Abstract
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new ...antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (M
pro
also known as 3CL
pro
) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the M
pro
Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the M
pro
as potential COVID-19 therapeutics for further testing and possibly clinical trials.
Summary
Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic ...laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well‐studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.
Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well‐studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.
Matrix metalloproteinases (MMPs) play central roles in vertebrate tissue development, remodeling, and repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate proteolytic ...activity by binding tightly to the MMP active site. While each of the four TIMPs can inhibit most MMPs, binding data reveal tremendous heterogeneity in affinities of different TIMP/MMP pairs, and the structural features that differentiate stronger from weaker complexes are poorly understood. Here we report the crystal structure of the comparatively weakly bound human MMP-10/TIMP-2 complex at 2.1 Å resolution. Comparison with previously reported structures of MMP-3/TIMP-1, MT1-MMP/TIMP-2, MMP-13/TIMP-2, and MMP-10/TIMP-1 complexes offers insights into the structural basis of binding selectivity. Our analyses identify a group of highly conserved contacts at the heart of MMP/TIMP complexes that define the conserved mechanism of inhibition, as well as a second category of diverse adventitious contacts at the periphery of the interfaces. The AB loop of the TIMP N-terminal domain and the contact loops of the TIMP C-terminal domain form highly variable peripheral contacts that can be considered as separate exosite interactions. In some complexes these exosite contacts are extensive, while in other complexes the AB loop or C-terminal domain contacts are greatly reduced and appear to contribute little to complex stability. Our data suggest that exosite interactions can enhance MMP/TIMP binding, although in the relatively weakly bound MMP-10/TIMP-2 complex they are not well optimized to do so. Formation of highly variable exosite interactions may provide a general mechanism by which TIMPs are fine-tuned for distinct regulatory roles in biology.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
One often observes small but measurable differences in the diffraction data measured from different crystals of a single protein. These differences might reflect structural differences in the protein ...and may reveal the natural dynamism of the molecule in solution. Partitioning these mixed‐state data into single‐state clusters is a critical step that could extract information about the dynamic behavior of proteins from hundreds or thousands of single‐crystal data sets. Mixed‐state data can be obtained deliberately (through intentional perturbation) or inadvertently (while attempting to measure highly redundant single‐crystal data). To the extent that different states adopt different molecular structures, one expects to observe differences in the crystals; each of the polystates will create a polymorph of the crystals. After mixed‐state diffraction data have been measured, deliberately or inadvertently, the challenge is to sort the data into clusters that may represent relevant biological polystates. Here, this problem is addressed using a simple multi‐factor clustering approach that classifies each data set using independent observables, thereby assigning each data set to the correct location in conformational space. This procedure is illustrated using two independent observables, unit‐cell parameters and intensities, to cluster mixed‐state data from chymotrypsinogen (ChTg) crystals. It is observed that the data populate an arc of the reaction trajectory as ChTg is converted into chymotrypsin.
The dynamics of proteins can be explored from polymorphs observed by the clustering of multiple data wedges.
PRSS3/mesotrypsin is an atypical isoform of trypsin, the upregulation of which has been implicated in promoting tumor progression. To date there are no mesotrypsin-selective pharmacological ...inhibitors which could serve as tools for deciphering the pathological role of this enzyme, and could potentially form the basis for novel therapeutic strategies targeting mesotrypsin. A virtual screen of the Natural Product Database (NPD) and Food and Drug Administration (FDA) approved Drug Database was conducted by high-throughput molecular docking utilizing crystal structures of mesotrypsin. Twelve high-scoring compounds were selected for testing based on lowest free energy docking scores, interaction with key mesotrypsin active site residues, and commercial availability. Diminazene (CID22956468), along with two similar compounds presenting the bis-benzamidine substructure, was validated as a competitive inhibitor of mesotrypsin and other human trypsin isoforms. Diminazene is the most potent small molecule inhibitor of mesotrypsin reported to date with an inhibitory constant (Ki) of 3.6±0.3 μM. Diminazene was subsequently co-crystalized with mesotrypsin and the crystal structure was solved and refined to 1.25 Å resolution. This high resolution crystal structure can now offer a foundation for structure-guided efforts to develop novel and potentially more selective mesotrypsin inhibitors based on similar molecular substructures.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open ...new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).
To take full advantage of advanced data collection techniques and high beam flux at next‐generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and ...align many samples per second. One approach is to use an acoustic ejector to eject crystal‐containing droplets onto a solid X‐ray transparent surface, which can then be positioned and rotated for data collection. Proof‐of‐concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide `conveyor belt'. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.
The highly automated macromolecular crystallography beamline AMX/17‐ID‐1 is an undulator‐based high‐intensity (>5 × 1012 photons s−1), micro‐focus (7 µm × 5 µm), low‐divergence (1 mrad × 0.35 mrad) ...energy‐tunable (5–18 keV) beamline at the NSLS‐II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17‐ID with the FMX beamline, the frontier micro‐focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro‐focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17‐ID‐1 as well as some plans for the near future are presented.
AMX (17‐ID‐1) is the highly automated macromolecular crystallography beamline at the NSLS‐II. Photon delivery system, beamline instrumentation, high‐performance computing environment and suites of applications are described for beamline scientists and users. AMX's primary mission is to support routine structure determination from the most challenging projects.
Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their ...zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.
Background: Chymotrypsin C (CTRC) targets specific regulatory cleavage sites within trypsinogens and procarboxypeptidases.
Results: The crystal structure of CTRC reveals the structural basis of substrate specificity.
Conclusion: Long-range electrostatic and hydrophobic complementarity drives CTRC association with preferred substrates.
Significance: The observations reveal the mechanistic basis for CTRC selectivity in digestive enzyme activation and degradation.
Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD+ is ...poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD+ ratio. Here we investigate the molecular mechanism for NADH/NAD+ sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD+, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40° closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.
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▸ A structural comparison of Rex in the NAD+/DNA-bound, apo-, and NADH-bound states ▸ A rigid-body rotation of Rex subunits permits DNA binding after NADH dissociation ▸ A single NAD+ binds the Rex dimer/DNA complex ▸ A triad of conserved residues are required for NADH/NAD+ sensing by Rex
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