Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end

Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently "poisoned" growth, yielding a wider range of growth rate compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP tubulin at the growing microtubule end may be an important early event determining catastrophe.

Parallel and Sequential Pathways of Molecular Recognition of a Tandem-Repeat Protein and Its Intrinsically Disordered Binding Partner

The Wnt signalling pathway plays an important role in cell proliferation, differentiation, and fate decisions in embryonic development and the maintenance of adult tissues. The twelve armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here, we investigated the interaction between β-catenin and the intrinsically disordered transcription factor TCF7L2, comprising a very long nanomolar-affinity interface of approximately 4800 Å2 that spans ten of the twelve ARM repeats of β-catenin. First, a fluorescence reporter system for the interaction was engineered and used to determine the kinetic rate constants for the association and dissociation. The association kinetics of TCF7L2 and β-catenin were monophasic and rapid (7.3 ± 0.1 × 107 M−1·s−1), whereas dissociation was biphasic and slow (5.7 ± 0.4 × 10−4 s−1, 15.2 ± 2.8 × 10−4 s−1). This reporter system was then combined with site-directed mutagenesis to investigate the striking variability in the conformation adopted by TCF7L2 in the three different crystal structures of the TCF7L2–β-catenin complex. We found that the mutation had very little effect on the association kinetics, indicating that most interactions form after the rate-limiting barrier for association. Mutations of the N- and C-terminal subdomains of TCF7L2 that adopt relatively fixed conformations in the crystal structures had large effects on the dissociation kinetics, whereas the mutation of the labile sub-domain connecting them had negligible effect. These results point to a two-site avidity mechanism of binding with the linker region forming a “fuzzy” complex involving transient contacts that are not site-specific. Strikingly, the two mutations in the N-terminal subdomain that had the largest effects on the dissociation kinetics showed two additional phases, indicating partial flux through an alternative dissociation pathway that is inaccessible to the wild type. The results presented here provide insights into the kinetics of the molecular recognition of a long intrinsically disordered region with an elongated repeat-protein surface, a process found to involve parallel routes with sequential steps in each.

Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

A Multiscale Model for Quantitative Prediction of Insulin Aggregation Nucleation Kinetics

We combined kinetic, thermodynamic, and structural information from single molecule (protein folding) and two molecule (protein association) explicit-solvent simulations for determination of kinetic parameters in protein aggregation nucleation with insulin as model protein. A structural bioinformatics approach was developed to account for heterogeneity of aggregation-prone species with the transition complex theory, developed for native protein-receptor interactions, found applicable in modeling association kinetics involving this non-native species. We show that a key simplification arises from presence of only a few relevant modes for non-native association kinetics and that it is necessary to explicitly account for conformational rearrangement of a diffusional intermediate leading to the formation of aggregation pathway dimer and small oligomers. The kinetic parameters thus obtained were used in a population balance model and very accurate predictions for aggregation nucleation time varying over two orders of magnitude with changes in concentration of insulin or an aggregation-inhibitor ligand were obtained while an empirical parameter set was not found to be transferable for prediction of ligand effects. This physically determined kinetic parameter set also provided several insights into the mechanism of aggregation nucleation. Finally we discuss a route for application of our approach in high-throughput computational screening of ligands for inhibiting aggregation.