Biology subjects

Jedrzejczak, R.

Publications and source records attributed to Jedrzejczak, R..

10 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of detergent-bound Vibrio cholerae MakA captures a candidate intermediate between the soluble form and mature assembly of an α-pore-forming toxin

The tripartite toxin MakA/MakB/MakE from Vibrio cholerae belongs to a family of -pore-forming toxins that undergo extensive conformational rearrangements upon interacting with membrane. While soluble structures of MakA, MakB and MakE have been determined, the structural basis of membrane association and pore assembly remains poorly understood. Here, we report a cryo-electron microscopy structure of a detergent-associated form of MakA at a global resolution of 3.13 Angstrom, with focused refinement of a flexible region improving the local resolution to 2.68 Angstrom. This structure consists of a trimer of MakA asymmetric homodimers, in which each subunit adopts a transmembrane -helical conformation distinct from the previously reported soluble structure. The asymmetric homodimer closely resembles the building block observed in the previously reported helical MakA assembly and is structurally similar to the homodimeric building blocks of the Aeromonas hydrophila AhlB and Serratia marcescens SmhA pores, and the heterodimeric building blocks of the Xenorhabdus nematophila XaxAB and Yersinia enterocolitica YaxAB pores, suggesting a conserved assembly principle among distantly related -pore-forming toxins. Complementary hydropathy analysis, AlphaFold2 modeling, and liposome-based assays reveal distinct roles for the three components, with MakA acting as the primary membrane-inserting subunit, MakE exhibiting low but consistent membrane-association, and MakB alone remaining largely soluble. Together, these results support a model in which the MakA asymmetric homodimer represents a plausible early membrane-insertion unit and building block for pore assembly, while the trimer of dimers represents an early assembly intermediate.

microbiology↗

bioRxiv2021

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

biophysics↗

bioRxiv2020

Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Methylation of RNA Cap in SARS-CoV-2 captured by serial crystallography

The genome of the SARS-CoV-2 coronavirus contains 29 proteins, of which 15 are nonstructural. Nsp10 and Nsp16 form a complex responsible for the capping of mRNA at the 5' terminus. In the methylation reaction the S-adenosyl-L-methionine serves as the donor of the methyl group that is transferred to Cap-0 at the first transcribed nucleotide to create Cap-1. The presence of Cap-1 makes viral RNAs mimic the host transcripts and prevents their degradation. To investigate the 2'-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We determined crystal structures of Nsp10/16 that revealed the states before and after the methylation reaction, for the first time illustrating coronavirus Nsp10/16 complexes with the m7GpppAm2'-O Cap-1, where 2'OH of ribose is methylated. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. This data provide important mechanistic insight and can be used to design small molecules that inhibit viral RNA maturation making SARS-CoV-2 sensitive to host innate response.

molecular biology↗

bioRxiv2020

Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors

The number of new cases world-wide for the COVID-19 disease is increasing dramatically, while efforts to contain Severe Acute Respiratory Syndrome Coronavirus 2 is producing varied results in different countries. There are three key SARS-CoV-2 enzymes potentially targetable with antivirals: papain-like protease (PLpro), main protease (Mpro), and RNA-dependent RNA polymerase. Of these, PLpro is an especially attractive target because it plays an essential role in several viral replication processes, including cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex (RTC), and disruption of host viral response machinery to facilitate viral proliferation and replication. Moreover, this enzyme is conserved across different coronaviruses and promising inhibitors have already been discovered for its SARS-CoV variant. Here we report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the enzyme from SARS-CoV-2 in both wild-type and mutant forms. These efforts include the first structures of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors, determined at 1.60-2.70 Angstroms. This collection of structures provides fundamental molecular and mechanistic insight to PLpro, and critically, illustrates details for inhibitors recognition and interactions. All presented compounds inhibit the peptidase activity of PLpro in vitro, and some molecules block SARS-CoV-2 replication in cell culture assays. These collated findings will accelerate further structure-based drug design efforts targeting PLpro, with the ultimate goal of identifying high-affinity inhibitors of clinical value for SARS-CoV-2.

molecular biology↗

bioRxiv2020

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

ABSTRACTSARS-CoV-2 Nsp15 is a uridylate-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family. It degrades the polyuridine extensions in (-) sense strand of viral RNA and some non-translated RNA on (+) sense strand. This activity seems to be responsible for the interference with the innate immune response and evasion of host pattern recognition. Nsp15 is highly conserved in coronaviruses suggesting that its activity is important for virus replication. Here we report first structures with bound nucleotides and show that SARS-CoV-2 Nsp15 specifically recognizes U in a pattern previously predicted for EndoU. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. Inhibitors of Nsp15 have been reported but not actively pursued into therapeutics. The current COVID-19 pandemic brought to attention the repurposing of existing drugs and the rapid identification of new antiviral compounds. Tipiracil is an FDA approved drug that is used with trifluridine in the treatment of colorectal cancer. Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme’s active site, providing basis for the uracil scaffold-based drug development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Crystal structures of SARS-CoV-2 ADP-ribose phosphatase (ADRP): from the apo form to ligand complexes

Among 15 nonstructural proteins (Nsps), the newly emerging SARS-CoV-2 encodes a large, multidomain Nsp3. One of its units is ADP-ribose phosphatase domain (ADRP, also known as macrodomain) which is believed to interfere with the host immune response. Such a function appears to be linked to the proteins ability to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remains unknown. Here, we have determined five, high resolution (1.07 - 2.01 [A]) crystal structures corresponding to the apo form of the protein and complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADPr. We show that the protein undergoes conformational changes to adapt to the ligand in a manner previously observed before for in close homologs from other viruses. We also identify a conserved water molecule that may participate in hydrolysis. This work builds foundations for future structure-based research of the ADRP, including search for potential antiviral therapeutics.

molecular biology↗

bioRxiv2020

The crystal structure of nsp10-nsp16 heterodimer from SARS CoV-2in complex with S-adenosylmethionine

SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 [A]. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

biochemistry↗

bioRxiv2020

Therapeutic genetic variation revealed in diverse Hsp104 homologs

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. We have engineered potentiated Hsp104 variants to antagonize proteotoxic misfolding linked to human neurodegenerative diseases. However, these Hsp104 variants can exhibit off-target toxicity, which may limit their therapeutic utility. Hsp104 is conserved among all nonmetazoan eukaryotes, which raises the possibility that natural variants might exist with enhanced, selective activity against neurodegenerative disease substrates. To assess this possibility, we screened a cross-kingdom collection of Hsp104 homologs in several yeast proteotoxicity models. We uncovered therapeutic genetic variation among several Hsp104 homologs that specifically antagonize TDP-43 or -synuclein condensate formation and toxicity in yeast, human cells, and C. elegans. Surprisingly, this variation manifested as increased passive chaperone activity, distinct from disaggregase activity, which neutralizes proteotoxicity of specific substrates. Thus, by exploring natural tuning of passive chaperone activity we elucidated enhanced, substrate-specific agents to counter proteotoxicity underlying neurodegenerative disease.

biochemistry↗

bioRxiv2020

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS- and MERS-CoVs the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report the high-resolution crystal structure of endoribonuclease Nsp15/NendoU from SARS-CoV-2 - a virus causing current world-wide epidemics. We compare this structure with previously reported models of Nsp15 from SARS and MERS coronaviruses.

molecular biology↗