Modeling reveals the strength of weak interactions in stacked-ring assembly.

Cells employ many large macromolecular machines for the execution and regulation of processes that are vital for cell and organismal viability. Interestingly, cells cannot synthesize these machines as functioning units. Instead, cells synthesize the molecular parts that must then assemble into the functional complex. Many important machines, including chaperones such as GroEL and proteases such as the proteasome, comprise protein rings that are stacked on top of one another. While there is some experimental data regarding how stacked-ring complexes such as the proteasome self-assemble, a comprehensive understanding of the dynamics of stacked-ring assembly is currently lacking. Here, we developed a mathematical model of stacked-trimer assembly and performed an analysis of the assembly of the stacked homomeric trimer, which is the simplest stacked-ring architecture. We found that stacked rings are particularly susceptible to a form of kinetic trapping that we term deadlock, in which the system gets stuck in a state where there are many large intermediates that are not the fully assembled structure but that cannot productively react. When interaction affinities are uniformly strong, deadlock severely limits assembly yield. We thus predicted that stacked rings would avoid situations where all interfaces in the structure have high affinity. Analysis of available crystal structures indicated that indeed the majority-if not all-of stacked trimers do not contain uniformly strong interactions. Finally, to better understand the origins of deadlock, we developed a formal pathway analysis and showed that, when all the binding affinities are strong, many of the possible pathways are utilized. In contrast, optimal assembly strategies utilize only a small number of pathways. Our work suggests that deadlock is a critical factor influencing the evolution of macromolecular machines and provides general principles for understanding the self-assembly efficiency of existing machines

Scaffolding, Multisite Phosphorylation and Other Aspects of Regulation in Signal Transduction

For cells to respond appropriately and timely to internal and external stimulus, they rely heavily on signaltransduction cascades to regulate gene and protein activity and response. Protein activity is oftenregulated by ligand binding, scaffolding, or by post-translational modifications such as multisitephosphorylation. The Mitogen-Activated Protein Kinases (MAPKs) are multisite proteins with critical rolesin development and various diseases. The c-Jun N-terminal Kinases (JNK) cascade is one of many MAPKcascades with multisite proteins. Here, we consider mathematical and experimental analyses tounderstand scaffolding and multisite modifications as protein regulation mechanisms.Scaffolding is a regulatory mechanism for signal transduction where scaffold proteins bind to manycomponents of a signaling pathway. MAPK scaffold proteins, such as IQ-motif-containing GTPaseactivatingprotein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases.Scaffolding is a regulatory mechanism for signal transduction where scaffold proteins bind to manycomponents of a signaling pathway. Thus, it important to know which domains on IQGPA1 bind to whichMAP kinases for developing new therapies. Here, we show with in vitro binding assays that the IQ domainof IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2. Additionally, we show that theWW domain is neither necessary nor sufficient for binding to ERK1 or ERK2. These findings prompt a reevaluationof how IQGAP1 regulates MAPK cascade proteins.Protein phosphorylation also regulates a substrate’s enzymatic activity, location, stability, and/orinteractions with other proteins. Moreover, proteins that are regulated in this way often contain multiplemodification sites. In the JNK pathway, transcription factor c-Jun is a multisite substrate shown to regulatecell responses such as cell proliferation, apoptosis, and DNA repair. Understanding kinase-substratespecificity in MAPKs is crucial to advancing medical therapies for diseases. Phosphoproteomic studieshave provided insights into the conservation of phosphosites and their evolution across species. However,not much is known about the constraints novel sites experience. Here, we demonstrate with in vitro kinaseassays that the Docking site (D-site) in c-Jun plays a significant role in the phosphorylation of all native andnovel sites of c-Jun. Results indicate that the D-site is necessary for phosphorylation of native and novelsites of c-Jun by JNK2 enzyme.Mathematical models are useful to study complex biological phenomena such as multisite proteins (i.e.,proteins with n > 1 modifications sites). Proteins with multiple sites on which they can be modified orbound by ligand have been observed to create an ultrasensitive dose response. Here, we consider thecontribution of the individual modification/binding sites to the activation process, and show how theirindividual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-Wyman-Changeux (MWC) model that allows for variable free energy contributions at distinct sites, andassociate a so-called activation parameter to each site. Our analysis shows that ultrasensitivity generallydecreases with increasing activation parameter values and depends on their mean and not on theirvariability. Additionally, results suggest that a protein can increase its ultrasensitivity by evolving newsites. These results provide insights into the performance objectives of multiple modification/binding sitesand thus help gain a greater understanding of signaling and its role in diseases. Together, mathematicaland experimental analyses show promising insights into signal transduction regulation throughscaffolding, multisite PTMs, and docking

Scaffolding, Multisite Phosphorylation and Other Aspects of Regulation in Signal Transduction

For cells to respond appropriately and timely to internal and external stimulus, they rely heavily on signaltransduction cascades to regulate gene and protein activity and response. Protein activity is oftenregulated by ligand binding, scaffolding, or by post-translational modifications such as multisitephosphorylation. The Mitogen-Activated Protein Kinases (MAPKs) are multisite proteins with critical rolesin development and various diseases. The c-Jun N-terminal Kinases (JNK) cascade is one of many MAPKcascades with multisite proteins. Here, we consider mathematical and experimental analyses tounderstand scaffolding and multisite modifications as protein regulation mechanisms.Scaffolding is a regulatory mechanism for signal transduction where scaffold proteins bind to manycomponents of a signaling pathway. MAPK scaffold proteins, such as IQ-motif-containing GTPaseactivatingprotein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases.Scaffolding is a regulatory mechanism for signal transduction where scaffold proteins bind to manycomponents of a signaling pathway. Thus, it important to know which domains on IQGPA1 bind to whichMAP kinases for developing new therapies. Here, we show with in vitro binding assays that the IQ domainof IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2. Additionally, we show that theWW domain is neither necessary nor sufficient for binding to ERK1 or ERK2. These findings prompt a reevaluationof how IQGAP1 regulates MAPK cascade proteins.Protein phosphorylation also regulates a substrate’s enzymatic activity, location, stability, and/orinteractions with other proteins. Moreover, proteins that are regulated in this way often contain multiplemodification sites. In the JNK pathway, transcription factor c-Jun is a multisite substrate shown to regulatecell responses such as cell proliferation, apoptosis, and DNA repair. Understanding kinase-substratespecificity in MAPKs is crucial to advancing medical therapies for diseases. Phosphoproteomic studieshave provided insights into the conservation of phosphosites and their evolution across species. However,not much is known about the constraints novel sites experience. Here, we demonstrate with in vitro kinaseassays that the Docking site (D-site) in c-Jun plays a significant role in the phosphorylation of all native andnovel sites of c-Jun. Results indicate that the D-site is necessary for phosphorylation of native and novelsites of c-Jun by JNK2 enzyme.Mathematical models are useful to study complex biological phenomena such as multisite proteins (i.e.,proteins with n > 1 modifications sites). Proteins with multiple sites on which they can be modified orbound by ligand have been observed to create an ultrasensitive dose response. Here, we consider thecontribution of the individual modification/binding sites to the activation process, and show how theirindividual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-Wyman-Changeux (MWC) model that allows for variable free energy contributions at distinct sites, andassociate a so-called activation parameter to each site. Our analysis shows that ultrasensitivity generallydecreases with increasing activation parameter values and depends on their mean and not on theirvariability. Additionally, results suggest that a protein can increase its ultrasensitivity by evolving newsites. These results provide insights into the performance objectives of multiple modification/binding sitesand thus help gain a greater understanding of signaling and its role in diseases. Together, mathematicaland experimental analyses show promising insights into signal transduction regulation throughscaffolding, multisite PTMs, and docking

Effect of magnitude and variability of energy of activation in multisite ultrasensitive biochemical processes.

Protein activity is often regulated by ligand binding or by post-translational modifications such as phosphorylation. Moreover, proteins that are regulated in this way often contain multiple ligand binding sites or modification sites, which can operate to create an ultrasensitive dose response. Here, we consider the contribution of the individual modification/binding sites to the activation process, and how their individual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-Wyman-Changeux (MWC) model that allows for variable conformational free energy contributions from distinct sites, and associate a so-called activation parameter to each site. Our analysis shows that the ultrasensitivity generally increases as the conformational free energy contribution from one or more sites is strengthened. Furthermore, ultrasensitivity depends on the mean of the activation parameters and not on their variability. In some cases, we find that the best way to maximize ultrasensitivity is to make the contribution from all sites as strong as possible. These results provide insights into the performance objectives of multiple modification/binding sites and thus help gain a greater understanding of signaling and its role in diseases

Effect of magnitude and variability of energy of activation in multisite ultrasensitive biochemical processes.

Protein activity is often regulated by ligand binding or by post-translational modifications such as phosphorylation. Moreover, proteins that are regulated in this way often contain multiple ligand binding sites or modification sites, which can operate to create an ultrasensitive dose response. Here, we consider the contribution of the individual modification/binding sites to the activation process, and how their individual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-Wyman-Changeux (MWC) model that allows for variable conformational free energy contributions from distinct sites, and associate a so-called activation parameter to each site. Our analysis shows that the ultrasensitivity generally increases as the conformational free energy contribution from one or more sites is strengthened. Furthermore, ultrasensitivity depends on the mean of the activation parameters and not on their variability. In some cases, we find that the best way to maximize ultrasensitivity is to make the contribution from all sites as strong as possible. These results provide insights into the performance objectives of multiple modification/binding sites and thus help gain a greater understanding of signaling and its role in diseases

Understanding the separation of timescales in bacterial proteasome core particle assembly.

The 20S proteasome core particle (CP) is a molecular machine that is a key component of cellular protein degradation pathways. Like other molecular machines, it is not synthesized in an active form but rather as a set of subunits that assemble into a functional complex. The CP is conserved across all domains of life and is composed of 28 subunits, 14 α and 14 β, arranged in four stacked seven-member rings (α7β7β7α7). While details of CP assembly vary across species, the final step in the assembly process is universally conserved: two half proteasomes (HPs; α7β7) dimerize to form the CP. In the bacterium Rhodococcus erythropolis, experiments have shown that the formation of the HP is completed within minutes, while the dimerization process takes hours. The N-terminal propeptide of the β subunit, which is autocatalytically cleaved off after CP formation, plays a key role in regulating this separation of timescales. However, the detailed molecular mechanism of how the propeptide achieves this regulation is unclear. In this work, we used molecular dynamics simulations to characterize HP conformations and found that the HP exists in two states: one where the propeptide interacts with key residues in the HP dimerization interface and likely blocks dimerization, and one where this interface is free. Furthermore, we found that a propeptide mutant that dimerizes extremely slowly is essentially always in the nondimerizable state, while the wild-type rapidly transitions between the two. Based on these simulations, we designed a propeptide mutant that favored the dimerizable state in molecular dynamics simulations. In vitro assembly experiments confirmed that this mutant dimerizes significantly faster than wild-type. Our work thus provides unprecedented insight into how this critical step in CP assembly is regulated, with implications both for efforts to inhibit proteasome assembly and for the evolution of hierarchical assembly pathways

The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2.

Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (Kd ) for this interaction of 8 μm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding

The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2.

Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (Kd ) for this interaction of 8 μm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding