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Poly(ethylene terephthalate) (PET) is the most common polyester plastic in the packaging industry and a major source of environmental pollution due to its single use. Several enzymes, termed PET hydrolases, have been found to hydrolyze this polymer at different temperatures, with the enzyme from Ideonella sakaiensis (IsPETase) having optimal catalytic activity at 30-35 degrees C. Crystal structures of IsPETase have revealed that the side chain of a conserved tryptophan residue within an active site loop (W185) shifts between three conformations to enable substrate binding and product release. This is facilitated by two residues unique to IsPETase, S214 and I218. When these residues are inserted into other PET hydrolases in place of the otherwise strictly conserved histidine and phenylalanine residues found at their respective positions, they enhance activity and decrease Topt. Herein, we combine molecular dynamics and well-tempered metadynamics simulations to investigate dynamic changes of the S214/I218 and H214/F218 variants of IsPETase, as well as three other mesophilic and thermophilic PET hydrolases, at their respective temperature and pH optima. Our simulations show that the S214/I218 insertion both increases the flexibility of active site loop regions harboring key catalytic residues and the conserved tryptophan and expands the conformational plasticity of this tryptophan side chain, enabling the conformational transitions that allow for substrate binding and product release in IsPETase. The observed catalytic enhancement caused by this substitution in other PET hydrolases appears to be due to conformational selection, by capturing the conformational ensemble observed in IsPETase.

Transcription factors are multidomain proteins with specific DNA binding and regulatory domains. In the human FoxP subfamily (FoxP1, FoxP2, FoxP3, and FoxP4) of transcription factors, a 90 residue-long disordered region links a Leucine Zipper (ZIP)—known to form coiled-coil dimers—and a Forkhead (FKH) domain—known to form domain swapping dimers. We used replica exchange discrete molecular dynamics simulations, single-molecule fluorescence experiments, and other biophysical tools to understand how domain tethering in FoxP1 impacts dimerization at ZIP and FKH domains and how DNA binding allosterically regulates their dimerization. We found that domain tethering promotes FoxP1 dimerization but inhibits a FKH domain-swapped structure. Furthermore, our findings indicate that the linker mediates the mutual organization and dynamics of ZIP and FKH domains, forming closed and open states with and without interdomain contacts, thus highlighting the role of the linkers in multidomain proteins. Finally, we found that DNA allosterically promotes structural changes that decrease the dimerization propensity of FoxP1. We postulate that, upon DNA binding, the interdomain linker plays a crucial role in the gene regulatory function of FoxP1.

In every domain of life, NusG-like proteins bind to the elongating RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes. Structures of factor-bound transcription elongation complexes (TECs) reveal similar contacts to RNAP, consistent with a shared mechanism of action. However, NusG homologs differ in their regulatory roles, modes of recruitment, and effects on RNA synthesis. Some of these differences could be due to conformational changes in RNAP and NusG-like proteins, which cannot be captured in static structures. Here, we employed hydrogen-deuterium exchange mass spectrometry to investigate changes in local and non-local structural dynamics of Escherichia coli NusG and its paralog RfaH, which have opposite effects on expression of xenogenes, upon binding to TEC. We found that NusG and RfaH regions that bind RNAP became solvent-protected in factor-bound TECs, whereas RNAP regions that interact with both factors showed opposite deuterium uptake changes when bound to NusG or RfaH. Additional changes far from the factor-binding site were observed only with RfaH. Our results provide insights into differences in structural dynamics exerted by NusG and RfaH during binding to TEC, which may explain their different functional outcomes and allosteric regulation of transcriptional pausing by RfaH.

Cyanobacteria possesses the simplest circadian clock, composed of three proteins that act as a phosphorylation oscillator: KaiA, KaiB, and KaiC. The timing of this oscillator is determined by the fold-switch of KaiB, a structural rearrangement of its C-terminal half that is accompanied by a change in the oligomerization state. During the day, KaiB forms a stable tetramer (gsKaiB), whereas it adopts a monomeric thioredoxin-like fold during the night (fsKaiB). Although the structures and functions of both native states are well studied, little is known about the sequence and structure determinants that control their structural interconversion. Here, we used confinement molecular dynamics (CCR-MD) and folding simulations using structure-based models to show that the dissociation of the gsKaiB dimer is a key energetic event for the fold-switch. Hydrogen-deuterium exchange mass spectrometry (HDXMS) recapitulates the local stability of protein regions reported by CCR-MD, with both approaches consistently indicating that the energy and backbone flexibility changes are solely associated with the region that fold-switches between gsKaiB and fsKaiB and that the localized regions that differentially stabilize gsKaiB also involve regions outside the dimer interface. Moreover, two mutants (R23C and R75C) previously reported to be relevant for altering the rhythmicity of the Kai clock were also studied by HDXMS. Particularly, R75C populates dimeric and monomeric states with a deuterium incorporation profile comparable to the one observed for fsKaiB, emphasizing the importance of the oligomerization state of KaiB for the fold-switch. These findings suggest that the information necessary to control the rhythmicity of the cyanobacterial biological clock is, to a great extent, encoded within the KaiB sequence.

The fields of click chemistry and biocatalysis have rapidly grown over the last two decades. The development of robust and active biocatalysts and the widespread use of straightforward click reactions led to significant interactions between these two fields. Therefore the name bio-click chemistry seems to be an accurate definition of chemoenzymatic reactions cooperating with click transformations. Bio-click chemistry can be understood as the approach towards molecules of high-value using a green and sustainable approach by exploiting the potential of biocatalytic enzyme activity combined with the reliable nature of click reactions. This review summarizes the principal bio-click chemistry reactions reported over the last two decades, with a special emphasis on small molecules. Contributions to the field of bio-click chemistry are manifold, but the synthesis of chiral molecules with applications in medicinal chemistry and sustainable syntheses will be especially highlighted.

The NusG protein family is structurally and functionally conserved in all domains of life. Its members directly bind RNA polymerases and regulate transcription processivity and termination. RfaH, a divergent sub‐family in its evolutionary history, is known for displaying distinct features than those in NusG proteins, which allows them to regulate the expression of virulence factors in enterobacteria in a DNA sequence‐dependent manner. A striking feature is its structural interconversion between an active fold, which is the canonical NusG three‐dimensional structure, and an autoinhibited fold, which is distinctively novel. How this novel fold is encoded within RfaH sequence to encode a metamorphic protein remains elusive. In this work, we used publicly available genomic RfaH protein sequences to construct a complete multiple sequence alignment, which was further augmented with metagenomic sequences and curated by predicting their secondary structure propensities using JPred. Coevolving pairs of residues were calculated from these sequences using plmDCA and GREMLIN, which allowed us to detect the enrichment of key metamorphic contacts after sequence filtering. Finally, we combined our coevolutionary predictions with molecular dynamics to demonstrate that these interactions are sufficient to predict the structures of both native folds, where coevolutionary‐derived non‐native contacts may play a key role in achieving the compact RfaH novel fold. All in all, emergent coevolutionary signals found within RfaH sequences encode the autoinhibited and active folds of this protein, shedding light on the key interactions responsible for the action of this metamorphic protein.

Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDECYT Fondecyt 1150460 1191321 Beca Doctoral Conicyt (CONICYT PhD Fellowships) 21120436 21151101 United States Department of Health & Human Services National Institutes of Health (NIH) - USA S10 OD016234

Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2. ispartof: AUTOPHAGY vol:17 issue:7 pages:1714-1728 ispartof: location:United States status: published

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60°C to 70°C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25°C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modelling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases. IMPORTANCE A myriad of consumer products contains polyethylene terephthalate (PET), a plastic that has accumulated as waste in the environment due to its long-term stability and poor waste management. One promising solution is the enzymatic biodegradation of PET, with most known enzymes only catalyzing this process at high temperatures. Here, we bioinformatically identified and biochemically characterized an enzyme from an Antarctic organism that degrades PET at 25°C with similar efficiency than the few PET-degrading enzymes active at moderate temperatures. Reasoning that Antarctica harbors other PET-degrading enzymes, we analyzed available data from Antarctic metagenomic samples and successfully identified other potential enzymes. Our findings contribute to increasing the repertoire of known PET-degrading enzymes that are currently being considered as biocatalysts for the biological recycling of plastic waste.

Indigoids are natural pigments obtained from plants by ancient cultures. Romans used them mainly as dyes, whereas Asian cultures applied these compounds as treatment agents for several diseases. In the modern era, the chemical industry has made it possible to identify and develop synthetic routes to obtain them from petroleum derivatives. However, these processes require high temperatures and pressures and large amounts of solvents, acids, and alkali agents. Thus, enzyme engineering and the development of bacteria as whole-cell biocatalysts emerges as a promising green alternative to avoid the use of these hazardous materials and consequently prevent toxic waste generation. In this research, we obtained two novel variants of phenylacetone monooxygenase (PAMO) by iterative saturation mutagenesis. Heterologous expression of these two enzymes, called PAMOHPCD and PAMOHPED, in E. coli was serendipitously found to produce indigoids. These interesting results encourage us to characterize the thermal stability and enzyme kinetics of these new variants and to evaluate indigo and indirubin production in a whole-cell system by HPLC. The highest yields were obtained with PAMOHPCD supplemented with L-tryptophan, producing ~3000 mg/L indigo and ~130.0 mg/L indirubin. Additionally, both enzymes could oxidize and produce several indigo derivatives from substituted indoles, with PAMOHPCD being able to produce the well-known Tyrian purple. Our results indicate that the PAMO variants described herein have potential application in the textile, pharmaceutics, and semiconductors industries, prompting the use of environmentally friendly strategies to obtain a diverse variety of indigoids.

RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) has gained popularity for the detection of SARS-CoV-2. The high specificity, sensitivity, simple protocols and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents and their distribution under cold chain shipping limits RT-LAMP’s applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus (M-MLV) Reverse Transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs, and thus we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) stalled at a DNA signal known as ops. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the ops exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD.Although RfaH transformation has been extensively characterized computationally, most studies employ tertiary biases towards each native state, hampering the analysis of sequence-encoded interactions on fold-switching. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and also by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.

ChemSusChem xx, 1-11 (2021). doi:10.1002/cssc.202101062, Our planet is flooded with plastics and the need for sustainable recycling strategies of polymers has become increasingly urgent. Enzyme-based hydrolysis of post-consumer plastic is an emerging strategy for closed-loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7 isolated from a compost metagenome completely hydrolyzed amorphous PET films, releasing 91 mg of terephthalic acid per hour and mg of enzyme. Degradation rates of the PET film of 6.8 ��m h -1 were monitored by vertical scanning interferometry. Structural analysis indicated the importance of leucine at position 210 for the extraordinarily high PET-hydrolyzing activity of PHL7. Within 24 h, 0.6 mg enzyme g PET -1 completely degraded post-consumer thermoform PET packaging in an aqueous buffer at 70��C without any energy-intensive pretreatments. Terephthalic acid recovered from the enzymatic hydrolysate was used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint., Published by Wiley-VCH, Weinheim

Forkhead box P (FoxP) proteins are unique transcription factors that spatiotemporally regulate gene expression by tethering two chromosome loci together via functional domain-swapped dimers formed through their DNA-binding domains. Further, the differential kinetics on this dimerization mechanism underlie an intricate gene regulation network at physiological conditions. Nonetheless, poor understanding of the structural dynamics and steps of the association process impedes to link the functional domain swapping to human-associated diseases. Here, we have characterized the DNA-binding domain of human FoxP1 by integrating single-molecule Förster Resonance Energy Transfer (FRET) and Hydrogen-Deuterium Exchange coupled with Mass Spectrometry (HDXMS) data with Molecular Dynamics (MD) simulations. Our results confirm the formation of a previously postulated domain-swapped (DS) FoxP1 dimer in solution and reveal the presence of highly populated, heterogeneous, and locally disordered dimeric intermediates along the dimer dissociation pathway. The unique features of FoxP1 provide a glimpse of how intrinsically disordered regions can facilitate domain swapping oligomerization and other tightly regulated association mechanisms relevant in biological processes.

Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra performance liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction.

The association of two or more proteins to adopt a quaternary complex is one of the most widespread mechanisms by which protein function is modulated. In this scenario, three-dimensional domain swapping (3D-DS) constitutes one plausible pathway for the evolution of protein oligomerization that exploits readily available intramolecular contacts to be established in an intermolecular fashion. However, analysis of the oligomerization kinetics and thermodynamics of most extant 3D-DS proteins shows its dependence on protein unfolding, obscuring the elucidation of the emergence of 3D-DS during evolution, its occurrence under physiological conditions, and its biological relevance. Here, we describe the human FoxP subfamily of transcription factors as a feasible model to study the evolution of 3D-DS, due to their significantly faster dissociation and dimerization kinetics and lower dissociation constants in comparison to most 3D-DS models. Through the biophysical and functional characterization of FoxP proteins, relevant structural aspects highlighting the evolutionary adaptations of these proteins to enable efficient 3D-DS have been ascertained. Most biophysical studies on FoxP suggest that the dynamics of the polypeptide chain are crucial to decrease the energy barrier of 3D-DS, enabling its fast oligomerization under physiological conditions. Moreover, comparison of biophysical parameters between human FoxP proteins in the context of their minute sequence differences suggests differential evolutionary strategies to favor homoassociation and presages the possibility of heteroassociations, with direct impacts in their gene regulation function.

Mutations in the gene encoding polycystin-1 (PC1) underlie autosomal dominant polycystic kidney disease (ADPKD). ADPKD patients present with multiple cardiovascular co-morbidities believed to be caused by renal dysfunction. LV hypertrophy and diastolic dysfunction can manifest during childhood or in young adults prior to a formal diagnosis of hypertension, and evidence suggests that LV function is impaired in ADPKD patients with normal or moderately reduced kidney function. These facts suggest that cardiomyocyte-autonomous effects may contribute to the cardiovascular abnormalities seen in ADPKD. Contractile function (systolic and diastolic) measured by echo was significantly reduced in PC1 cKO ( Pkd1 F/F ;αMHC-Cre) mice compared with controls ( Pkd1 F/F ). PC1 cKO cardiomyocytes manifest impaired contractility and smaller and slower Ca 2+ transients. Using a multidimensional approach, we discovered that cardiomyocytes lacking PC1 have shorter action potentials (APD50/90) and decreased SERCA activity. These alterations impair EC-coupling and decrease SR Ca 2+ loading during pacing. Remarkably, square pulses under voltage clamp (-80 to +10 mV) produced Ca 2+ transients with similar amplitude between genotypes, which highlights that alterations in action potential (AP) duration drive most of the EC-coupling changes. PC1-deficient cardiomyocytes manifested an increase in outward K + currents (I to , I Kslow1/2 and I ss ) but not in inward currents (I K1 ). PC1 over-expression in HEK293T cells reduced the currents of heterologously expressed Kv4.3/2.1/1.5 channels. The inhibitory effects of PC1 on Kv4.3 currents were mediated by PC1-CT (C-terminus) through its coiled-coil domain (CCD). Interestingly, a naturally occurring human mutant PC1 R4228X , located in the CCD, manifested no suppressive effects on Kv4.3 channel. Finally, to begin to test for relevance to human pathology, we found that PC1 ablation reduces AP duration, and PC1-CT over-expression had the opposite effect in human stem cell-derived cardiomyocytes. Our findings uncover a novel role for PC1 controlling action potential duration and SERCA. PC1-deficient cardiomyocytes manifest impaired contractility, likely contributing to contractile dysfunction in ADPKD patients.

BACKGROUND: Polycystin-1 (PC1) is a transmembrane protein originally identified in autosomal dominant polycystic kidney disease where it regulates the calcium-permeant cation channel polycystin-2. Autosomal dominant polycystic kidney disease patients develop renal failure, hypertension, left ventricular hypertrophy, and diastolic dysfunction, among other cardiovascular disorders. These individuals harbor PC1 loss-of-function mutations in their cardiomyocytes, but the functional consequences are unknown. PC1 is ubiquitously expressed, and its experimental ablation in cardiomyocyte-specific knockout mice reduces contractile function. Here, we set out to determine the pathophysiological role of PC1 in cardiomyocytes. METHODS: Wild-type and cardiomyocyte-specific PC1 knockout mice were analyzed by echocardiography. Excitation-contraction coupling was assessed in isolated cardiomyocytes and human embryonic stem cell-derived cardiomyocytes, and functional consequences were explored in heterologous expression systems. Protein-protein interactions were analyzed biochemically and by means of ab initio calculations. RESULTS: PC1 ablation reduced action potential duration in cardiomyocytes, decreased Ca2+ transients, and myocyte contractility. PC1-deficient cardiomyocytes manifested a reduction in sarcoendoplasmic reticulum Ca2+ stores attributable to a reduced action potential duration and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) activity. An increase in outward K+ currents decreased action potential duration in cardiomyocytes lacking PC1. Overexpression of full-length PC1 in HEK293 cells significantly reduced the current density of heterologously expressed Kv4.3, Kv1.5 and Kv2.1 potassium channels. PC1 C terminus inhibited Kv4.3 currents to the same degree as full-length PC1. Additionally, PC1 coimmunoprecipitated with Kv4.3, and a modeled PC1 C-terminal structure suggested the existence of 2 docking sites for PC1 within the N terminus of Kv4.3, supporting a physical interaction. Finally, a naturally occurring human mutant PC1R4228X manifested no suppressive effects on Kv4.3 channel activity. CONCLUSIONS: Our findings uncover a role for PC1 in regulating multiple Kv channels, governing membrane repolarization and alterations in SERCA activity that reduce cardiomyocyte contractility.

A regulatory factor RfaH, present in many Gram-negative bacterial pathogens, is required for transcription and translation of long operons encoding virulence determinants. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA sequence within these operons. Upon recruitment, the C-terminal domain of this two-domain protein refolds from an α-hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound β-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (α-hairpin) and active (β-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state (∼20% deuteron incorporation), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the β-folded state. Computationally-predicted ΔGs agree with these results by displaying similar anisotropic stability within the tip of the α-hairpin and on neighboring N-terminal domain residues. Remarkably, the β-folded state shows comparable stability to non-metamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins, and for devising targeted strategies to combat bacterial diseases.SignificanceInfections caused by Gram-negative bacteria are a worldwide health threat due to rapid acquisition of antibiotic resistance. RfaH, a protein essential for virulence in several Gram-negative pathogens, undergoes a large-scale structural rearrangement in which one RfaH domain completely refolds. Refolding transforms RfaH from an inactive state that restricts RfaH recruitment to a few target genes into an active state that binds to, and couples, transcription and translation machineries to elicit dramatic activation of gene expression. However, the molecular basis of this unique conformational change is poorly understood. Here, we combine molecular dynamics and structural biology to unveil the hotspots that differentially stabilize both states of RfaH. Our findings provide novel insights that will guide design of inhibitors blocking RfaH action.

Cullin-RING E3 ligases (CRLs) are elongated and bowed protein complexes that transfer ubiquitin over 60Å to proteins targeted for proteasome degradation. One such CRL contains the ankyrin repeat and SOCS box protein 9 (ASB9), which binds to and partially inhibits creatine kinase (CK). While current models for the ASB9-CK complex contain some known interface residues, the overall structure andprecise interface of the ASB9-CK complex remains unknown. Through an integrative modeling approach, we report a third-generation model that reveals precisely the interface interactions and also fits the shape of the ASB9-CK complex as determined by small-angle X-ray scattering. We constructed an atomic model for the entire CK-targeting CRL to uncover dominant modes of motion that could permit ubiquitin transfer. Remarkably, only the correctly docked CK-containing E3 ligase and not incorrectly docked structures permitted close approach of ubiquitin to the CK substrate.

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Forkhead box P (FoxP) proteins are members of the versatile Fox transcription factors, which control the timing and expression of multiple genes for eukaryotic cell homeostasis. Compared to other Fox proteins, they can form domain-swapped dimers through their DNA-binding –forkhead– domains, enabling spatial reorganization of distant chromosome elements by tethering two DNA molecules together. Yet, domain swapping stability and DNA binding affinity varies between different FoxP proteins. Experimental evidence suggests that the protonation state of a histidine residue conserved in all Fox proteins is responsible for pH-dependent modulation of these interactions. Here, we explore the consequences of the protonation state of another histidine (H59), only conserved within FoxM/O/P subfamilies, on folding and dimerization of the forkhead domain of human FoxP1. Dimer dissociation kinetics and equilibrium unfolding experiments demonstrate that protonation of H59 leads to destabilization of the domain-swapped dimer due to an increase in free energy difference between the monomeric and transition states. This pH–dependence is abolished when H59 is mutated to alanine. Furthermore, anisotropy measurements and molecular dynamics evidence that H59 has a direct impact in the local stability of helix H3. Altogether, our results highlight the relevance of H59 in domain swapping and folding stability of FoxP1.

10 p.-9 fig.-2 tab., During evolution, some homologs proteins appear with different connectivity between secondary structures (different topology) but conserving the tridimensional arrangement of them (same architecture). These events can produce two types of arrangements; circular permutation or non-cyclic permutations. The first one results in the N and C terminus transferring to a different position on a protein sequence while the second refers to a more complex arrangement of the structural elements. In ribokinase superfamily, two different topologies can be identified, which are related to each other as a non-cyclic permutation occurred during the evolution. Interestingly, this change in topology is correlated with the nucleotide specificity of its members. Thereby, the connectivity of the secondary elements allows us to distinguish an ATP-dependent and an ADP-dependent topology. Here we address the impact of introducing the topology of a homologous ATP-dependent kinase in an ADP-dependent kinase (Thermococcus litoralis glucokinase) in the structure, nucleotide specificity, and substrate binding order of the engineered enzyme. Structural evidence demonstrates that rewiring the topology of TlGK leads to an active and soluble enzyme without modifications on its three-dimensional architecture. The permuted enzyme (PerGK) retains the nucleotide preference of the parent TlGK enzyme but shows a change in the substrate binding order. Our results illustrate how the rearrangement of the protein folding topology during the evolution of the ribokinase superfamily enzymes may have dictated the substrate-binding order in homologous enzymes of this superfamily., This research was supported by Fondo Nacional de Desarrollo Científico y Tecnológico from Chile (Grant 1150460, 3160373) and the Spanish Ministry of Economy, Industry and Competitiveness (CTQ2015-66206-C2-2-R), co-funded with European Union ERDF funds (European Regional Development Fund), and CSIC (PIE-201620E064) to M.C.V.

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis , termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 A resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.

The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP(-) and was activated by free Mg(2+). On the other hand, HXE mutants did not affect the KM for MgADP(-), were still inhibited by free Mg(2+), and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP-AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.

Natural sweeteners, such as stevia and thaumatin, exert their sweet taste by specifically binding to sweet taste receptors. However, the molecular basis of their sweetening power remains to be ascertained. In the present study, we built a comparative model of the hT1R2 and hT1R3 subunits in order to characterize their interactions with natural, non-caloric sweeteners – from glycosylated terpenoids to sweet proteins – at the molecular level. The binding free energy between hT1R2-hT1R3 and sweeteners of different families shows a strong correlation with their sweetness intensity for both, small sweeteners (r = −0.89) and sweet proteins (r = −0.97). The correlation is further improved and generalized throughout all families of sweeteners evaluated, when EC50 values are used instead of relative intensities (r = −0.91). Altogether, these results contribute to a better understanding of the sweetness perception of these sweeteners, and promote the use of docking for better prediction of resulting sweetness.

Detailed molecular mechanisms underpinning enzymatic reactions are still a central problem in biochemistry. The need for active site flexibility to sustain catalytic activity constitutes a notion of wide acceptance, although its direct influence remains to be fully understood. With the aim of studying the relationship between structural dynamics and enzyme catalysis, the cellulase Cel5A from Bacillus agaradherans is used as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility are related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of Cel5A are selected for protein engineering via site-directed mutagenesis. Three Cel5A variants are generated, N141L, A137Y and I102A/A137Y, showing a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes. These results are interpreted in structural terms by molecular dynamics simulations, showing that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility. These results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations.

Spatiotemporal control of the cAMP signaling pathway is governed by both hormonal stimulation of cAMP generation by adenylyl cyclases (activation phase) and cAMP hydrolysis by phosphodiesterases (PDEs) (termination phase). The termination phase is initiated by PDEs actively targeting the protein kinase A (PKA) R-subunit through formation of a PDE-PKAR-cyclic adenosine monophosphate (cAMP) complex (the termination complex). Our results using PDE8 as a model PDE, reveal that PDEs mediate active hydrolysis of cAMP bound to its receptor RIα by enhancing the enzymatic activity. This accelerated cAMP turnover occurs via formation of a stable PDE8-RIα complex, where the protein-protein interface forms peripheral contacts and the central ligand cements this ternary interaction. The basis for enhanced catalysis is active translocation of cAMP from its binding site on RIα to the hydrolysis site on PDE8 through direct “channeling.” Our results reveal cAMP channeling in the PDE8-RIα complex and a molecular description of how this channel facilitates processive hydrolysis of unbound cAMP. Thus, unbound cAMP maintains the PDE8-RIα complex while being hydrolyzed, revealing an undiscovered mode for amplification of PKA activity by cAMP-mediated sequestration of the R-subunit by PDEs. This novel regulatory mode explains the paradox of cAMP signal amplification by accelerated PDE-mediated cAMP turnover. This highlights how target effector proteins of small-molecule ligands can promote enzyme-mediated ligand hydrolysis by scaffolding effects. Enhanced activity of the PDE8-RIα complex facilitates robust desensitization, allowing the cell to respond to dynamic levels of cAMP rather than steady-state levels. The PDE8-RIα complex represents a new class of PDE-based complexes for specific drug discovery targeting the cAMP signaling pathway.

Cold shock proteins (Csp) constitute a family of ubiquitous small proteins that act as RNA-chaperones to avoid cold-induced termination of translation. All members contain two subdomains composed of 2 and 3 β-strands, respectively, which are connected by a hinge loop and fold into a β-barrel. Bacillus caldolyticus Csp (BcCsp) is one of the most studied members of the family in terms of its folding, function, and structure. This protein has been described as a monomer in solution, although a recent crystal structure showed dimerization via domain swapping (DS). In contrast, other cold shock proteins of the same fold are known to dimerize in a nonswapped arrangement. Hypothesizing that reducing the size of the hinge loop may promote swapping as in several other DS proteins with different folds we deleted two residues from these region (BcCsp∆36-37), leading to a protein in monomer–dimer equilibrium with similar folding stability to that of the wild-type. Strikingly, the crystal structure of BcCsp∆36-37 revealed a nonswapped dimer with its interface located at the nucleic acid-binding surface, showing that the deletion led to structural consequences far from the perturbation site. Concomitantly, circular dichroism experiments on BcCsp∆36-37 demonstrated that binding of the oligonucleotide hexathymidine disrupts the dimer. Additionally, HDXMS shows a protective effect on the protein structure upon dimerization, where the resulting interactions between ligand-binding surfaces in the dimer reduced the extent of exchange throughout the whole protein. Our work provides evidence of the complex interplay between conformational dynamics, deletions, and oligomerization within the Csp protein family. Databases Structural data are available in the Protein Data Bank under accession number 5JX4.

This report summarizes the scientific content and activities of the second edition of the Latin American Symposium (LA-SCS), organized by the Student Council (SC) of the International Society for Computational Biology (ISCB), held in conjunction with the Fourth Latin American conference from the International Society for Computational Biology (ISCB-LA 2016) in Buenos Aires, Argentina, on November 19, 2016. Fil: Monzón, Alexander. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Hasenahuer, Marcia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Mancini, Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centre for Genomic Regulation; España Fil: Coimbra, Nilson. Universidade Federal de Minas Gerais; Brasil Fil: Cravero, Fiorella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; Argentina Fil: Cáceres Molina, Javier Ignacio. Universidad Andrés Bello; Chile Fil: Ramírez Sarmiento, César A.. Pontificia Universidad Católica de Chile; Chile Fil: Palopoli, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Meysman, Pieter. Universiteit Antwerp; Bélgica Fil: Parra, Rodrigo Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Max Planck Institute for Biophysical Chemistry; Alemania

Synthesis of β-ionone in recombinant Saccharomyces cerevisiae is limited by the efficiency of Carotenoid Cleavage Dioxygenases (CCD), membrane-tethered enzymes catalyzing the last step in the pathway. We performed in silico design and membrane affinity analysis, focused on single-point mutations of PhCCD1 to improve membrane anchoring. The resulting constructs were tested in a β-carotene hyper-producing strain by comparing colony pigmentation against colonies transformed with native PhCCD1 and further analyzed by β-ionone quantification via RP-HPLC. Two single-point mutants increased β-ionone yields almost 3-fold when compared to native PhCCD1. We also aimed to improve substrate accessibility of PhCCD1 through the amino-terminal addition of membrane destination peptides directed towards the endoplasmic reticulum or plasma membrane. Yeast strains expressing peptide-PhCCD1 constructs showed β-ionone yields up to 4-fold higher than the strain carrying the native enzyme. Our results demonstrate that protein engineering of CCDs significantly increases the yield of β-ionone synthesized by metabolically engineered yeast.

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened β-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 μM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240–309. These residues correspond to most of the β-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the β-meander module is not affected by monomerizing mutations.

Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single-molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic adenine diphosphate (ADP)-dependent glucokinase from Thermococcus litoralis triggered by the sequential binding of substrates. In the absence of substrates, the mechanical unfolding of TlGK shows an intermediate 1, which is stabilized in the presence of Mg·ADP(-), the first substrate to bind to the enzyme. However, in the presence of this substrate, an additional unfolding event is observed, intermediate 1*. Finally, in the presence of both substrates, the unfolding force of intermediates 1 and 1* increases as a consequence of the domain closure. These results show that SMFS can be used as a powerful experimental tool to investigate binding mechanisms of different enzymes with more than one ligand, expanding the repertoire of protocols traditionally used in enzymology.

RfaH is a bacterial virulence factor that belongs to the universally conserved NusG family of transcription factors and is commonly referred as the first example of a transformer protein. In contrast to other NusG proteins, the C-terminal domain (CTD) of RfaH folds into an α-helical hairpin that is stabilized by interactions with the N-terminal domain (NTD), which in turn masks an RNA polymerase (RNAp) binding site and thus constitutes an autoinhibited state.This autoinhibition is relieved when binding to a specific DNA element in the transcription elongation complex triggers domain dissociation, allowing the NTD to interact with RNAp to facilitate transcription while the CTD dramatically refolds into a NusG-like β-barrel that interacts with the ribosome to activate translation, thus changing both the structure and function of RfaH.Since this phenomenon is experimentally challenging, their structural details remain to be elucidated. Here, we explore the mechanism of the conformational switching of RfaH in the full-length protein using a dual-basin structure-based model. Our simulations capture several features described experimentally, such as the requirement of disruption of interdomain contacts to trigger the dramatic α-to-β transformation of RfaH, confirms the roles of previously indicated residues E48 and R138, and suggests a new important role for F130, in the stability of the interdomain interaction. These native basins are connected through an intermediate state that builds up upon binding to the NTD and shares features from both folds. We also examine the competitive binding between RfaH-CTD and RNAp for the NTD, showing that RNAp binding favors the β fold.Our study shows that native-biased models are appropriate for interrogating detailed mechanisms of the structural rearrangements during the native state interconversion of RfaH. FUNDING: Fondecyt 11140601.

Increasing evidence has shown that the forkhead DNA-binding domain of human FoxP proteins forms domain-swapped dimers. During domain swapping, identical segments are exchanged between two subunits, thus leading to an intertwined oligomer where interactions of the native monomer are reconstituted intermolecularly. Domain swapping in FoxP enables formation of long-range chromosomal interactions of high relevance in gene transcription, with several disease-causing mutations related to impairment of this process.

Pyridoxal 5'-phosphate is the active form of vitamin B6 and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we analyse the catalytic and regulatory mechanisms of hPLK as a function of a precise determination of the species involved in metal-nucleotide equilibriums and describe new regulatory mechanisms for this enzyme. hPLK activity is supported by several metals, being Zn(2+) the most effective, although the magnitude of the effect observed is highly dependent on the relative concentrations of metal and nucleotide used. The true substrate for the reaction catalyzed by hPLK is the metal nucleotide complex, while ATP(4-) and HATP(3-) did not affect the activity. The enzyme presents substrate inhibition by both pyridoxal (PL) and ZnATP(2-), although the latter behaves as a weakly inhibitor. Our study also established, for the first time, a dual role for free Zn(2+); as an activator at low concentrations (19 μM optimal concentration) and as a potent inhibitor with a IC50 of 37 μM. These results highlighted the importance of an accurate estimation of the actual concentration of the species involved in metal-nucleotide equilibriums in order to obtain reliable values for the kinetic parameters, and for determine the true regulators of the PLK activity. They also help to explain the dissimilar kinetic parameters reported in the literature for this enzyme.