12 research outputs found
High Potency of a Bivalent Human VH Domain in SARS-CoV-2 Animal Models
Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH
domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with
high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized
mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic
and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively
small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all
three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VHFc
ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent
neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to
SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic
HP1 protein Chp2 selectively recruits nucleosome remodeler through non-canonical interaction
The establishment and maintenance of heterochromatic regions within the genome is an important factor for chromosome stability and con- trolled gene expression. It is dependent on many protein factors that are highly conserved from yeast to human. Among them are HP1 pro- teins that recognize heterochromatin-specific methylation marks and are involved in the recruitment of effector proteins. In my thesis, I will focus on the fission yeast HP1 protein Chp2 and its interaction with the Snf2/HDAC- containing remodeling complex SHREC, a homolog of NuRD complexes in higher organisms. Previously, Chp2 was found to be functionally and biochemically associated with the chromatin remodeler Mit1, a subunit of the SHREC complex. However, details of this interaction were unknown. I was able to solve the structure of the Chp2-Mit1 complex with high resolution and could show that an extensive interface between the two proteins provides high-affinity binding. The data reveals an unusual mode of HP1-client interaction and provides an example how specificity between different HP1 proteins canbe achieved. My work adds to the current knowledge about the highly conserved class of HP1 proteins and deepens our understanding of the molecular basis of their function
Glycan reactive anti‑HIV‑1 antibodies bind the SARS‑CoV‑2 spike protein but do not block viral entry
The SARS-CoV-2 spike glycoprotein is a focal point for vaccine immunogen and therapeutic antibody
design, and also serves as a critical antigen in the evaluation of immune responses to COVID-19. A
common feature amongst enveloped viruses such as SARS-CoV-2 and HIV-1 is the propensity for
displaying host-derived glycans on entry spike proteins. Similarly displayed glycosylation motifs can
serve as the basis for glyco-epitope mediated cross-reactivity by antibodies, which can have important
implications on virus neutralization, antibody-dependent enhancement (ADE) of infection, and the
interpretation of antibody titers in serological assays. From a panel of nine anti-HIV-1 gp120 reactive
antibodies, we selected two (PGT126 and PGT128) that displayed high levels of cross-reactivity with
the SARS-CoV-2 spike. We report that these antibodies are incapable of neutralizing pseudoviruses
expressing SARS-CoV-2 spike proteins and are unlikely to mediate ADE via FcÎłRII receptor
engagement. Nevertheless, ELISA and other immunoreactivity experiments demonstrate these
antibodies are capable of binding the SARS-CoV-2 spike in a glycan-dependent manner. These results
contribute to the growing literature surrounding SARS-CoV-2 S cross-reactivity, as we demonstrate
the ability for cross-reactive antibodies to interfere in immunoassays.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofReviewedFacultyPostdoctoralGraduat
Transcriptional gene silencing requires dedicated interaction between HP1 protein Chp2 and chromatin remodeler Mit1.
Heterochromatin protein 1 (HP1) proteins are key factors of eukaryotic heterochromatin that coordinate chromatin compaction and transcriptional gene silencing. Through their multivalency they act as adaptors between histone H3 Lys9 di/trimethyl marks in chromatin and effector complexes that bind to the HP1 chromoshadow domain. Most organisms encode for multiple HP1 isoforms and the molecular mechanisms that underpin their diverse functions in genome regulation remain poorly understood. In fission yeast, the two HP1 proteins Chp2 and Swi6 assume distinct roles and Chp2 is tightly associated with the nucleosome remodeling and deacetylation complex SHREC. Here we show that Chp2 directly engages the SHREC nucleosome remodeler subunit Mit1. The crystal structure of the interaction interface reveals an extraordinarily extensive and specific interaction between the chromoshadow domain of Chp2 and the N terminus of Mit1. The integrity of this interface is critical for high affinity binding and for heterochromatin formation. Comparison with Swi6 shows that the Chp2-Mit1 interface is highly selective and thereby provides the molecular basis for the functional specialization of an HP1 isoform
The Role of Djp1 in Import of the Mitochondrial Protein Mim1 Demonstrates Specificity between a Cochaperone and Its Substrate Protein
A Ponceau S Staining-Based Dot Blot Assay for Rapid Protein Quantification of Biological Samples
Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive
Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies
The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious
than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Ă… resolution cryo-electron microscopy (cryo-EM) structure of the
complex between the ACE2 receptor and N501Y spike protein ectodomains that shows
Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant,
and likely contributes to its increased infectivity. However, this mutation does not result in
large structural changes, enabling important neutralization epitopes to be retained in the
spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent
neutralizing antibody fragments.Medicine, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofReviewedFacultyResearcherPostdoctoralGraduat
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Cryo-EM structure determination of small therapeutic protein targets at 3 Ă…-resolution using a rigid imaging scaffold.
Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Ă…. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases