Mechanism Of Allosteric Regulation Research Articles

The Speed of Allosteric Signaling Within a Single-Domain Protein.

While much is known about different allosteric regulation mechanisms, the nature of the allosteric signal and the time scale on which it propagates remains elusive. The PDZ3 domain from postsynaptic density-95 protein is a small protein domain with a terminal third α-helix, i.e., the α3-helix, which is known to be allosterically active. By cross-linking the allosteric helix with an azobenzene moiety, we obtained a photocontrollable PDZ3 variant. Photoswitching triggers its allosteric transition, resulting in a change in binding affinity of a peptide to the remote binding pocket. Using time-resolved infrared and UV/vis spectroscopy, we follow the allosteric signal transduction and reconstruct the timeline in which the allosteric signal propagates through the protein within 200 ns.

Slow Protein Turnover Explains Limited Protein-Level Response to Diurnal Transcriptional Oscillations in Cyanobacteria.

Metabolically engineered cyanobacteria have the potential to mitigate anthropogenic CO2 emissions by converting CO2 into renewable fuels and chemicals. Yet, better understanding of metabolic regulation in cyanobacteria is required to develop more productive strains that can make industrial scale-up economically feasible. The aim of this study was to find the cause for the previously reported inconsistency between oscillating transcription and constant protein levels under day-night growth conditions. To determine whether translational regulation counteracts transcriptional changes, Synechocystis sp. PCC 6803 was cultivated in an artificial day-night setting and the level of transcription, translation and protein was measured across the genome at different time points using mRNA sequencing, ribosome profiling and quantitative proteomics. Furthermore, the effect of protein turnover on the amplitude of protein oscillations was investigated through in silico simulations using a protein mass balance model. Our experimental analysis revealed that protein oscillations were not dampened by translational regulation, as evidenced by high correlation between translational and transcriptional oscillations (r = 0.88) and unchanged protein levels. Instead, model simulations showed that these observations can be attributed to a slow protein turnover, which reduces the effect of protein synthesis oscillations on the protein level. In conclusion, these results suggest that cyanobacteria have evolved to govern diurnal metabolic shifts through allosteric regulatory mechanisms in order to avoid the energy burden of replacing the proteome on a daily basis. Identification and manipulation of such mechanisms could be part of a metabolic engineering strategy for overproduction of chemicals.

Decoding allosteric regulation by the acyl carrier protein

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522-542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., Chem. Biol. 17, 776-785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein-protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528-547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein-protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

Allosteric regulation of histone lysine methyltransferases: from context-specific regulation to selective drugs.

Histone lysine methyltransferases (HKMTs) are key regulators of many cellular processes. By definition, HKMTs catalyse the methylation of lysine residues in histone proteins. The enzymatic activities of HKMTs are under precise control, with their allosteric regulation emerging as a prevalent paradigm. We review the molecular mechanisms of allosteric regulation of HKMTs using well-studied histone H3 (K4, K9, K27 and K36) methyltransferases as examples. We discuss the current advances and future potential in targeting allosteric sites of HKMTs for drug development.

Exploring the Distinct Binding and Activation Mechanisms for Different CagA Oncoproteins and SHP2 by Molecular Dynamics Simulations.

CagA is a major virulence factor of Helicobacter pylori. H. pylori CagA is geographically subclassified into East Asian CagA and Western CagA, which are characterized by the presence of a EPIYA-D or EPIYA-C segment. The East Asian CagA is more closely associated with gastric cancer than the Western CagA. In this study, molecular dynamic (MD) simulations were performed to investigate the binding details of SHP2 and EPIYA segments, and to explore the allosteric regulation mechanism of SHP2. Our results show that the EPIYA-D has a stronger binding affinity to the N-SH2 domain of SHP2 than EPIYA-C. In addition, a single EPIYA-D binding to N-SH2 domain of SHP2 can cause a deflection of the key helix B, and the deflected helix B could squeeze the N-SH2 and PTP domains to break the autoinhibition pocket of SHP2. However, a single EPIYA-C binding to the N-SH2 domain of SHP2 cannot break the autoinhibition of SHP2 because the secondary structure of the key helix B is destroyed. However, the tandem EPIYA-C not only increases its binding affinity to SHP2, but also does not significantly break the secondary structure of the key helix B. Our study can help us better understand the mechanism of gastric cancer caused by Helicobacter pylori infection.

Dynamic Network Modeling of Allosteric Interactions and Communication Pathways in the SARS-CoV-2 Spike Trimer Mutants: Differential Modulation of Conformational Landscapes and Signal Transmission via Cascades of Regulatory Switches.

The rapidly growing body of structural and biochemical studies of the SARS-CoV-2 spike glycoprotein has revealed a variety of distinct functional states with radically different arrangements of the receptor-binding domain, highlighting a remarkable function-driven conformational plasticity and adaptability of the spike proteins. In this study, we examined molecular mechanisms underlying conformational and dynamic changes in the SARS-CoV-2 spike mutant trimers through the lens of dynamic analysis of allosteric interaction networks and atomistic modeling of signal transmission. Using an integrated approach that combined coarse-grained molecular simulations, protein stability analysis, and perturbation-based modeling of residue interaction networks, we examined how mutations in the regulatory regions of the SARS-CoV-2 spike protein can differentially affect dynamics and allosteric signaling in distinct functional states. The results of this study revealed key functional regions and regulatory centers that govern collective dynamics, allosteric interactions, and control signal transmission in the SARS-CoV-2 spike proteins. We found that the experimentally confirmed regulatory hotspots that dictate dynamic switching between conformational states of the SARS-CoV-2 spike protein correspond to the key hinge sites and global mediating centers of the allosteric interaction networks. The results of this study provide a novel insight into allosteric regulatory mechanisms of SARS-CoV-2 spike proteins showing that mutations at the key regulatory positions can differentially modulate distribution of states and determine topography of signal communication pathways operating through state-specific cascades of control switch points. This analysis provides a plausible strategy for allosteric probing of the conformational equilibrium and therapeutic intervention by targeting specific hotspots of allosteric interactions and communications in the SARS-CoV-2 spike proteins.

Structural basis for allosteric regulation of pyruvate kinase M2 by phosphorylation and acetylation

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pHi) from ∼7.1 (in normal cells) to ∼7.5 (in cancer cells). Biochemical studies of WT PKM2 (wtPKM2) and the two mimetic variants demonstrated that the activity decreases as the pH is increased from 7.0 to 8.0, and wtPKM2 is optimally active and amenable to FBP-mediated allosteric regulation at pHi 7.5. However, the PTM mimetics exist as a mixture of tetramer and dimer, indicating that physiologically dimeric fraction is important and might be necessary for the modified PKM2 to translocate into the nucleus. Thus, our findings provide insight into how PTMs and pH regulate PKM2 and offer a broader understanding of its intricate allosteric regulation mechanism by phosphorylation or acetylation.

Exploring the Allosteric Mechanism of Src Homology-2 Domain-Containing Protein Tyrosine Phosphatase 2 (SHP2) by Molecular Dynamics Simulations.

The Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2, encoded by PTPN11) is a critical allosteric phosphatase for many signaling pathways. Programmed cell death 1 (PD-1) could be phosphorylated at its immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and can bind to SHP2 to initiate T cell inactivation. Although the interaction of SHP2-PD-1 plays an important role in the immune process, the complex structure and the allosteric regulation mechanism remain unknown. In this study, molecular dynamics (MD) simulations were performed to study the binding details of SHP2 and PD-1, and explore the allosteric regulation mechanism of SHP2. The results show that ITIM has a preference to bind to the N-SH2 domain and ITSM has almost the same binding affinity to the N-SH2 and C-SH2 domain. Only when ITIM binds to the N-SH2 domain and ITSM binds to the C-SH2 domain can the full activation of SHP2 be obtained. The binding of ITIM and ITSM could change the motion mode of SHP2 and switch it to the activated state.

A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

Allosteric Regulation at the Crossroads of New Technologies: Multiscale Modeling, Networks, and Machine Learning.

Allosteric regulation is a common mechanism employed by complex biomolecular systems for regulation of activity and adaptability in the cellular environment, serving as an effective molecular tool for cellular communication. As an intrinsic but elusive property, allostery is a ubiquitous phenomenon where binding or disturbing of a distal site in a protein can functionally control its activity and is considered as the “second secret of life.” The fundamental biological importance and complexity of these processes require a multi-faceted platform of synergistically integrated approaches for prediction and characterization of allosteric functional states, atomistic reconstruction of allosteric regulatory mechanisms and discovery of allosteric modulators. The unifying theme and overarching goal of allosteric regulation studies in recent years have been integration between emerging experiment and computational approaches and technologies to advance quantitative characterization of allosteric mechanisms in proteins. Despite significant advances, the quantitative characterization and reliable prediction of functional allosteric states, interactions, and mechanisms continue to present highly challenging problems in the field. In this review, we discuss simulation-based multiscale approaches, experiment-informed Markovian models, and network modeling of allostery and information-theoretical approaches that can describe the thermodynamics and hierarchy allosteric states and the molecular basis of allosteric mechanisms. The wealth of structural and functional information along with diversity and complexity of allosteric mechanisms in therapeutically important protein families have provided a well-suited platform for development of data-driven research strategies. Data-centric integration of chemistry, biology and computer science using artificial intelligence technologies has gained a significant momentum and at the forefront of many cross-disciplinary efforts. We discuss new developments in the machine learning field and the emergence of deep learning and deep reinforcement learning applications in modeling of molecular mechanisms and allosteric proteins. The experiment-guided integrated approaches empowered by recent advances in multiscale modeling, network science, and machine learning can lead to more reliable prediction of allosteric regulatory mechanisms and discovery of allosteric modulators for therapeutically important protein targets.

Allosteric Mechanism of the Hsp90 Chaperone Interactions with Cochaperones and Client Proteins by Modulating Communication Spines of Coupled Regulatory Switches: Integrative Atomistic Modeling of Hsp90 Signaling in Dynamic Interaction Networks.

Conformational landscapes of the Hsp90 chaperones have revealed that the intrinsic plasticity and functional adaptability of these molecular chaperones to a large cohort of cochaperones and a vast protein clientele can be regulated by a number of single switch points broadly dispersed in the chaperone structure. A detailed dynamic view of the allosteric changes mediated by conformational switches and the mechanism of their coupling during the ATPase cycle remains poorly understood and presents an important area of investigation. In this work, we employed integrative computational modeling that included evolutionary and coevolutionary analyses, experiment-guided protein docking and structure modeling, molecular simulations, energetic analysis, and network modeling to perform a systematic characterization of molecular and network signatures of conformational switch points and dissect their allosteric cross-talk in the Hsp90 complexes with cochaperones and client proteins. Using a hierarchical modeling of dynamic interaction networks, we show that the allosteric regulation of the Hsp90 interactions with p23 and Aha1 cochaperones and p53 client protein may be determined by the intramolecular communication "spines" of spatially separated and allosterically coupled regulatory switches. Using a battery of computational approaches, we examined how p23, Aha1, and p53 proteins can modulate signal transmission in the Hsp90 by exploiting communication spines of regulatory switches in the global allosteric network. This study proposes a community-chain mechanism of allosteric coupling between conformational switch centers and identifies key regulatory control points that mediate long-range interactions and communications in the Hsp90 chaperone. The results of this investigation provide novel insights into the nature of allosteric regulation mechanisms in the Hsp90 chaperones and offer a simple mechanistic model of the Hsp90 communications and adaptation to binding partners during the functional cycle.

Structural Determinants of Phosphopeptide Binding to the N-Terminal Src Homology 2 Domain of the SHP2 Phosphatase.

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11, plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in PTPN11 are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. The SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics (MD) simulations of the N-SH2 domain complexed to 12 different peptides to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position −2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3, and +5 in an apolar groove of the domain and interaction of residue −2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues −1, +1, +2, and +4. Finally, negatively charged residues at positions +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific for the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core β sheet and the loop that closes the pY binding pocket.

Mechanistic insights explain the transforming potential of the T507K substitution in the protein-tyrosine phosphatase SHP2

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

Multimeric Proteins that Can Come Apart, Change Shape, and Reassemble Differently with Functional Consequences ‐ Morpheeins

Herein we describe progress in a class of shape‐shifting proteins originally called morpheeins. These are homomultimers that can dissociate, change conformation in the dissociated state, and reassemble into a structurally and functionally distinct multimer. The original observation was of an octamer/dimer/dimer*/hexamer equilibrium wherein the conformational change was a hinge motion between folded domains of porphobilinogen synthase (PBGS). This phenomenon is established as a mechanism for allosteric regulation, druggable, and the structural basis for an inborn error of metabolism.Many aspects of the original observations in the early 2000s were unexpected and peculiar; suggesting a structural metastability. At the time, vocabulary to describe such multi‐structural and multi‐functional protein dynamics were limited. We defined morpheeins as (homo‐multimeric) proteins that can come apart, change shape and reassemble differently with functional consequences. A Wikipedia page introduced a dice model of a morpheein (see image), and showed how multimer‐specific surface cavities can provide a general mechanism for drug action. In the beginning, multimeric proteins with more than one shape (in the absence of refolding) strongly defied the one sequence, one structure, one function paradigm. Two decades later, there are a growing number of examples of multimeric proteins with multiple structures and other proteins with multiple functions, many in the absence of any chemical modification or significant refolding.The quaternary structure dynamics of PBGS have now been extensively described. Interestingly, one consequence of our focus on PBGS is that the literature began equated the specific properties of PBGS with all of the factors that must be in place for a protein to be classified as a morpheein. This is akin to saying that in order to be considered a mammal, an organism must have all of the characteristics of a rat. Herein, we set straight the definition of a morpheein; it is a protein that can come apart, change conformation in the dissociated state, and reassemble to a structurally and functionally distinct multimer. Alternate assemblies need not have different stoichiometries. Assembly interchange need not be triggered by ligand binding or catalysis. Functional difference need not be high activity and low activity (as in allosteric regulation of enzyme action), but can be moonlighting activities. Often the conformational change is a repositioning of folded domains relative to each other.An excellent example of a moonlighting protein that appears to be a morpheein, and has also been called a transformer, is the VP40 protein of the Ebola virus. It too exists as dimers or hexamer or octamer and each alternate assembly is responsible for an alternate function in the viral life cycle. The individual assemblies are stabilized through interaction with a “third party”. A third likely example is the family of alternate assemblies of ribonucleotide reductase, whose timely interchange amongst alternate assemblies appears essential to the regulation of nucleotide pools. A fourth example is HIV integrase, whose three domains reposition relative to each other as part of viral integration. These examples will be illustrated.Support or Funding InformationNIH grants 5R01 NS100081 and P30 CA006927Dice image depicting an interchange of morpheein forms and a ligand that stabilizes one form.Figure 1

Allosteric Modulation of SIRT1 Activity Affects Substrate Recognition and Protein Conformation

SIRT1 is a lysine deacetylase that plays an important role in regulating various cellular pathways such as neurodegeneration and insulin secretion. As such, it is of great interest to modulate SIRT1 activity as a therapeutic approach for diseases such as Alzheimer’s disease and diabetes. Several small‐molecule allosteric regulators of SIRT1, known as STACs (Sirtuin Activating Compounds) are studied, such as resveratrol. It is known that these regulators bind to the STAC Binding Domain (SBD) of SIRT1, distal from the enzyme active site, and regulates SIRT1 activity through allosteric effects. However, the molecular mechanism of these allosteric effects is not well understood, impeding more specific designs of SIRT1 modulator compounds. In this study we take a systematic approach to study the changes in Michaelis‐Menten kinetics and overall conformation of SIRT1 when its activity is allosterically decreased and increased. A previous end‐point screen has found that the effect of resveratrol on SIRT1 is substrate‐sequence dependent, where resveratrol can increase, decrease, or have no effect on the activity of SIRT1 against different subsets of peptide substrates. We have chosen several peptides from each of the above‐mentioned categories and determined the Michaelis‐Menten kinetics of SIRT1 against these substrates with and without resveratrol. Upon comparison, we find that the allosteric effect of resveratrol mainly changes the substrate recognition (KM) of SIRT1 for these peptides. In addition, we used Small Angle X‐Ray Scattering (SAXS) to monitor the overall conformation of SIRT1 in complex with the different peptide substrates with and without resveratrol and found that the protein conformation is altered upon addition of resveratrol. Combining these findings, we have a better understanding of the mechanism for allosteric regulation of SIRT1, which could shed light on other allosteric regulations in the cell and aide in guided design of small molecule SIRT1 modulators.Support or Funding InformationNational Institutes of Health NIGMS SC2GM132000‐01

Defining the Regulatory Roles of Glucan Phosphatases in Transitory Starch Degradation

Plants generate starch during the day and metabolize it at night to store and use for cellular energy. The glucan phosphatase Starch EXcess4 (SEX4) plays a crucial role in transitory starch degradation via its involvement in the reversible starch phosphorylation mechanism. Previous structural studies have shown that SEX4 preferentially binds to and dephosphorylates starch at the C6 position of glucosyl residues, but there remains little data on how SEX4 navigates and interacts with the complex starch granule. Further, enzyme kinetics and regulatory mechanisms of SEX4 are currently not well understood. We employed both generic and substrate specific phosphatase assays to determine kinetics and substrate binding mechanisms of SEX4. Upon use of a generic phosphatase substrate, p‐nitro phenyl phosphate (p‐NPP), SEX4 followed Michaelis‐Menten kinetics. A sigmoidal curve observed for the physiological substrate amylopectin suggests a non‐ Michaelis‐Menten behavior for SEX4 and provides experimental evidence for a possible allosteric regulatory mechanism of SEX4. The enhanced dephosphorylation rates obtained for generic phosphatase assays with SEX4 preincubated with different domains of starch structure strongly suggest a conformational change within the active site of SEX4 upon glucan binding. Collectively, our finding suggests a novel posttranslational regulatory mechanism of SEX4 that is necessary for the regulation of transitory starch degradation at night.

A combined experimental and modelling approach for the Weimberg pathway optimisation

The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.

Structure and function of crocodilian hemoglobins and allosteric regulation by chloride, ATP, and CO2.

Hemoglobins (Hbs) of crocodilians are reportedly characterized by unique mechanisms of allosteric regulatory control, but there are conflicting reports regarding the importance of different effectors, such as chloride ions, organic phosphates, and CO2. Progress in understanding the unusual properties of crocodilian Hbs has also been hindered by a dearth of structural information. Here, we present the first comparative analysis of blood properties and Hb structure and function in a phylogenetically diverse set of crocodilian species. We examine mechanisms of allosteric regulation in the Hbs of 13 crocodilian species belonging to the families Crocodylidae and Alligatoridae. We also report new amino acid sequences for the α- and β-globins of these taxa, which, in combination with structural analyses, provide insights into molecular mechanisms of allosteric regulation. All crocodilian Hbs exhibited a remarkably strong sensitivity to CO2, which would permit effective O2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O2-affinities, there is considerable variation in sensitivity to Cl- ions and ATP, which appears to be at least partly attributable to variation in the extent of NH2-terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs.

An Allosteric Gating Mechanism of TMEM16A Calcium-Activated Chloride Channel

The Ca2+-activated chloride channel (CaCC) TMEM16A is widely expressed throughout the body with diverse functions ranging from trans-epithelial fluid transport, smooth muscle contraction, to nociception. Activation of TMEM16A requires binding of intracellular Ca2+ whereas binding of the phosphatidylinositol PIP2 facilitates channel opening. Assembled as a homodimer, each TMEM16A monomer is consisted of ten transmembrane (TM) segments in which TMs 3 to 8 form an independent chloride-permeable pore. Owing to their direct contributions to the permeation pathway, TMs 3 to 8 have been extensively studied. Our recent work suggests that TMs 3-8 comprise of two functionally distinct modules in which TMs 6-8 constitute the Ca2+-binding module that controls Ca2+-dependent channel activation while TMs 3-5 form a regulatory module that mediates PIP2-dependent modulation of channel opening. However, it remains unknown whether the remaining TMs, namely TMs 1, 2, 9, and 10, all of which are distant from the permeation pathway, also contribute to channel gating. Here we combine electrophysiological analyses with mutagenesis to demonstrate that TM2 and TM10 interaction allosterically controls TMEM16A gating. These studies not only provide the molecular underpinnings of allosteric regulatory mechanism in TMEM16A but will also facilitate our mechanistic understanding of the gating mechanism in TMEM16 Ca2+-activated ion channels and lipid scramblases.

Computational Modeling and Engineering of Allosteric Regulatory Mechanisms in Signaling Proteins: Integration of Multiscale Simulations, Network Biology and Machine Learning

The allosteric interactions and regulation of molecular chaperones and protein kinases allow for molecular communication and event coupling in signal transduction networks. We report the results of integrative systems biology studies of the Hsp90 chaperone and protein kinases with an atomic level analysis of the communication pathways regulating conformational equilibrium of theses protein systems in signaling networks. Biophysical modeling of allosteric regulation in the protein kinases has offered additional insights into organizing principles of kinase activation by molecular chaperones that may be orchestrated by a cross-talk between key regulatory regions. The results of biophysical and computational systems biology analyses combined with proteomics experiments have been integrated into a graph-based network model of allosteric regulation. The evolution of protein structure networks in molecular chaperones and protein kinases during allosteric activation has revealed the increased cooperativity reflecting a preferential attachment of allosterically interacting functional sites. Among our primary findings is the emerging evidence that a small number of functional motifs may be utilized by the chaperone and protein kinases to act collectively as central regulators of the intermolecular communications, ATP hydrolysis, and protein client binding in signaling networks. Integration of computational systems biology and machine learning analysis of the Hsp90 interactions with oncogenic kinase mutants is then used to construct models of allosteric regulation of oncogenic proteins by molecular chaperones in signaling cascades. A computational synthetic biology framework is proposed for design and re-engineering signal transduction networks and pathways that involve cross-talk between molecular chaperones and protein kinase clients. We have also analyzed mechanisms by which kinase drugs and allosteric Hsp90 modulators can act synergistically and exert their pharmacological effect by depriving the client kinase of access to the molecular chaperone.