitors.