Structural and Functional Studies of Polyketide Synthases

Polyketides, natural products produced by multi-domain polyketide synthases (PKSs), have proven to be excellent starting points for drug discovery. Rational engineering of PKSs holds much promise for the generation of novel polyketide pharmaceuticals, however to enable this we need to gain a better understanding of how the mature polyketides are generated and how individual modules within a polyketide synthase assemble and interact. Here, work was performed to investigate three polypeptides from natural product indanomycin and rhizoxin biosynthesis, including the candidate polyketide cyclase IdmH, the fourth subunit of the indanomycin megasynthase, IdmO, and the branching module from the rhizoxin PKS. Indanomycin needs to undergo several transformations by post-PKS tailoring enzymes. One such enzyme, IdmH, has been hypothesised to act as a cyclase and catalyse the formation of the indane ring via a Diels-Alder reaction. Crystal structure of the wild-type IdmH was determined to 2.7 Å resolution and the interactions between IdmH and its proposed product indanomycin were characterised using NMR spectroscopy and in silico methods. Fully-reducing IdmO module was successfully expressed and purified. Characterisation by negative-stain electron microscopy resulted in a low-resolution model of IdmO, while attempts to carry out cryo-electron microscopy (cryo-EM) analysis revealed a number of difficulties associated with the denaturation of this large complex during cryo-EM grid preparation. A similar cryo-EM approach was utilised to study the branching module from the rhizoxin PKS. A 3.7 Å resolution map was determined for this module containing the ketosynthase, branching and acyl carrier protein (ACP) domains. Two ACP binding sites were identified, which can help explain the unorthodox activity of this module. This research has provided valuable insights into different aspects of PKS biology ranging from polyketide tailoring and branching to the assembly of the intact modules and forms a solid basis for future studies of these fascinating biosynthetic machines

Neutralizing antibodies reveal cryptic vulnerabilities and interdomain crosstalk in the porcine deltacoronavirus spike protein

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that has recently been detected in humans. Despite this zoonotic concern, the antigenic structure of PDCoV remains unknown. The virus relies on its spike (S) protein for cell entry, making it a prime target for neutralizing antibodies. Here, we generate and characterize a set of neutralizing antibodies targeting the S protein, shedding light on PDCoV S interdomain crosstalk and its vulnerable sites. Among the four identified antibodies, one targets the S1A domain, causing local and long-range conformational changes, resulting in partial exposure of the S1B domain. The other antibodies bind the S1B domain, disrupting binding to aminopeptidase N (APN), the entry receptor for PDCoV. Notably, the epitopes of these S1B-targeting antibodies are concealed in the prefusion S trimer conformation, highlighting the necessity for conformational changes for effective antibody binding. The binding footprint of one S1B binder entirely overlaps with APN-interacting residues and thus targets a highly conserved epitope. These findings provide structural insights into the humoral immune response against the PDCoV S protein, potentially guiding vaccine and therapeutic development for this zoonotic pathogen.</p

Sialoglycan binding triggers spike opening in a human coronavirus

Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion 1-5. Spike opening exposes domain S1 B, allowing it to bind to proteinaceous receptors 6,7, and is also thought to enable protein refolding during membrane fusion 4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1 A. This binding triggers the transition of S1 B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain, N-terminal domain and S2 region, distal to the ACE2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity.The MANCO project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 101003651). This work made use of the Dutch national e infrastructure with the support of the SURF Cooperative using grant no. EINF-2453. This research was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) - 398066876/GRK 2485/1; BMBF (Federal Ministry of Education and Research) project entitled RAPID (Risk assessment in re-pandemic respiratory infectious diseases), 01KI1723G, Ministry of Science and Culture of Lower Saxony in Germany (14 - 76103-184 CORONA-15/20)N

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other Variants of Concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and/or hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs, and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity.The MANCO project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 101003651). This work made use of the Dutch national e-infrastructure with the support of the SURF Cooperative using grant no. EINF-2453. This research was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) -398066876/GRK 2485/1; BMBF (Federal Ministry of Education and Research) project entitled RAPID (Risk assessment in re-pandemic respiratory infectious diseases), 01KI1723G, Ministry of Science and Culture of Lower Saxony in Germany (14 - 76103-184 CORONA-15/20)Peer reviewe

The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19)

Reunalistoituksen toiminnan laadun kehittäminen

Tässä opinnäytetyössä käsitellään Isku Teollisuus Oy:n levykalustetehtaan reunalistoituslinjaston toiminnan laadun kehittämistä. Työn tavoitteena oli tutkia ajankäyttötutkimuksien avulla mahdollisuuksia parantaa reunalistoituslinjaston tehokkuutta. Tavoitteena oli myös tutkia mahdollisuuksia lyhentää asetteen tekoon kuluvaa aikaa. Nykyisin ajettavat sarjakoot ovat suhteellisen pieniä ja asetteita joudutaan vaihtamaan usein. Työhön kuului myös tutkia reunanauhojen vähentämisellä saatavia hyötyjä ja tarkastella mahdollisuuksia poistaa joitakin nykyisin käytettäviä reunanauhoja. Teoriaosuudessa on käsitelty erilaisia ajankäyttötutkimusmenetelmiä ja asetusaikoihin liittyvää teoriaa. Työnmittaukset toteutin havainnointitutkimuksena, joka oli sopivin tapa saada halutut tutkimustulokset. Työnmittaus tehtiin reunalistoituslinjastolla kellottaen sekuntikellon kanssa eri työvaiheisiin kuluvaa aikaa. Koska linjastolla työskentelee vuorossa yleensä kolme henkilöä, niin sen takia tutkimuksessa keskityttiin linjan toimintaan ja toimimattomuuteen, eikä yksittäisten työntekijöiden tekemisiin. Työnmittauksilla selvisi eri työvaiheisiin kuluva aika, ja tuloksista voidaan huomata myös turhaan työhön käytettävä aika. Tutkimuksesta selviää, että reunanauhojen vähentämisestä saatavia suurimpia hyötyjä olisi aseteaikojen lyhentyminen. Erityisesti hienosäätöön ja kappaleen tarkastamiseen kuluva aika tulisi lyhentymään. Tutkimuksen perusteella saa hyvän kuvan siitä, mihin reunalistoituslinjastolla työaika kuluu, ja tuloksista voidaan myös tehdä päätöksiä jatkotoimenpiteistä.The aim of the thesis was to find ways to improve efficiency in the edge banding production line. The thesis was commissioned by Isku Teollisuus Oy. The measurements for the work were based on time study methods and theories, especially work survey. The methods are presented in the theory part. Work survey was performed in the edge banding production line, where every work stage was observed by taking time with a stopwatch. The results of the work survey provide a good picture of how the working hours in the edge banding production line are spent, and it gives a good basis to make decisions on further actions to improve efficiency in the edge banding production line. One of the goals for this thesis was to examine possibilities to shorten the time that is used in setup operations between product series. Nowadays the product series made on the edge banding production line are quite small, and employees have to change setups approximately 15 times per shift, which takes a big part of working hours. That is why finding the ways to reduce time used in setup operations was an important part of the study. Different ways to reduce time used in setup operations were examined by using the SMED method, which is a LEAN production method used for reducing waste in a manufacturing process. Part of this work was to study possibilities to reduce some of the most used edge bands. Results of study for reducing some of the edge bands can be used for making decisions on how many and which edge bands the company should reduce without big changes on the product line. The major benefits of reducing some of the edge bands are that the time used in setup operations will decrease, because then different products can be manufactured using the same edge bands. That significantly decreases times spent on setup operations, especially in fine adjustment and product checkups

Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans

The human betacoronaviruses HKU1 and OC43 (subgenus Embecovirus) arose from separate zoonotic introductions, OC43 relatively recently and HKU1 apparently much longer ago. Embecovirus particles contain two surface projections called spike (S) and haemagglutinin-esterase (HE), with S mediating receptor binding and membrane fusion, and HE acting as a receptor-destroying enzyme. Together, they promote dynamic virion attachment to glycan-based receptors, specifically 9-O-acetylated sialic acid. Here we present the cryo-EM structure of the ~80 kDa, heavily glycosylated HKU1 HE at 3.4 Å resolution. Comparison with existing HE structures reveals a drastically truncated lectin domain, incompatible with sialic acid binding, but with the structure and function of the esterase domain left intact. Cryo-EM and mass spectrometry analysis reveals a putative glycan shield on the now redundant lectin domain. The findings further our insight into the evolution and host adaptation of human embecoviruses, and demonstrate the utility of cryo-EM for studying small, heavily glycosylated proteins