Biology subjects

Anastas, A.

Publications and source records attributed to Anastas, A..

3 recordsLinked to original sources

bioRxiv2026

Mapping SARS-CoV-2 Nucleocapsid Function with Nanobodies

The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the proteins structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential. ImportanceSARS-CoV-2 and emerging coronaviruses remain a major global health threat, yet critical gaps persist in our understanding of their molecular pathogenesis. The nucleocapsid (N) protein, the most abundantly expressed SARS-CoV-2 antigen, plays essential roles beyond genome packaging, including immune evasion and intracellular organization. Here, we generate and characterize a panel of domain-specific nanobodies (VHHs) that enable precise dissection of Ns functional architecture. Using integrated biochemical, structural, and virological approaches, we uncover distinct mechanisms of viral inhibition, including disruption of phase condensation through a conserved linker region. These findings address long-standing knowledge gaps about a multifunctional viral protein and establish VHHs as powerful, modular tools for probing coronavirus biology, with broad potential for therapeutic, diagnostic, and cell biology applications.

microbiology↗

bioRxiv2024

An effective method of measuring nanobody binding kinetics and competition-based epitope mapping using biolayer interferometry

Protein-protein interactions (PPI) underpin nearly all biological processes, and understanding the molecular mechanisms governing these interactions is crucial for the progress of biomedical sciences. The emergence of AI-driven computational tools can help reshape the methods in structural biology, however model data often quires empirical validation. The large scale of predictive modeling data will therefore benefit from optimized methodologies for the high-throughput biochemical characterization of PPIs. Biolayer interferometry (BLI) is one of very few approaches that can determine the rate of biomolecular interactions, called kinetics, and of the commonly available kinetic measurement techniques, it is the most suitable for high-throughput experimental designs. Here, we provide step-by-step instructions on how to perform kinetics experiments using BLI. We further describe the basis and execution of competition and epitope binning experiments, which are particularly useful for antibody and nanobody screening applications. The procedure requires 3 hours to complete and is suitable for users with minimal experience with biochemical techniques.

biochemistry↗

bioRxiv2023

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

biochemistry↗