Biology subjects

Pappu, R. V.

Publications and source records attributed to Pappu, R. V..

7 recordsLinked to original sources

bioRxiv2018

Evolved sequence features within the intrinsically disordered tail influence FtsZ assembly and bacterial cell division

Intrinsically disordered regions (IDRs) challenge the well-established sequence-structure-function paradigm for describing protein function and evolution. Here, we direct a combination of biophysical and cellular studies to further our understanding of how the intrinsically disordered C-terminal tail of FtsZ contributes to cell division in rod-shaped bacteria. FtsZ is a modular protein that encompasses a conserved GTPase domain and a highly variable intrinsically disordered C-terminal tail (CTT). The CTT is essential for forming the cytokinetic Z-ring. Despite poor sequence conservation of the CTT, the patterning of oppositely charged residues, which refers to the extent of linear mixing / segregation of oppositely charged residues within CTT sequences is bounded within a narrow range. To assess the impact of evolutionary bounds on charge patterning within CTT sequences we performed experiments, aided by sequence design, to quantify the impact of changing the patterning of oppositely charged residues within the CTT on the functions of FtsZ from B. subtilis. Z-ring formation is robust if and only if the extent of linear mixing / segregation of oppositely charged residues within the CTT sequences is within evolutionarily observed bounds. Otherwise, aberrant, CTT-mediated, FtsZ assemblies impair Z-ring formation. The complexities of CTT sequences also have to be above a threshold value because FtsZ variants with low complexity CTTs are not tolerated in cells. Taken together, our results suggest that CTT sequences have evolved to be \"just right\" and that this is achieved through an optimal extent of charge patterning while maintaining the sequence complexity above a threshold value.

biophysics↗

bioRxiv2018

Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

Proper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Forster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.

cell biology↗

bioRxiv2017

A deep mutational scan of an acidic activation domain

Transcriptional activation domains are intrinsically disordered peptides with little primary sequence conservation. These properties have made it difficult to identify the sequence features that define activation domains. For example, although acidic activation domains were discovered 30 years ago, we still do not know what role, if any, acidic residues play in these peptides. To address this question we designed a rational mutagenesis scheme to independently test four sequence features theorized to control the strength of activation domains: acidity (negative charge), hydrophobicity, intrinsic disorder, and short linear motifs. To test enough mutants to deconvolve these four features we developed a method to quantify the activities of thousands of activation domain variants in parallel. Our results with Gcn4, a classic acidic activation domain, suggest that acidic residues in particular regions keep two hydrophobic motifs exposed to solvent. We also found that the specific activity of the Gcn4 activation domain increases during amino acid starvation. Our results suggest that Gcn4 may have evolved to have low activity but high inducibility. Our results also demonstrate that high-throughput rational mutation scans will be powerful tools for unraveling the properties that control how intrinsically disordered proteins function.

systems biology↗

bioRxiv2017

Tadpole-like conformations of huntingtin exon 1 with expanded polyglutamine engenders novel interactions in cells

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntingtons disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.

biochemistry↗

bioRxiv2017

Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins

Many intracellular membraneless bodies appear to form via reversible phase transitions of multivalent proteins. Two relevant types of phase transitions are sol-gel transitions (gelation) and phase separation plus gelation. Gelation refers to the formation of a system spanning molecular network. This can either be enabled by phase separation or it can occur independently. Despite relevance for the formation and selectivity of compositionally distinct protein and RNA assemblies, the determinants of gelation as opposed to phase separation plus gelation remain unclear. Here, we focus on linear multivalent proteins that consist of interaction domains that are connected by disordered linkers. Using results from computer simulations and theoretical analysis we show that the lengths and sequence-specific features of disordered linkers determine the coupling between phase separation and gelation. Thus, the precise nature of phase transitions for linear multivalent proteins should be biologically tunable through genetic encoding of or post-translational modifications to linker sequences.

cell biology↗

bioRxiv2017

Inert and seed-competent tau monomers elucidate the structural origins of aggregation

Tauopathies feature progressive accumulation of tau amyloids. Pathology may begin when these amplify from a protein template, or seed, whose structure is unknown. We have purified and characterized distinct forms of tau monomer--inert (Mi) and seed-competent (Ms). Recombinant Ms triggered intracellular tau aggregation, induced tau fibrillization in vitro, and self-assembled. Ms from Alzheimers disease also seeded aggregation and self-assembled in vitro to form seed-competent multimers. We used crosslinking with mass spectrometry to probe structural differences in Mi vs. Ms. Crosslinks informed models of local peptide structure within the repeat domain which suggest relative inaccessibility of residues that drive aggregation (VQIINK/VQIVYK) in Mi, and exposure in Ms. Limited proteolysis supported this idea. Although tau monomer has been considered to be natively unstructured, our findings belie this assumption and suggest that initiation of pathological aggregation could begin with conversion of tau monomer from an inert to a seed-competent form.

neuroscience↗

bioRxiv2017

Endocytosis caused by liquid-liquid phase separation of proteins

Membrane invagination and vesicle formation are key steps in endocytosis and cellular trafficking. Here, we show that endocytic coat proteins with prion-like domains (PLDs) form hemispherical puncta in the budding yeast, S. cerevisiae. These puncta have the hallmarks of biomolecular condensates and enable membrane remodeling to drive actin-independent endocytosis. The puncta, which we refer to as endocytic condensates, form and dissolve reversibly in response to changes in temperature and solution conditions. The condensates are organized around dynamic protein-protein interaction networks, which involve interactions among PLDs with high glutamine contents. The endocytic coat protein Sla1 is at the hub of the protein-protein interaction network. Using active rheology, we indirectly characterized the material properties of endocytic condensates. These experiments show that endocytic condensates are viscoelastic materials and allow us to estimate the interfacial tension between endocytic condensates and their surroundings. We then adapt the physics of contact mechanics, specifically the contact theory of Hertz, to develop a quantitative framework for describing how interfacial tensions among condensates, the membrane, and the cytosol can deform the plasma membrane to enable actin independent endocytosis.

cell biology↗