A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

The CLIP-170 N-terminal domain binds directly to both F-actin and microtubules in a mutually exclusive manner.

The cooperation between the actin and microtubule (MT) cytoskeletons is important for cellular processes such as cell migration and muscle cell development. However, a full understanding of how this cooperation occurs has yet to be sufficiently developed. The MT plus-end tracking protein CLIP-170 has been implicated in this actin-MT coordination by associating with the actin-binding signaling protein IQGAP1 and by promoting actin polymerization through binding with formins. Thus far, the interactions of CLIP-170 with actin were assumed to be indirect. Here, we demonstrate using high-speed cosedimentation assays that CLIP-170 can bind to filamentous actin (F-actin) directly. We found that the affinity of this binding is relatively weak but strong enough to be significant in the actin-rich cortex, where actin concentrations can be extremely high. Using CLIP-170 fragments and mutants, we show that the direct CLIP-170-F-actin interaction is independent of the FEED domain, the region that mediates formin-dependent actin polymerization, and that the CLIP-170 F-actin-binding region overlaps with the MT-binding region. Consistent with these observations, in vitro competition assays indicate that CLIP-170-F-actin and CLIP-170-MT interactions are mutually exclusive. Taken together, these observations lead us to speculate that direct CLIP-170-F-actin interactions may function to reduce the stability of MTs in actin-rich regions of the cell, as previously proposed for MT end-binding protein 1.

Measuring Tau-microtubule affinity through cosedimentation assays.

Microtubule cosedimentation assays have long been used to study the affinity of interactions between Tau protein and microtubules. While these assays are very useful for characterizing and comparing the effects of alterations to either Tau or the microtubule filaments, they can also be problematic. We provide a set of straightforward instructions for performing these assays and point out a number of challenges and pitfalls that can complicate their interpretation.

Interactions between Tau and Different Conformations of Tubulin: Implications for Tau Function and Mechanism.

Tau is a multifaceted neuronal protein that stabilizes microtubules (MTs), but the mechanism of this activity remains poorly understood. Questions include whether Tau binds MTs laterally or longitudinally and whether Tau's binding affinity depends on the nucleotide state of tubulin. We observed that Tau binds tightly to Dolastatin-10 tubulin rings and promotes the formation of Dolastatin-10 ring stacks, implying that Tau can crosslink MT protofilaments laterally. In addition, we found that Tau prefers GDP-like tubulin conformations, which implies that Tau binding to the MT surface is biased away from the dynamic GTP-rich MT tip. To investigate the potential impact of these Tau activities on MT stabilization, we incorporated them into our previously developed dimer-scale computational model of MT dynamics. We found that lateral crosslinking activities have a much greater effect on MT stability than do longitudinal crosslinking activities, and that introducing a bias toward GDP tubulin has little impact on the observed MT stabilization. To address the question of why Tau is GDP-tubulin-biased, we tested whether Tau might affect MT binding of the +TIP EB1. We confirmed recent reports that Tau binds directly to EB1 and that Tau competes with EB1 for MT binding. Our results lead to a conceptual model where Tau stabilizes the MT lattice by strengthening lateral interactions between protofilaments. We propose that Tau's GDP preference allows the cell to independently regulate the dynamics of the MT tip and the stability of the lattice.

Interactions between the Microtubule Binding Protein EB1 and F-Actin.

Many cellular processes including cell division and cell migration require coordination between the actin and microtubule (MT) cytoskeletons. This coordination is as-yet poorly understood, but proteins such as formins and IQGAP1 are known to be involved. We show that the MT binding protein EB1 (end-binding protein 1), a key regulator of MT dynamics, can bind directly to filamentous actin (F-actin) F-actin. We determined that the EB1:F-actin interaction is salt sensitive and weak under physiological salt concentrations but might be relevant in contexts where the local concentration of actin is high. Using bioinformatics and mutagenesis, we found that the EB1:F-actin binding site partially overlaps the well-characterized EB1:MT binding interface. Congruently, competition experiments indicate that EB1 can bind to F-actin or MTs but not both simultaneously. These observations suggest that EB1:F-actin interactions may negatively regulate EB1:MT interactions, and we speculate that this interaction may assist cells in differentially regulating MT stability in the actin-rich cortex as opposed to the cell interior.

Promoting microtubule assembly: A hypothesis for the functional significance of the +TIP network.

Regulation of microtubule (MT) dynamics is essential for many cellular processes, but the machinery that controls MT dynamics remains poorly understood. MT plus-end tracking proteins (+TIPs) are a set of MT-associated proteins that dynamically track growing MT ends and are uniquely positioned to govern MT dynamics. +TIPs associate with each other in a complex array of inter- and intra-molecular interactions known as the "+TIP network." Why do so many +TIPs bind to other +TIPs? Typical answers include the ideas that these interactions localize proteins where they are needed, deliver proteins to the cortex, and/or create regulatory pathways. We propose an additional and more mechanistic hypothesis: that +TIPs bind each other to create a superstructure that promotes MT assembly by constraining the structural fluctuations of the MT tip, thus acting as a polymerization chaperone.

Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin.

Regulation of microtubule dynamic instability is crucial for cellular processes, ranging from mitosis to membrane transport. Stathmin (also known as oncoprotein 18/Op18) is a prominent microtubule destabilizer that acts preferentially on microtubule minus ends. Stathmin has been studied intensively because of its association with multiple types of cancer, but its mechanism of action remains controversial. Two models have been proposed. One model is that stathmin promotes microtubule catastrophe indirectly, and does so by sequestering tubulin; the other holds that stathmin alters microtubule dynamics by directly destabilizing growing microtubules. Stathmin's sequestration activity is well established, but the mechanism of any direct action is mysterious because stathmin binds to microtubules very weakly. To address these issues, we have studied interactions between stathmin and varied tubulin polymers. We show that stathmin binds tightly to Dolastatin-10 tubulin rings, which mimic curved tubulin protofilaments, and that stathmin depolymerizes stabilized protofilament-rich polymers. These observations lead us to propose that stathmin promotes catastrophe by binding to and acting upon protofilaments exposed at the tips of growing microtubules. Moreover, we suggest that stathmin's minus-end preference results from interactions between stathmin's N terminus and the surface of α-tubulin that is exposed only at the minus end. Using computational modeling of microtubule dynamics, we show that these mechanisms could account for stathmin's observed activities in vitro, but that both the direct and sequestering activities are likely to be relevant in a cellular context. Taken together, our results suggest that stathmin can promote catastrophe by direct action on protofilament structure and interactions.

Using MTBindingSim as a tool for experimental planning and interpretation.

MTBindingSim is a program that enables users to simulate experiments in which proteins or other ligands (e.g., drugs) bind to microtubules or other polymers under various binding models. The purpose of MTBindingSim is to help researchers and students gain an intuitive understanding of binding behavior and design experiments to distinguish between different binding mechanisms. MTBindingSim is open-source, freely available software and can be found at bindingtutor.org/mtbindingsim. This chapter first describes the capabilities of MTBindingSim, including the experimental designs and protein-binding models that it simulates, and then discusses two examples in which MTBindingSim is utilized in an experimental context. In the first, MTBindingSim is used to investigate potential explanations for unusual behavior observed in the binding of the neuronal protein Tau to microtubules, demonstrating that some potential explanations are incompatible with the experimental data. In the second example, MTBindingSim is used to design experiments to examine the question of whether the plus-end tracking protein EB1 binds preferentially to the microtubule seam.

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam.

EB1 is a highly conserved microtubule (MT) plus end tracking protein (+TIP) involved in regulating MT dynamics, but the mechanisms of its effects on MT polymerization remain undefined. Resolving this question requires understanding how EB1 interacts with MTs. Previous electron microscopy of the S. pombe EB1 homolog Mal3p suggested that Mal3p binds specifically to the MT seam, implying that EB1 family members promote MT polymerization by stabilizing the seam. However, more recent electron microscopy indicates that Mal3p binds everywhere except the seam. Neither set of experiments investigated the behavior of human EB1, or provided an explanation for why these studies arrived at different answers. To resolve these questions, we have used a combination of MT-binding assays and theoretical modeling with MTBindingSim. Our results indicate that human EB1 binds to the lattice, consistent with the recent Mal3p results, and show that Mal3p-binding assays that were previously interpreted as evidence for preferential seam binding are equally consistent with weak lattice binding. In addition, we used analytical ultracentrifugation to investigate the possibility that the EB1 monomer-dimer equilibrium might contribute to EB1 binding behavior, and determined that the EB1 dimerization dissociation constant is approximately 90 nM. We and others find that the cellular concentration of EB1 is on the order of 200 nM, suggesting that a portion of EB1 may be monomeric at physiological concentrations. These observations lead us to suggest that regulation of EB1 dimerization might play a role in controlling EB1 function.