Biology subjects

Kozielski, F. G.

Publications and source records attributed to Kozielski, F. G..

6 recordsLinked to original sources

bioRxiv2023

Oligomeric state of β-coronavirus non-structural protein 10 stimulators studied by OmniSEC and Small Angle X-ray Scattering

Members of the {beta}-coronavirus family such as SARS-CoV-2, SARS, and MERS have caused pandemics over the last 20 years. Future pandemics are likely and studying the coronavirus family members is necessary for their understanding and treatment. Coronaviruses possess 16 non-structural proteins, many of which are involved in viral replication and other vital functions. Non-structural protein 10 (nsp10) is an essential stimulator of nsp14 and nsp16, modulating RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to understanding its multifunctional role. We report the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. Proteins were subjected to a combination of OmniSEC and SEC-MALS to characterise their oligomeric state. Full-length nsp10s were predominantly monomeric in solution, while truncated versions of nsp10 have a higher tendency to oligomerise. Small angle X-ray scattering (SAXS) experiments reveal a globular shape of nsp10 which is conserved in all three coronaviruses, including MERS nsp10, which diverges most from SARS and SARS-CoV-2 nsp10s. In conclusion, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 are globular and predominantly monomeric in solution. Additionally, we describe for the first time a functional role of the C-terminus of nsp10 for tight binding to nsp14.

biochemistry↗

bioRxiv2023

High-confidence placement of difficult-to-fit fragments into electron density by using anomalous signals - a case study using hits targeting SARS-CoV-2 non-structural protein 1

The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands1. Significantly less is even known about multiple binding orientations for fragments (< 300 Da) although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tuneable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to advance the rational optimisation of SARS-CoV-2 nsp1 targeting fragment hits.

biochemistry↗

bioRxiv2022

Emerging variants of SARS-CoV-2 NSP10 highlight strong functional conservation of its binding to two non-structural proteins, NSP14 and NSP16

The coronavirus SARS-CoV-2 protects its RNA from being recognized by host immune responses by methylation of its 5 end, also known as capping. This process is carried out by two enzymes, non-structural protein 16 (NSP16) containing 2-O-methyltransferase and NSP14 through its N7 methyltransferase activity, which are essential for the replication of the viral genome as well as evading the hosts innate immunity. NSP10 acts as a crucial cofactor and stimulator of NSP14 and NSP16. To further understand the role of NSP10, we carried out a comprehensive analysis of >13 million globally collected whole-genome sequences (WGS) of SARS-CoV-2 obtained from the Global Initiative Sharing All Influenza Data (GISAID) and compared it with the reference genome Wuhan/WIV04/2019 to identify all currently known variants in NSP10. T12I, T102I, and A104V in NSP10 have been identified as the three most frequent variants and characterized using X-ray crystallography, biophysical assays and enhanced sampling simulations. In contrast to other proteins such as spike and NSP6, NSP10 is significantly less prone to mutation due to its crucial role in replication. The functional effects of the variants were examined for their impact on the binding affinity and stability of both NSP14-NSP10 and NSP16-NSP10 complexes. These results highlight the limited changes induced by variant evolution in NSP10 and reflect on the critical roles NSP10 plays during the SARS-CoV-2 life cycle. These results also indicate that there is limited capacity for the virus to overcome inhibitors targeting NSP10 via the generation of variants in inhibitor binding pockets. Significance StatementThe SARS-CoV-2 proteins have constantly been evolving. These variants assist the virus to survive, adapt and evade the host immune responses. While the main focus has been on structural proteins like Spike, there is very limited structural and functional information on the effects of emerging mutations on other essential non-structural viral proteins. One such protein is NSP10, an essential cofactor for NSP14 and NSP16. This study demonstrates that NSP10 is more resistant to genetic variations than other SARS-CoV-2 non-structural proteins and that the presence of mutations conserve structural and dynamic changes in NSP10. The effects of naturally occurring mutations reflect the evolutionary relationship between structurally conserved essential cofactors, their function and the role they play in the survival of the virus.

biophysics↗

bioRxiv2022

Cytomotive actins and tubulins share a polymerisation switch mechanism conferring robust dynamics

Protein filaments are used in myriads of ways to organise other molecules in space and time within cells. Some filament-forming proteins couple the hydrolysis of nucleotides to their polymerisation cycle, thus powering the directed movement of other molecules. These filaments are termed cytomotive. Only members of the actin and tubulin protein superfamilies are known to form cytomotive filaments. We sought to examine the basis of cytomotivity via structural studies of the polymerisation cycles of actin and tubulin homologues from across the tree of life. We analysed published data and performed new structural experiments designed to disentangle functional components of these complex filament systems. In sum, our analysis demonstrates the existence of shared subunit polymerisation switches amongst both cytomotive actins and tubulins, i.e. the conformation of subunits switches upon assembly into filaments. Such cytomotive switches explain filament robustness, by enabling the coupling of kinetic and structural polarities required for useful cytomotive behaviours, and by ensuring that single cytomotive filaments do not fall apart.

molecular biology↗

bioRxiv2022

The structure of the humanised A33 Fab C226S variant, an immunotherapy candidate for colorectal cancer

Colorectal cancer (CRC) causes the second highest cancer-related deaths worldwide. The human A33 antigen is a validated immunotherapy target, which is homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. In this article, we report the structure of a humanised antigen-binding fragment A33 (A33 Fab), a therapeutic antibody candidate, in two different crystal forms. Insights into the structural features of A33 Fab are provided with a focus on the grafted complementarity-determining regions (CDRs) and the switch linker between the variable and the constant regions. SynopsisThe crystal structure of humanised A33 Fab, targeting colorectal cancer related antigen, was determined in two different space groups.

biophysics↗

bioRxiv2022

Two ligand-binding sites on SARS-CoV-2 non-structural protein 1 revealed by fragment-based x-ray screening

The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 through suppressing its replication and or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.95 [A]). Binding affinities of hits against nsp1 were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal sift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medially relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1.

biochemistry↗