Biology subjects

Kushwah, M. S.

Publications and source records attributed to Kushwah, M. S..

3 recordsLinked to original sources

bioRxiv2023

Cooperativity and induced oligomerisation control the interaction of SARS- CoV-2 with its cellular receptor and patient-derived antibodies

Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV-2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant-dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets. One-Sentence SummaryMultivalent interactions between viral proteins, cell-surface receptors, and anti-viral antibodies regulate infection and inhibition.

biophysics↗

bioRxiv2023

Short oligomers rather than rings of human RAD52 promote single-strand annealing

Genome maintenance and stability rely on the repair of DNA double-strand breaks. The break repair can be mediated by the single-strand annealing protein RAD52. RAD52 forms rings that are thought to promote annealing. However, RAD52s annealing activity decreases with increasing concentrations that favor ring formation. Thus, which oligomeric form and how RAD52 anneals DNA strands and detects sequence homology is unclear. We combine mass photometry with biochemical assays to quantify oligomeric states of human RAD52 with and without DNA and put forward an alternative mechanism illustrating the critical role of short oligomers for single-stranded DNA annealing. We found that while truncated RAD52 formed undecameric rings at nanomolar concentrations, full-length RAD52 was mostly monomeric at lower nanomolar, physiological concentrations. At higher concentrations, it formed rings with a variable stoichiometry from heptamers to tridecamers. At low concentrations, with hardly any rings present, RAD52 already promoted single-strand annealing. Rings and short oligomers could bind at least two single DNA strands, but if complementary strands were both bound to rings annealing was inhibited. Our findings suggest that single-strand annealing and homology detection is mediated by short oligomers of RAD52 instead of rings.

molecular biology↗

bioRxiv2021

Single Molecule Mass Photometry Reveals Dynamic Oligomerization of Plant and Human Peroxiredoxins for Functional Conservation and Diversification

Single molecule mass photometry was used to study the dynamic equilibria of the ubiquitous and highly abundant 2-Cysteine peroxiredoxins (2-CysPRX). 2-CysPRXs adopt distinct functions in all cells dependent on their oligomeric conformation ranging from dimers to decamers and high molecular weight aggregates (HMW). The oligomeric state depends on the redox state of their catalytic cysteinyl residues. To which degree they interconvert, how the interconversion is regulated, and how the oligomerisation propensity is organism specific remains, however, poorly understood. The dynamics differs between wild-type and single point mutants affecting the oligomerization interfaces, with concomitant changes to function. Titrating concentration and redox state of Arabidopsis thaliana and human 2-CysPRXs revealed features conserved among all 2-CysPRX and clear differences concerning oligomer transitions, the occurrence of transition states and the formation of HMW which are associated with chaperone activity or storage. The results indicate functional differentiation of human 2-CysPRXs. Our results point to a diversified functionality of oligomerization for 2-CysPRXs and illustrate the power of mass photometry to non-invasively quantify oligomer distributions in a redox environment. This knowledge is important to fully address and model PRX function in cell redox signaling e.g., in photosynthesis, cardiovascular and neurological diseases or carcinogenesis.

biochemistry↗