Current Research in Structural Biology最新文献

{"title":"Protein conformation and biomolecular condensates","authors":"Diego S. Vazquez,&nbsp;Pamela L. Toledo,&nbsp;Alejo R. Gianotti,&nbsp;Mario R. Ermácora","doi":"10.1016/j.crstbi.2022.09.004","DOIUrl":"10.1016/j.crstbi.2022.09.004","url":null,"abstract":"<div><p>Protein conformation and cell compartmentalization are fundamental concepts and subjects of vast scientific endeavors. In the last two decades, we have witnessed exciting advances that unveiled the conjunction of these concepts. An avalanche of studies highlighted the central role of biomolecular condensates in membraneless subcellular compartmentalization that permits the spatiotemporal organization and regulation of myriads of simultaneous biochemical reactions and macromolecular interactions. These studies have also shown that biomolecular condensation, driven by multivalent intermolecular interactions, is mediated by order-disorder transitions of protein conformation and by protein domain architecture. Conceptually, protein condensation is a distinct level in protein conformational landscape in which collective folding of large collections of molecules takes place. Biomolecular condensates arise by the physical process of phase separation and comprise a variety of bodies ranging from membraneless organelles to liquid condensates to solid-like conglomerates, spanning lengths from mesoscopic clusters (nanometers) to micrometer-sized objects. In this review, we summarize and discuss recent work on the assembly, composition, conformation, material properties, thermodynamics, regulation, and functions of these bodies. We also review the conceptual framework for future studies on the conformational dynamics of condensed proteins in the regulation of cellular processes.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 285-307"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/1b/ed/main.PMC9508354.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40377924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Binding properties of the anti-TB drugs bedaquiline and TBAJ-876 to a mycobacterial F-ATP synthase","authors":"Alexander Krah ,&nbsp;Gerhard Grüber ,&nbsp;Peter J. Bond","doi":"10.1016/j.crstbi.2022.09.001","DOIUrl":"10.1016/j.crstbi.2022.09.001","url":null,"abstract":"<div><p>Tuberculosis (TB), the deadly disease caused by <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>), kills more people worldwide than any other bacterial infectious disease. There has been a recent resurgence of TB drug discovery activities, resulting in the identification of a number of novel enzyme inhibitors. Many of these inhibitors target the electron transport chain complexes and the F₁F_O-ATP synthase; these enzymes represent new target spaces for drug discovery, since the generation of ATP is essential for the bacterial pathogen's physiology, persistence, and pathogenicity. The anti-TB drug bedaquiline (BDQ) targets the <em>Mtb</em> F-ATP synthase and is used as salvage therapy against this disease. Medicinal chemistry efforts to improve the physio-chemical properties of BDQ resulted in the discovery of 3,5-dialkoxypyridine (DARQ) analogs to which TBAJ-876 belongs. TBAJ-876, a clinical development candidate, shows attractive <em>in vitro</em> and <em>in vivo</em> antitubercular activity. Both BDQ and TBAJ-876 inhibit the mycobacterial F₁F_O-ATP synthase by stopping rotation of the <em>c</em>-ring turbine within the F_O domain, thereby preventing proton translocation and ATP synthesis to occur. While structural data for the BDQ bound state are available, no structural information about TBAJ-876 binding have been described. In this study, we show how TBAJ-876 binds to the F_O domain of the <em>M. smegmatis</em> F₁F_O-ATP synthase. We further calculate the binding free energy of both drugs bound to their target and predict an increased affinity of TBAJ-876 for the F_O domain. This approach will be useful in future efforts to design new and highly potent DARQ analogs targeting F-ATP synthases of <em>Mtb</em>, nontuberculosis mycobacteria (NTM) as well as the <em>M. leprosis</em> complex.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 278-284"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9516385/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40388872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolically-incorporated deuterium in myelin localized by neutron diffraction and identified by mass spectrometry","authors":"Anne Baumann ,&nbsp;Andrew R. Denninger ,&nbsp;Marek Domin ,&nbsp;Bruno Demé ,&nbsp;Daniel A. Kirschner","doi":"10.1016/j.crstbi.2022.06.003","DOIUrl":"10.1016/j.crstbi.2022.06.003","url":null,"abstract":"&lt;div&gt;&lt;p&gt;Myelin is a natural and dynamic multilamellar membrane structure that continues to be of significant biological and neurological interest, especially with respect to its biosynthesis and assembly during its normal formation, maintenance, and pathological breakdown. To explore the usefulness of neutron diffraction in the structural analysis of myelin, we investigated the use of &lt;em&gt;in vivo&lt;/em&gt; labeling by metabolically incorporating non-toxic levels of deuterium (&lt;sup&gt;2&lt;/sup&gt;H; D) via drinking water into a pregnant dam (D-dam) and her developing embryos. All of the mice were sacrificed when the pups (D-pups) were 55 days old. Myelinated sciatic nerves were dissected, fixed in glutaraldehyde and examined by neutron diffraction. Parallel samples that were unfixed (trigeminal nerves) were frozen for mass spectrometry (MS). The diffraction patterns of the nerves from deuterium-fed mice (D-mice) vs. the controls (H-mice) had major differences in the intensities of the Bragg peaks but no appreciable differences in myelin periodicity. Neutron scattering density profiles showed an appreciable increase in density at the center of the lipid-rich membrane bilayer. This increase was greater in D-pups than in D-dam, and its localization was consistent with deuteration of lipid hydrocarbon, which predominates over transmembrane protein in myelin. MS analysis of the lipids isolated from the trigeminal nerves demonstrated that in the pups the percentage of lipids that had one or more deuterium atoms was uniformly high across lipid species (97.6% ​± ​2.0%), whereas in the mother the lipids were substantially less deuterated (60.6% ​± ​26.4%) with levels varying among lipid species and subspecies. The mass distribution pattern of deuterium-containing isotopologues indicated the fraction (in %) of each lipid (sub-)species having one or more deuteriums incorporated: in the D-pups, the pattern was always bell-shaped, and the average number of D atoms ranged from a low of ∼4 in fatty acid to a high of ∼9 in cerebroside. By contrast, in D-dam most lipids had more complex, overlapping distributions that were weighted toward a lower average number of deuteriums, which ranged from a low of ∼3–4 in fatty acid and in one species of sulfatide to a high of 6–7 in cerebroside and sphingomyelin. The consistently high level of deuteration in D-pups can be attributed to their &lt;em&gt;de novo&lt;/em&gt; lipogenesis during gestation and rapid, postnatal myelination. The widely varying levels of deuteration in D-dam, by contrast, likely depends on the relative metabolic stability of the particular lipid species during myelin maintenance. Our current findings demonstrate that stably-incorporated D label can be detected and localized using neutron diffraction in a complex tissue such as myelin; and moreover, that MS can be used to screen a broad range of deuterated lipid species to monitor differential rates of lipid turnover. In addition to helping to develop a comprehensive understand","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 231-245"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/4b/d9/main.PMC9356250.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40704011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A cryptic phosphate-binding pocket on the SPFH domain of human stomatin that regulates a novel fibril-like self-assembly","authors":"Koki Kataoka ,&nbsp;Shota Suzuki ,&nbsp;Takeshi Tenno ,&nbsp;Natsuko Goda ,&nbsp;Emi Hibino ,&nbsp;Atsunori Oshima ,&nbsp;Hidekazu Hiroaki","doi":"10.1016/j.crstbi.2022.05.002","DOIUrl":"10.1016/j.crstbi.2022.05.002","url":null,"abstract":"<div><p>Human stomatin (hSTOM) is a component of the membrane skeleton of erythrocytes that maintains the membrane's shape and stiffness through interconnecting spectrin and actin. hSTOM is a member of the protein family that possesses a single stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of the molecule. Although SPFH domain proteins are widely distributed from archaea to mammals, the detailed function of the domain remains unclear. In this study, we first determined the solution structure of the SPFH domain of hSTOM (hSTOM(SPFH)) via NMR. The solution structure of hSTOM(SPFH) is essentially identical to the already reported crystal structure of the STOM SPFH domain (mSTOM(SPFH)) of mice, except for the existence of a small hydrophilic pocket on the surface. We identified this pocket as a phosphate-binding site by comparing its NMR spectra with and without phosphate ions. Meanwhile, during the conventional process of protein NMR analysis, we eventually discovered that hSTOM(SPFH) formed a unique solid material after lyophilization. This lyophilized hSTOM(SPFH) sample was moderately slowly dissolved in a physiological buffer. Interestingly, it was resistant to dissolution against the phosphate buffer. We then found that the lyophilized hSTOM(SPFH) formed a fibril-like assembly under electron microscopy. Finally, we succeeded in reproducing this fibril-like assembly of hSTOM(SPFH) using a centrifugal ultrafiltration device, thus demonstrating that the increased protein concentration may promote self-assembly of hSTOM(SPFH) into fibril forms. Our observations may help understand the molecular function of the SPFH domain and its involvement in protein oligomerization as a component of the membrane skeleton. (245 words).</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 158-166"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X22000149/pdfft?md5=d55d18fabd98379509d79e7d642039e1&pid=1-s2.0-S2665928X22000149-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49613120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Energy landscape of the SARS-CoV-2 reveals extensive conformational heterogeneity","authors":"Ghoncheh Mashayekhi ,&nbsp;John Vant ,&nbsp;Abhigna Polavarapu ,&nbsp;Abbas Ourmazd ,&nbsp;Abhishek Singharoy","doi":"10.1016/j.crstbi.2022.02.001","DOIUrl":"10.1016/j.crstbi.2022.02.001","url":null,"abstract":"<div><p>Cryo-electron microscopy (cryo-EM) has produced a number of structural models of the SARS-CoV-2 spike, already prompting biomedical outcomes. However, these reported models and their associated electrostatic potential maps represent an unknown admixture of conformations stemming from the underlying energy landscape of the spike protein. As with any protein, some of the spike's conformational motions are expected to be biophysically relevant, but cannot be interpreted only by static models. Using experimental cryo-EM images, we present the energy landscape of the glycosylated spike protein, and identify the diversity of low-energy conformations in the vicinity of its open (so called 1RBD-up) state. The resulting atomic refinement reveal global and local molecular rearrangements that cannot be inferred from an average 1RBD-up cryo-EM model. Here we report varied degrees of “openness” in global conformations of the 1RBD-up state, not revealed in the single-model interpretations of the density maps, together with conformations that overlap with the reported models. We discover how the glycan shield contributes to the stability of these low-energy conformations. Five out of six binding sites we analyzed, including those for engaging ACE2, therapeutic mini-proteins, linoleic acid, two different kinds of antibodies, switch conformations between their known apo- and holo-conformations, even when the global spike conformation is 1RBD-up. This apo-to-holo switching is reminiscent of a conformational preequilibrium. We found only one binding site, namely that of AB-C135 remains in apo state within all the sampled free energy-minimizing models, suggesting an induced fit mechanism for the docking of this antibody to the spike.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 68-77"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/4f/91/main.PMC8902891.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10487902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanism of RNA recognition by a Musashi RNA-binding protein","authors":"Jinan Wang ,&nbsp;Lan Lan ,&nbsp;Xiaoqing Wu ,&nbsp;Liang Xu ,&nbsp;Yinglong Miao","doi":"10.1016/j.crstbi.2021.12.002","DOIUrl":"10.1016/j.crstbi.2021.12.002","url":null,"abstract":"<div><p>The Musashi RNA-binding proteins (RBPs) regulate translation of target mRNAs and maintenance of cell stemness and tumorigenesis. Musashi-1 (MSI1), long considered as an intestinal and neural stem cell marker, has been more recently found to be over expressed in many cancers. It has served as an important drug target for treating acute myeloid leukemia and solid tumors such as ovarian, colorectal and bladder cancer. One of the reported binding targets of MSI1 is Numb, a negative regulator of the Notch signaling. However, the dynamic mechanism of Numb RNA binding to MSI1 remains unknown, largely hindering effective drug design targeting this critical interaction. Here, we have performed extensive all-atom microsecond-timescale simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which successfully captured multiple times of spontaneous and highly accurate binding of the Numb RNA from bulk solvent to the MSI1 protein target site. GaMD simulations revealed that Numb RNA binding to MSI1 involved largely induced fit in both the RNA and protein. The simulations also identified important low-energy intermediate conformational states during RNA binding, in which Numb interacted mainly with the β2-β3 loop and C terminus of MSI1. The mechanistic understanding of RNA binding obtained from our GaMD simulations is expected to facilitate rational structure-based drug design targeting MSI1 and other RBPs.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 10-20"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ec/3e/main.PMC8695263.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39649771","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HPMPdb: A machine learning-ready database of protein molecular phenotypes associated to human missense variants","authors":"Daniele Raimondi ,&nbsp;Francesco Codicè ,&nbsp;Gabriele Orlando ,&nbsp;Joost Schymkowitz ,&nbsp;Frederic Rousseau ,&nbsp;Yves Moreau","doi":"10.1016/j.crstbi.2022.04.004","DOIUrl":"10.1016/j.crstbi.2022.04.004","url":null,"abstract":"<div><p>Current human Single Amino acid Variants (SAVs) databases provide a link between a SAVs and their effect on the carrier individual phenotype, often dividing them into Deleterious/Neutral variants. This is a very coarse-grained description of the genotype-to-phenotype relationship because it relies on un-realistic assumptions such as the perfect Mendelian behavior of each SAV and considers only dichotomic phenotypes. Moreover, the link between the effect of a SAV on a protein (its molecular phenotype) and the individual phenotype is often very complex, because multiple level of biological abstraction connect the protein and individual level phenotypes. Here we present HPMPdb, a manually curated database containing human SAVs associated with the detailed description of the molecular phenotype they cause on the affected proteins. With particular regards to machine learning (ML), this database can be used to let researchers go beyond the existing Deleterious/Neutral prediction paradigm, allowing them to build molecular phenotype predictors instead. Our class labels describe in a succinct way the effects that each SAV has on 15 protein molecular phenotypes, such as protein-protein interaction, small molecules binding, function, post-translational modifications (PTMs), sub-cellular localization, mimetic PTM, folding and protein expression. Moreover, we provide researchers with all necessary means to re-producibly train and test their models on our database. The webserver and the data described in this paper are available at hpmp.esat.kuleuven.be.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 167-174"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X22000125/pdfft?md5=d27cdb5d3a4326b327b8edd15547ebac&pid=1-s2.0-S2665928X22000125-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41376490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bridging the N-terminal and middle domains in FliG of the flagellar rotor","authors":"Dagnija Tupiņa ,&nbsp;Alexander Krah ,&nbsp;Jan K. Marzinek ,&nbsp;Lorena Zuzic ,&nbsp;Adam A. Moverley ,&nbsp;Chrystala Constantinidou ,&nbsp;Peter J. Bond","doi":"10.1016/j.crstbi.2022.02.002","DOIUrl":"10.1016/j.crstbi.2022.02.002","url":null,"abstract":"<div><p>Flagella are necessary for bacterial movement and contribute to various aspects of virulence. They are complex cylindrical structures built of multiple molecular rings with self-assembly properties. The flagellar rotor is composed of the MS-ring and the C-ring. The FliG protein of the C-ring is central to flagellar assembly and function due to its roles in linking the C-ring with the MS-ring and in torque transmission from stator to rotor. No high-resolution structure of an assembled C-ring has been resolved to date, and the conformation adopted by FliG within the ring is unclear due to variations in available crystallographic data. Here, we use molecular dynamics (MD) simulations to study the conformation and dynamics of FliG in different states of assembly, including both in physiologically relevant and crystallographic lattice environments. We conclude that the linker between the FliG N-terminal and middle domain likely adopts an extended helical conformation in vivo, in contrast with the contracted conformation observed in some previous X-ray studies. We further support our findings with integrative model building of full-length FliG and a FliG ring model that is compatible with cryo-electron tomography (cryo-ET) and electron microscopy (EM) densities of the C-ring. Collectively, our study contributes to a better mechanistic understanding of the flagellar rotor assembly and its function.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 59-67"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665928X22000046/pdfft?md5=54167321814b4f9235b624adb9db5f3a&pid=1-s2.0-S2665928X22000046-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45296238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans","authors":"Danielle L. Sexton ,&nbsp;Steffen Burgold ,&nbsp;Andreas Schertel ,&nbsp;Elitza I. Tocheva","doi":"10.1016/j.crstbi.2021.12.001","DOIUrl":"10.1016/j.crstbi.2021.12.001","url":null,"abstract":"<div><p>Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium <em>Deinococcus radiodurans</em>. Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of <em>D. radiodurans</em> cells, opening new lines of investigation into the physiology and evolution of the bacterium.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 1-9"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8688812/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39869480","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering enhanced thermostability into the Geobacillus pallidus nitrile hydratase","authors":"Jennifer C. Van Wyk ,&nbsp;B. Trevor Sewell ,&nbsp;Michael J. Danson ,&nbsp;Tsepo L. Tsekoa ,&nbsp;Muhammed F. Sayed ,&nbsp;Don A. Cowan","doi":"10.1016/j.crstbi.2022.07.002","DOIUrl":"10.1016/j.crstbi.2022.07.002","url":null,"abstract":"<div><p>Nitrile hydratases (NHases) are important biocatalysts for the enzymatic conversion of nitriles to industrially-important amides such as acrylamide and nicotinamide. Although thermostability in this enzyme class is generally low, there is not sufficient understanding of its basis for rational enzyme design. The gene expressing the Co-type NHase from the moderate thermophile, <em>Geobacillus pallidus</em> RAPc8 (NRRL B-59396), was subjected to random mutagenesis. Four mutants were selected that were 3 to 15-fold more thermostable than the wild-type NHase, resulting in a 3.4–7.6 ​kJ/mol increase in the activation energy of thermal inactivation at 63 ​°C. High resolution X-ray crystal structures (1.15–1.80 ​Å) were obtained of the wild-type and four mutant enzymes. Mutant 9E, with a resolution of 1.15 ​Å, is the highest resolution crystal structure obtained for a nitrile hydratase to date. Structural comparisons between the wild-type and mutant enzymes illustrated the importance of salt bridges and hydrogen bonds in enhancing NHase thermostability. These additional interactions variously improved thermostability by increased intra- and inter-subunit interactions, preventing cooperative unfolding of α-helices and stabilising loop regions. Some hydrogen bonds were mediated via a water molecule, specifically highlighting the significance of structured water molecules in protein thermostability. Although knowledge of the mutant structures makes it possible to rationalize their behaviour, it would have been challenging to predict in advance that these mutants would be stabilising.</p></div>","PeriodicalId":10870,"journal":{"name":"Current Research in Structural Biology","volume":"4 ","pages":"Pages 256-270"},"PeriodicalIF":2.8,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a0/5c/main.PMC9465369.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40360201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}