Biology subjects

Haas-Neill, L.

Publications and source records attributed to Haas-Neill, L..

4 recordsLinked to original sources

bioRxiv2024

An all-atom view into the disordered interaction interface of the TRIM5α PRYSPRY domain and the HIV capsid

Tripartite motif-containing protein 5, alpha isoform (TRIM5) is an innate immune factor that provides rhesus macaques with immunity to HIV. Despite high sequence similarity to rhesus TRIM5, human TRIM5 weakly restricts HIV without the introduction of mutations. The structural underpinnings of this functional difference are poorly understood because the interaction interface between TRIM5 and its target, the HIV capsid, involves intrinsically disordered regions. Here, we use all-atom molecular dynamics simulations to study several TRIM5 variants: rhesus TRIM5, human TRIM5, and human TRIM5 with an R332P mutation (a mutation known to enhance HIV restriction). Our data reveal differences in the conformational ensembles of wild-type and R332P human TRIM5, including a significant increase in the formation of a turn that includes the mutation site residue. We also carried out simulations of rhesus TRIM5 in complex with the HIV capsid protein. Our results indicate that the variable loops of TRIM5 are highly flexible, both in solution and in the complex with the capsid, indicative of a fuzzy interaction interface. Simulations of the complex, as well as experimental infections, indicate the basis for weak HIV restriction by human TRIM5 that is improved by an R332P mutation: replacing the positively charged arginine with a neutral residue decreases electrostatic repulsion with residue R82 of the capsid protein, which can be phenocopied by neutralizing mutations at capsid residue R82. Overall, our simulations provide a first view of the atomistic details of the HIV capsid-TRIM5 binding interface and a molecular mechanism for the observed functional differences between variants of the TRIM5 protein. Significance StatementHumans and viruses are engaged in an evolutionary arms race, which involves evolutionary cycles in which viruses escape host defense and hosts evolve to chase after them. The protein TRIM5 is a crucial part of the immune systems defense against viruses, and the region of TRIM5 that interacts with viral capsids is evolving rapidly. While a wealth of structural information is separately available for the HIV capsid protein and TRIM5, the interaction interface between these two proteins remains poorly understood because it involves multiple disordered regions. We present a first atomistic view of the TRIM5-capsid interaction interface using all-atom molecular dynamics simulations. We use our model of the interface and the probabilistic description of interactions to explain the improved HIV restriction of a mutation in the human TRIM5 protein, which has been proposed for human gene therapy, in terms of specific electrostatic interactions. Finally, we validate our findings with experimental infections. Our data provide detailed structural insight into how TRIM5 evolves improved restriction against its retroviral targets.

biophysics↗

bioRxiv2024

The structural influence of the oncogenic driver mutation N642H in the STAT5B SH2 domain

The point mutation N642H of the signal transducer and activator of transcription 5B (STAT5B) protein is associated with aggressive and drug-resistant forms of leukemia. This mutation is thought to promote cancer due to hyperactivation of STAT5B caused by increased stability of the active, parallel dimer state. However, the molecular mechanism leading to this stabilization is not well understood as there is currently no structure of the parallel dimer. To investigate the mutations mechanism of action, we conducted extensive all-atom molecular dynamics simula-tions of multiple oligomeric forms of both STAT5B and STAT5BN642H, including a model for the parallel dimer. The N642H mutation directly affects the hydrogen bonding network within the phosphotyrosine (pY)-binding pocket of the parallel dimer, enhancing the pY-binding in-teraction. The simulations indicate that apo STAT5B is highly flexible, exploring a diverse conformational space. In contrast, apo STAT5BN642H accesses two distinct conformational states, one of which resembles the conformation of the parallel dimer. The simulation predic-tions of the effects of the mutation on structure and dynamics are supported by the results of hydrogen-deuterium exchange (HDX) mass spectrometry measurements carried out on STAT5B and STAT5BN642H in which a phosphopeptide was used to mimic the effects of parallel dimer-ization on the SH2 domain. The molecular-level information uncovered in this work contributes to our understanding of STAT5B hyperactivation by the N642H mutation and could help pave the way for novel therapeutic strategies targeting this mutation.

biophysics↗

bioRxiv2024

PEG-mCherry interactions beyond classical macromolecular crowding

The dense cellular environment influences bio-macromolecular structure, dynamics, interactions and function. Despite advancements in understanding protein-crowder interactions, predicting their precise effects on protein structure and function remains challenging. Here, we elucidate the effects of PEG-induced crowding on the fluorescent protein mCherry using molecular dynamics simulations and fluorescence-based experiments. We identify and characterize specific PEG-induced structural and dynamical changes in mCherry. Importantly, we find interactions in which PEG molecules wrap around specific surface-exposed residues in a binding mode previously observed in protein crystal structures. Fluorescence correlation spectroscopy experiments capture PEG-induced changes, including aggregation, suggesting a potential role for the specific PEG-mCherry interactions identified in simulations. Additionally, mCherry fluorescence lifetimes are influenced by PEG and not by the bulkier crowder dextran or by another linear polymer, polyvinyl alcohol, highlighting the importance of crowder-protein soft interactions. This work augments our understanding of macromolecular crowding effects on protein structure and dynamics.

biophysics↗

bioRxiv2023

Fluorescent protein lifetimes report increased local densities and phases of nuclear condensates during embryonic stem cell differentiation

Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1 (HP1) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1 condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.

biophysics↗