Biophysical Rationale for the Selective Inhibition of PTP1B over TCPTP by Nonpolar Terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L-11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a ß site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the ß site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).

Analysis of neutral mutational drift in an allosteric enzyme.

Neutral mutational drift is an important source of biological diversity that remains underexploited in fundamental studies of protein biophysics. This study uses a synthetic transcriptional circuit to study neutral drift in protein tyrosine phosphatase 1B (PTP1B), a mammalian signaling enzyme for which conformational changes are rate limiting. Kinetic assays of purified mutants indicate that catalytic activity, rather than thermodynamic stability, guides enrichment under neutral drift, where neutral or mildly activating mutations can mitigate the effects of deleterious ones. In general, mutants show a moderate activity-stability tradeoff, an indication that minor improvements in the activity of PTP1B do not require concomitant losses in its stability. Multiplexed sequencing of large mutant pools suggests that substitutions at allosterically influential sites are purged under biological selection, which enriches for mutations located outside of the active site. Findings indicate that the positional dependence of neutral mutations within drifting populations can reveal the presence of allosteric networks and illustrate an approach for using synthetic transcriptional systems to explore these mutations in regulatory enzymes.

A biophysical rationale for the selective inhibition of PTP1B over TCPTP by nonpolar terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including type 2 diabetes, obesity, and cancer. However, a high degree of structural similarity between the catalytic domains of these enzymes has made the development of selective pharmacological inhibitors an enormous challenge. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over TCPTP, two PTPs with high sequence conservation. Here, we use molecular modeling with experimental validation to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations indicate that PTP1B and TCPTP contain a conserved h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically influential WPD loop, which it links to the L-11 loop and α 3 and α 7 helices-the C-terminal side of the catalytic domain. Terpenoid binding to either of two proximal allosteric sites-an α site and a ß site-can disrupt the allosteric network. Interestingly, binding to the α site forms a stable complex with only PTP1B; in TCPTP, where two charged residues disfavor binding at the α site, the terpenoids bind to the ß site, which is conserved between the two proteins. Our findings indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate, more broadly, how minor differences in the conservation of neighboring-yet functionally similar-allosteric sites can have very different implications for inhibitor selectivity.

Ketosynthase mutants enable short-chain fatty acid biosynthesis in E. coli.

Cells build fatty acids in tightly regulated assembly lines, or fatty acid synthases (FASs), in which ß-ketoacyl-acyl carrier protein (ACP) synthases (KSs) catalyze sequential carbon-carbon bond forming reactions that generate acyl-ACPs of varying lengths-precursors for a diverse set of lipids and oleochemicals. To date, most efforts to control fatty acid synthesis in engineered microbes have focused on modifying termination enzymes such as acyl-ACP thioesterases, which release free fatty acids from acyl-ACPs. Changes to the substrate specificity of KSs provide an alternative-and, perhaps, more generalizable-approach that focuses on controlling the acyl-ACPs available for downstream products. This study combines mutants of FabF and FabB, the two elongating KSs of the E. coli FAS, with in vitro and in vivo analyses to explore the use of KS mutants to control fatty acid synthesis. In vitro, single amino acid substitutions in the gating loop and acyl binding pocket of FabF shifted the product profiles of reconstituted FASs toward short chains and showed that KS mutants, alone, can cause large shifts in average length (i.e., 6.5-13.5). FabB, which is essential for unsaturated fatty acid synthesis, blunted this effect in vivo, but exogenously added cis-vaccenic acid (C18:1) enabled sufficient transcriptional repression of FabB to restore it. Strikingly, a single mutant of FabB afforded titers of octanoic acid as high as those generated by an engineered thioesterase. Findings indicate that fatty acid synthesis must be decoupled from microbial growth to resolve the influence of KS mutants on fatty acid profiles but show that these mutants offer a versatile approach for tuning FAS outputs.

Genetically Encoded Detection of Biosynthetic Protease Inhibitors.

Proteases are an important class of drug targets that continue to drive inhibitor discovery. These enzymes are prone to resistance mutations, yet their promise for treating viral diseases and other disorders continues to grow. This study develops a general approach for detecting microbially synthesized protease inhibitors and uses it to screen terpenoid pathways for inhibitory compounds. The detection scheme relies on a bacterial two-hybrid (B2H) system that links protease inactivation to the transcription of a swappable reporter gene. This system, which can accomodate multiple biochemical outputs (i.e., luminescence and antibiotic resistance), permitted the facile incorporation of four disease-relevant proteases. A B2H designed to detect the inactivation of the main protease of severe acute respiratory syndrome coronavirus 2 enabled the identification of a terpenoid inhibitor of modest potency. An analysis of multiple pathways that make this terpenoid, however, suggested that its production was necessary but not sufficient to confer a survival advantage in growth-coupled assays. This finding highlights an important challenge associated with the use of genetic selection to search for inhibitors─notably, the influence of pathway toxicity─and underlines the value of including multiple pathways with overlapping product profiles in pathway screens. This study provides a detailed experimental framework for using microbes to screen libraries of biosynthetic pathways for targeted protease inhibitors.

Allosteric Inhibition of PTP1B by a Nonpolar Terpenoid.

Protein tyrosine phosphatases (PTPs) are promising drug targets for treating a wide range of diseases such as diabetes, cancer, and neurological disorders, but their conserved active sites have complicated the design of selective therapeutics. This study examines the allosteric inhibition of PTP1B by amorphadiene (AD), a terpenoid hydrocarbon that is an unusually selective inhibitor. Molecular dynamics (MD) simulations carried out in this study suggest that AD can stably sample multiple neighboring sites on the allosterically influential C-terminus of the catalytic domain. Binding to these sites requires a disordered α7 helix, which stabilizes the PTP1B-AD complex and may contribute to the selectivity of AD for PTP1B over TCPTP. Intriguingly, the binding mode of AD differs from that of the most well-studied allosteric inhibitor of PTP1B. Indeed, biophysical measurements and MD simulations indicate that the two molecules can bind simultaneously. Upon binding, both inhibitors destabilize the α7 helix by disrupting interactions at the α3-α7 interface and prevent the formation of hydrogen bonds that facilitate closure of the catalytically essential WPD loop. These findings indicate that AD is a promising scaffold for building allosteric inhibitors of PTP1B and illustrate, more broadly, how unfunctionalized terpenoids can engage in specific interactions with protein surfaces.

A kinetic framework for modeling oleochemical biosynthesis in Escherichia coli.

Microorganisms build fatty acids with biocatalytic assembly lines, or fatty acid synthases (FASs), that can be repurposed to produce a broad set of fuels and chemicals. Despite their versatility, the product profiles of FAS-based pathways are challenging to adjust without experimental iteration, and off-target products are common. This study uses a detailed kinetic model of the Escherichia coli FAS as a foundation to model nine oleochemical pathways. These models provide good fits to experimental data and help explain unexpected results from in vivo studies. An analysis of pathways for alkanes and fatty acid ethyl esters (FAEEs), for example, suggests that reductions in titer caused by enzyme overexpression-an experimentally consistent phenomenon-can result from shifts in metabolite pools that are incompatible with the substrate specificities of downstream enzymes, and a focused examination of multiple alcohol pathways indicates that coordinated shifts in enzyme concentrations provide a general means of tuning the product profiles of pathways with promiscuous components. The study concludes by integrating all models into a graphical user interface. The models supplied by this work provide a versatile kinetic framework for studying oleochemical pathways in different biochemical contexts.

Evolution-Guided Biosynthesis of Terpenoid Inhibitors.

Terpenoids, the largest and most structurally diverse group of natural products, include a striking variety of biologically active compounds, from flavors to medicines. Despite their well-documented biochemical versatility, the evolutionary processes that generate new functional terpenoids are poorly understood and difficult to recapitulate in engineered systems. This study uses a synthetic biochemical objective─a transcriptional system that links the inhibition of protein tyrosine phosphatase 1B (PTP1B), a human drug target, to the expression of a gene for antibiotic resistance in Escherichia coli (E. coli)─to evolve a terpene synthase to produce enzyme inhibitors. Site saturation mutagenesis of poorly conserved residues on γ-humulene synthase (GHS), a promicuous enzyme, yielded mutants that improved fitness (i.e., the antibiotic resistance of E. coli) by reducing GHS toxicity and/or by increasing inhibitor production. Intriguingly, a combination of two mutations enhanced the titer of a minority product─a terpene alcohol that inhibits PTP1B─by over 50-fold, and a comparison of similar mutants enabled the identification of a site where mutations permit efficient hydroxylation. Findings suggest that the plasticity of terpene synthases enables an efficient sampling of structurally distinct starting points for building new functional molecules and provide an experimental framework for exploiting this plasticity in activity-guided screens.

Kinetically guided, ratiometric tuning of fatty acid biosynthesis.

Cellular metabolism is a nonlinear reaction network in which dynamic shifts in enzyme concentration help regulate the flux of carbon to different products. Despite the apparent simplicity of these biochemical adjustments, their influence on metabolite biosynthesis tends to be context-dependent, difficult to predict, and challenging to exploit in metabolic engineering. This study combines a detailed kinetic model with a systematic set of in vitro and in vivo analyses to explore the use of enzyme concentration as a control parameter in fatty acid synthesis, an essential metabolic process with important applications in oleochemical production. Compositional analyses of a modeled and experimentally reconstituted fatty acid synthase (FAS) from Escherichia coli indicate that the concentration ratio of two native enzymes-a promiscuous thioesterase and a ketoacyl synthase-can tune the average length of fatty acids, an important design objective of engineered pathways. The influence of this ratio is sensitive to the concentrations of other FAS components, which can narrow or expand the range of accessible chain lengths. Inside the cell, simple changes in enzyme concentration can enhance product-specific titers by as much as 125-fold and elicit shifts in overall product profiles that rival those of thioesterase mutants. This work develops a kinetically guided approach for using ratiometric adjustments in enzyme concentration to control the product profiles of FAS systems and, broadly, provides a detailed framework for understanding how coordinated shifts in enzyme concentration can afford tight control over the outputs of nonlinear metabolic pathways.

Analysis of Three Architectures for Controlling PTP1B with Light.

Photosensory domains are powerful tools for placing proteins under optical control, but their integration into light-sensitive chimeras is often challenging. Many designs require structural iterations, and direct comparisons of alternative approaches are rare. This study uses protein tyrosine phosphatase 1B (PTP1B), an influential regulatory enzyme, to compare three architectures for controlling PTPs with light: a protein fusion, an insertion chimera, and a split construct. All three designs permitted optical control of PTP1B activity in vitro (i.e., kinetic assays of purified enzyme) and in mammalian cells; photoresponses measured under both conditions, while different in magnitude, were linearly correlated. The fusion- and insertion-based architectures exhibited the highest dynamic range and maintained native localization patterns in mammalian cells. A single insertion architecture enabled optical control of both PTP1B and TCPTP, but not SHP2, where the analogous chimera was active but not photoswitchable. Findings suggest that PTPs are highly tolerant of domain insertions and support the use of in vitro screens to evaluate different optogenetic designs.

Microbially Guided Discovery and Biosynthesis of Biologically Active Natural Products.

The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge-the inhibition of a human drug target-into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identified two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors appear to target an allosteric site, which confers selectivity, and can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded alternative PTP-specific detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely defined biochemical activities yet possess unanticipated structures and/or binding sites.

Optogenetic Analysis of Allosteric Control in Protein Tyrosine Phosphatases.

Allosteric regulation enables dynamic adjustments to protein function that permit tight control over cellular biochemistry. Discrepancies in the allosteric systems of related proteins can thus reveal important differences in their susceptibilities to influential stimuli (e.g., allosteric ligands, mutations, or post-translational modifications). This study uses an optogenetic actuator as a tool to compare the allosteric systems of two structurally related regulatory proteins: protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP). It begins with an interesting observation: The fusion of a protein light switch to the allosterically influential α7 helix of PTP1B permits optical modulation of its catalytic activity, but a similar fusion to TCPTP does not. A subsequent analysis of different PTP chimeras shows that replacing regions of TCPTP with homologous regions from PTP1B can enhance photocontrol; as TCPTP becomes more "PTP1B-like", its photosensitivity increases. Interestingly, the structural changes required for photocontrol also enhance the sensitivity of TCPTP to other allosteric inputs, notably, an allosteric inhibitor and a newly reported activating mutation. Our findings indicate that the allosteric functionality of the α7 helix of PTP1B is not conserved across the PTP family and highlight residues necessary to transfer this functionality to other PTPs. More broadly, our results suggest that simple gene fusion events can strengthen allosteric communication within individual protein domains and describe an intriguing application for optogenetic actuators as structural probes-a sort of physically disruptive "ratchet"-for studying protein allostery.

Optogenetic interrogation and control of cell signaling.

Signaling networks control the flow of information through biological systems and coordinate the chemical processes that constitute cellular life. Optogenetic actuators - genetically encoded proteins that undergo light-induced changes in activity or conformation - are useful tools for probing signaling networks over time and space. They have permitted detailed dissections of cellular proliferation, differentiation, motility, and death, and enabled the assembly of synthetic systems with applications in areas as diverse as photography, chemical synthesis, and medicine. In this review, we provide a brief introduction to optogenetic systems and describe their application to molecular-level analyses of cell signaling. Our discussion highlights important research achievements and speculates on future opportunities to exploit optogenetic systems in the study and assembly of complex biochemical networks.

A kinetic rationale for functional redundancy in fatty acid biosynthesis.

Cells build fatty acids with biocatalytic assembly lines in which a subset of enzymes often exhibit overlapping activities (e.g., two enzymes catalyze one or more identical reactions). Although the discrete enzymes that make up fatty acid pathways are well characterized, the importance of catalytic overlap between them is poorly understood. We developed a detailed kinetic model of the fatty acid synthase (FAS) of Escherichia coli and paired that model with a fully reconstituted in vitro system to examine the capabilities afforded by functional redundancy in fatty acid synthesis. The model captures-and helps explain-the effects of experimental perturbations to FAS systems and provides a powerful tool for guiding experimental investigations of fatty acid assembly. Compositional analyses carried out in silico and in vitro indicate that FASs with multiple partially redundant enzymes enable tighter (i.e., more independent and/or broader range) control of distinct biochemical objectives-the total production, unsaturated fraction, and average length of fatty acids-than FASs with only a single multifunctional version of each enzyme (i.e., one enzyme with the catalytic capabilities of two partially redundant enzymes). Maximal production of unsaturated fatty acids, for example, requires a second dehydratase that is not essential for their synthesis. This work provides a kinetic, control-theoretic rationale for the inclusion of partially redundant enzymes in fatty acid pathways and supplies a valuable framework for carrying out detailed studies of FAS kinetics.

Minimally disruptive optical control of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)-an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer-and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

Evolutionarily Conserved Allosteric Communication in Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are an important class of regulatory enzymes that exhibit aberrant activities in a wide range of diseases. A detailed mapping of allosteric communication in these enzymes could, thus, reveal the structural basis of physiologically relevant-and, perhaps, therapeutically informative-perturbations (i.e., mutations, post-translational modifications, or binding events) that influence their catalytic states. This study combines detailed biophysical studies of protein tyrosine phosphatase 1B (PTP1B) with bioinformatic analyses of the PTP family to examine allosteric communication in this class of enzymes. Results of X-ray crystallography, molecular dynamics simulations, and sequence-based statistical analyses indicate that PTP1B possesses a broadly distributed allosteric network that is evolutionarily conserved across the PTP family, and findings from both kinetic studies and mutational analyses show that this network is functionally intact in sequence-diverse PTPs. The allosteric network resolved in this study reveals new sites for targeting allosteric inhibitors of PTPs and helps explain the functional influence of a diverse set of disease-associated mutations.

Abietane-Type Diterpenoids Inhibit Protein Tyrosine Phosphatases by Stabilizing an Inactive Enzyme Conformation.

Protein tyrosine phosphatases (PTPs) contribute to a striking variety of human diseases, yet they remain vexingly difficult to inhibit with uncharged, cell-permeable molecules; no inhibitors of PTPs have been approved for clinical use. This study uses a broad set of biophysical analyses to evaluate the use of abietane-type diterpenoids, a biologically active class of phytometabolites with largely nonpolar structures, for the development of pharmaceutically relevant PTP inhibitors. Results of nuclear magnetic resonance analyses, mutational studies, and molecular dynamics simulations indicate that abietic acid can inhibit protein tyrosine phosphatase 1B, a negative regulator of insulin signaling and an elusive drug target, by binding to its active site in a non-substrate-like manner that stabilizes the catalytically essential WPD loop in an inactive conformation; detailed kinetic studies, in turn, show that minor changes in the structures of abietane-type diterpenoids (e.g., the addition of hydrogens) can improve potency (i.e., lower IC50) by 7-fold. These findings elucidate a previously uncharacterized mechanism of diterpenoid-mediated inhibition and suggest, more broadly, that abietane-type diterpenoids are a promising source of structurally diverse-and, intriguingly, microbially synthesizable-molecules on which to base the design of new PTP-inhibiting therapeutics.

The Molecular Origin of Enthalpy/Entropy Compensation in Biomolecular Recognition.

Biomolecular recognition can be stubborn; changes in the structures of associating molecules, or the environments in which they associate, often yield compensating changes in enthalpies and entropies of binding and no net change in affinities. This phenomenon-termed enthalpy/entropy (H/S) compensation-hinders efforts in biomolecular design, and its incidence-often a surprise to experimentalists-makes interactions between biomolecules difficult to predict. Although characterizing H/S compensation requires experimental care, it is unquestionably a real phenomenon that has, from an engineering perspective, useful physical origins. Studying H/S compensation can help illuminate the still-murky roles of water and dynamics in biomolecular recognition and self-assembly. This review summarizes known sources of H/ S compensation (real and perceived) and lays out a conceptual framework for understanding and dissecting-and, perhaps, avoiding or exploiting-this phenomenon in biophysical systems.

Analysis of Interdependent Kinetic Controls of Fatty Acid Synthases.

Biocatalytic systems (e.g., multienzyme pathways or complexes) enable the conversion of simple sugars into complex products under ambient conditions and, thus, represent promising platforms for the synthesis of renewable fuels and chemicals. Unfortunately, to date, many of these systems have proven difficult to engineer without a detailed understanding of the kinetic relationships that regulate the concerted action of their constituent enzymes. This study develops a mechanistic kinetic model of the fatty acid synthase (FAS) of Escherichia coli and uses that model to determine how different FAS components work together to control the production of free fatty acids-precursors to a wide range of oleochemicals. Perturbational analyses indicate that the modification or overexpression of a single FAS component can depress fatty acid production (a commonly observed phenomenon) by sequestering the proteins with which it interacts and/or by depleting common substrate pools. Compositional studies, in turn, suggest that simple changes in the ratios of FAS components can alter the average length of fatty acids but show that specialized enzymes (i.e., highly specific ketoacyl synthases or thioesterases) are required for narrow product profiles. Intriguingly, a sensitivity analysis indicates that two components primarily influence-and, thus, enable fine control over-total production, but suggests that the enzymes that regulate product profile are more broadly influential. Findings thus reveal the general importance of kinetic considerations in efforts to engineer fatty acid biosynthesis and provide strategies-and a kinetic model-for incorporating those considerations into FAS designs.