Structural Basis of High-Precision Protein Ligation and Its Application.

Enzyme-catalyzed protein modifications have become invaluable in diverse applications, outperforming chemical methods in terms of precision, conjugation efficiency, and biological compatibility. Despite significant advances in ligases, such as sortase A and OaAEP1, their use in heterogeneous biological environments remains constrained by limited target sequence specificity. In 2021, Lupas' group introduced Connectase, a family of repurposed archaeal proteases for protein ligations, but its low processivity and lack of structural information have impeded further engineering for practical biological and biophysical applications.

Inhibition of falcilysin from Plasmodium falciparum by interference with its closed-to-open dynamic transition.

In the absence of an efficacious vaccine, chemotherapy remains crucial to prevent and treat malaria. Given its key role in haemoglobin degradation, falcilysin constitutes an attractive target. Here, we reveal the mechanism of enzymatic inhibition of falcilysin by MK-4815, an investigational new drug with potent antimalarial activity.

The Phlebovirus Ribonucleoprotein: An Overview.

In negative strand RNA viruses, ribonucleoproteins, not naked RNA, constitute the template used by the large protein endowed with polymerase activity for replicating and transcribing the viral genome. Here we give an overview of the structures and functions of the ribonucleoprotein from phleboviruses. The nucleocapsid monomer, which constitutes the basic structural unit, possesses a flexible arm allowing for a conformational switch between a closed monomeric state and the formation of a polymeric filamentous structure competent for viral RNA binding and encapsidation in the open state of N.

Identification of an inhibitory pocket in falcilysin provides a new avenue for malaria drug development.

Identification of new druggable protein targets remains the key challenge in the current antimalarial development efforts. Here we used mass-spectrometry-based cellular thermal shift assay (MS-CETSA) to identify potential targets of several antimalarials and drug candidates. We found that falcilysin (FLN) is a common binding partner for several drug candidates such as MK-4815, MMV000848, and MMV665806 but also interacts with quinoline drugs such as chloroquine and mefloquine.

Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848.

About 247 million cases of malaria occurred in 2021 with Plasmodium falciparum accounting for the majority of 619,000 deaths. In the absence of a widely available vaccine, chemotherapy remains crucial to prevent, treat, and contain the disease. The efficacy of several drugs currently used in the clinic is likely to suffer from the emergence of resistant parasites.

Employment of a high throughput functional assay to define the critical factors that influence vaccine induced cross-variant neutralizing antibodies for SARS-CoV-2.

The scale and duration of neutralizing antibody responses targeting SARS-CoV-2 viral variants represents a critically important serological parameter that predicts protective immunity for COVID-19. In this study, we describe the development and employment of a new functional assay that measures neutralizing antibodies for SARS-CoV-2 and present longitudinal data illustrating the impact of age, sex and comorbidities on the kinetics and strength of vaccine-induced antibody responses for key variants in an Asian volunteer cohort. We also present an accurate quantitation of serological responses for SARS-CoV-2 that exploits a unique set of in-house, recombinant human monoclonal antibodies targeting the viral Spike and nucleocapsid proteins and demonstrate a reduction in neutralizing antibody titres across all groups 6 months post-vaccination.

Defining neutralization and allostery by antibodies against COVID-19 variants.

The changing landscape of SARS-CoV-2 Spike protein is linked to the emergence of variants, immune-escape and reduced efficacy of the existing repertoire of anti-viral antibodies. The functional activity of neutralizing antibodies is linked to their quaternary changes occurring as a result of antibody-Spike trimer interactions. Here, we reveal the conformational dynamics and allosteric perturbations linked to binding of novel human antibodies and the viral Spike protein.

A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation.

Enzymatic peptide ligation holds great promise in the study of protein functions and development of protein therapeutics. Owing to their high catalytic efficiency and a minimal tripeptide recognition motif, peptidyl asparaginyl ligases (PALs) are particularly useful tools for bioconjugation. However, as an inherent limitation of transpeptidases, PAL-mediated ligation is reversible, requiring a large excess of one of the ligation partners to shift the reaction equilibrium in the forward direction.

Substrate-binding glycine residues are major determinants for hydrolase and ligase activity of plant legumains.

Peptide asparaginyl ligases (PALs) are useful tools for precision modifications of proteins and live-cell surfaces by ligating peptides after Asn/Asp (Asx). They share high sequence and structural similarity to plant legumains that are generally known as asparaginyl endopeptidases (AEPs), thus making it challenging to identify PALs from AEPs. In this study, we investigate 875 plant species from algae to seed plants with available sequence data in public databases to identify new PALs.

On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation.

Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell-surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes.

Engineering SARS-CoV-2 specific cocktail antibodies into a bispecific format improves neutralizing potency and breadth.

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce emergence of antibody resistance. Here we engineer two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD).

Structural basis for proenzyme maturation, substrate recognition, and ligation by a hyperactive peptide asparaginyl ligase.

Peptide ligases are versatile enzymes that can be utilized for precise protein conjugation for bioengineering applications. Hyperactive peptide asparaginyl ligases (PALs), such as butelase-1, belong to a small class of enzymes from cyclotide-producing plants that can perform site-specific, rapid ligation reactions after a target peptide asparagine/aspartic acid (Asx) residue binds to the active site of the ligase. How PALs specifically recognize their polypeptide substrates has remained elusive, especially at the prime binding side of the enzyme.

PAL-Mediated Ligation for Protein and Cell-Surface Modification.

Peptidyl Asx-specific ligases (PALs) effect peptide ligation by catalyzing transpeptidation reactions at Asn/Asp-peptide bonds. Owing to their high efficiency and mild aqueous reaction conditions, these ligases have emerged as powerful biotechnological tools for protein manipulation in recent years. PALs are enzymes of the asparaginyl endopeptidase (AEP) superfamily but have predominant transpeptidase activity as opposed to typical AEPs which are predominantly hydrolases.

Engineering SARS-CoV-2 cocktail antibodies into a bispecific format improves neutralizing potency and breadth.

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce antibody resistance. We engineered two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD).

Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold.

Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs.

Vypal2: A Versatile Peptide Ligase for Precision Tailoring of Proteins.

The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years.

Crystal structure and functional analysis of mycobacterial erythromycin resistance methyltransferase Erm38 reveals its RNA-binding site.

Erythromycin resistance methyltransferases (Erms) confer resistance to macrolide, lincosamide, and streptogramin antibiotics in Gram-positive bacteria and mycobacteria. Although structural information for ErmAM, ErmC, and ErmE exists from Gram-positive bacteria, little is known about the Erms in mycobacteria, as there are limited biochemical data and no structures available. Here, we present crystal structures of Erm38 from Mycobacterium smegmatis in apoprotein and cofactor-bound forms.

Asparaginyl Endopeptidase-Mediated Protein C-Terminal Hydrazinolysis for the Synthesis of Bioconjugates.

Asparaginyl endopeptidases (AEPs) are cysteinyl enzymes naturally catalyzing the hydrolysis and transpeptidation reactions at Asx-Xaa bonds. These reactions go by a common acyl-enzyme thioester intermediate, which is either attacked by water (for a protease-AEP) or by a peptidic amine nucleophile (for a ligase-AEP) to form the respective hydrolysis or aminolysis product. Herein, we show that hydrazine and hydroxylamine, two α-effect nucleophiles, are capable of resolving the thioester intermediate to yield peptide and protein products containing a C-terminal hydrazide and hydroxamic acid functionality, respectively.

A conserved arginine in NS5 binds genomic 3' stem-loop RNA for primer-independent initiation of flavivirus RNA replication.

The commitment to replicate the RNA genome of flaviviruses without a primer involves RNA-protein interactions that have been shown to include the recognition of the stem-loop A (SLA) in the 5' untranslated region (UTR) by the nonstructural protein NS5. We show that DENV2 NS5 arginine 888, located within the carboxy-terminal 18 residues, is completely conserved in all flaviviruses and interacts specifically with the top-loop of 3'SL in the 3'UTR which contains the pentanucleotide 5'-CACAG-3' previously shown to be critical for flavivirus RNA replication. We present virological and biochemical data showing the importance of this Arg 888 in virus viability and de novo initiation of RNA polymerase activity in vitro.

Purification of Dengue and Zika Virus Non-structural Protein 5 for Crystallization and Screening of Antivirals.

Dengue Virus (DENV) and ZIKA Virus (ZIKV) are two important human pathogens that belong to the Flavivirus genus of positive strand RNA viruses. Symptoms of DENV infections range from asymptomatic or mild fever to life-threatening forms, while ZIKV can lead to teratogenic effects such as microcephaly in newborns and neurological disease like the Guillain-Barré syndrome.Non-Structural Protein 5 (NS5) is the largest and most conserved enzyme across flaviviruses and hence constitutes a prime target for developing pan-flavivirus antiviral inhibitors.

Optimal flexibility of the linker region of Zika virus NS5 methyltransferase-polymerase is critical for virus replication.

The flavivirus NS5 protein contains an N-terminal methyl-transferase (MTase) connected through a flexible linker with a C-terminal RNA-dependent RNA-polymerase (RdRp) domain, that work cooperatively to replicate and methylate the viral genome. In this study we probed the importance of an evolutionary-conserved hydrophobic residue (Val266) located at the start of the ten-residue interdomain linker of Zika virus (ZIKV) NS5. In flavivirus NS5 crystal structures, the start of the linker forms a 3 helix when NS5 adopts a compact conformation, but becomes disordered or extended in open conformations.

N -Hydroxyasparagine: A Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases.

Peptidyl asparaginyl ligases (PALs) are powerful tools for peptide macrocyclization. Herein, we report that a derivative of Asn, namely N -hydroxyasparagine or Asn(OH), is an unnatural P1 substrate of PALs. By Asn(OH)-mediated cyclization, we prepared cyclic peptides as new matrix metalloproteinase 2 (MMP2) inhibitors displaying the hydroxamic acid moiety of Asn(OH) as the key pharmacophore.

Amidoxime prodrugs convert to potent cell-active multimodal inhibitors of the dengue virus protease.

The flavivirus genus of the Flaviviridae family comprises Dengue, Zika and West-Nile viruses which constitute unmet medical needs as neither appropriate antivirals nor safe vaccines are available. The dengue NS2BNS3 protease is one of the most promising validated targets for developing a dengue treatment however reported protease inhibitors suffer from toxicity and cellular inefficacy. Here we report SAR on our previously reported Zika-active carbazole scaffold, culminating prodrug compound SP-471P (EC 1.