Insulin-regulated aminopeptidase (IRAP) is a zinc-dependent metalloenzyme identified as a novel target for combating diabetes-induced diseases due to its crucial role in glucose metabolism and insulin sensitivity regulation. IRAP's catalytic domain catalyzes the N-terminal peptide bond hydrolysis of natural substrate oxytocin, a neuroactive peptide linked to improved cognition and other elemental brain functions. Angiotensin IV and similar peptides are recognized as cognitive enhancers due to their ability to competitively inhibit IRAP's proteolytic activity, thereby mitigating natural neuropeptide degradation. Despite a very similar binding complex between the substrate and the inhibitor with IRAP, particularly around the scissile bond, it is unclear why the enzyme metabolizes oxytocin but does not efficiently degrade angiotensin IV. We employed enhanced sampling quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations and higher-level QM/MM calculations to explore the reaction of these two peptides in IRAP. The calculated energy barrier for oxytocin cleavage was in very good agreement with the experimental data. A significantly higher energy barrier for the formation of the oxyanion tetrahedral intermediate (TI) and a higher overall barrier for the peptide cleavage were observed for the reaction with angiotensin IV. Comprehensive electronic structure analysis utilizing NBO and NCI methods unveiled the molecular basis for different reactivity, a stabilizing interaction between the sigma hole of the N-terminus disulfide bond and the hybridizing lone pair of the scissile peptide nitrogen in oxytocin. The interplay between a weak noncovalent spodium bond and strong bidentate coordination of the catalytic Zn2+ by angiotensin IV caused a larger deviation of the valine C-Cα-Cβ angle from ideal tetrahedral geometry, consequently destabilizing the TI. These results underscore the critical importance of analyzing the dynamics, interactions, and electronic properties of reaction intermediates and transition states in enzymatic processes. Our findings have significant implications for the rational design and development of IRAP inhibitors as potential therapeutic agents for memory disorders, neurodegenerative diseases, and diabetes.

Insulin-regulated aminopeptidase (IRAP) is a zinc-dependent metalloenzyme involved in the regulation of glucose metabolism and insulin sensitivity identified as a novel target for combating diabetes-induced diseases. IRAP’s catalytic domain catalyzes the N-terminal peptide bond hydrolysis of the natural substrate oxytocin, a neuroactive pep-tide linked to improved cognition and other elemental brain functions. Angiotensin IV and similar peptides are recognized as cognitive enhancers due to their ability to competitively inhibit proteolytic activity of IRAP, reducing the degradation of natural neuropeptides. Despite a very similar binding complex between the substrate and the inhibitor with IRAP, especially around the scissile bond, it is unclear why the enzyme metabolizes oxytocin but does not efficiently degrade angiotensin IV. We employed enhanced sampling QM/MM molecular dynamics simulations to explore free energy landscapes for reaction of these two peptides in IRAP. A significantly higher energy barrier for the formation of the oxyanion tetrahedral intermediate (TI) and higher overall barrier for the peptide cleavage was observed for the reaction of angiotensin IV. Electronic structure analysis (NBO and NCI) revealed the reasons for different reactivity, including stabilization of the on the sigma hole of the N-terminus disulfide in oxytocin by the hybridizing lone pair of the scissile peptide nitrogen. The interplay between weak non-covalent spodium bond and strong bi-dentate coordination of the catalytic Zn2+ by angiotensin IV caused larger deviation of valine C-Cα-Cβ angle from the ideal tetrahedral, which destabilizes the TI. The results emphasise the importance of analysing dynamics, interactions and electronic properties of reaction intermediates and transition states in enzymes, and have implications for the de-sign and development of IRAP inhibitors for the treatment of memory disorders, neurodegenerative diseases, and diabetes.

We test a range of standard generalized Born (GB) models and protein force fields for a set of five experimentally characterized, designed peptides comprising alternating blocks of glutamate and lysine, which have been shown to differ significantly in α-helical content. Sixty-five combinations of force fields and GB models are evaluated in >800 μs of molecular dynamics simulations. GB models generally do not reproduce the experimentally observed α-helical content, and none perform well for all five peptides. These results illustrate that these models are not usefully predictive in this context. These peptides provide a useful test set for simulation methods.

De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally.

Zinc metalloproteins are ubiquitous, with protein zinc centers of structural and functional importance, involved in interactions with ligands and substrates and often of pharmacological interest. Biomolecular simulations are increasingly prominent in investigations of protein structure, dynamics, ligand interactions, and catalysis, but zinc poses a particular challenge, in part because of its versatile, flexible coordination. A computational workflow generating reliable models of ligand complexes of biological zinc centers would find broad application. Here, we evaluate the ability of alternative treatments, using (nonbonded) molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) at semiempirical (DFTB3) and density functional theory (DFT) levels of theory, to describe the zinc centers of ligand complexes of six metalloenzyme systems differing in coordination geometries, zinc stoichiometries (mono- and dinuclear), and the nature of interacting groups (specifically the presence of zinc-sulfur interactions). MM molecular dynamics (MD) simulations can overfavor octahedral geometries, introducing additional water molecules to the zinc coordination shell, but this can be rectified by subsequent semiempirical (DFTB3) QM/MM MD simulations. B3LYP/MM geometry optimization further improved the accuracy of the description of coordination distances, with the overall effectiveness of the approach depending upon factors, including the presence of zinc-sulfur interactions that are less well described by semiempirical methods. We describe a workflow comprising QM/MM MD using DFTB3 followed by QM/MM geometry optimization using DFT (e.g., B3LYP) that well describes our set of zinc metalloenzyme complexes and is likely to be suitable for creating accurate models of zinc protein complexes when structural information is more limited.

We test a range of standard implicit solvent models and protein forcefields for a set of 5 experimentally characterized, designed α-helical peptides. 65 combinations of forcefield and implicit solvent models are evaluated in >800 µs of molecular dynamics simulations. The data show that implicit solvent models generally fail to reproduce the experimentally observed secondary structure content, and none performs well for all 5 peptides. The results show that these models are not usefully predictive.

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide-lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.

MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the "last-resort" antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.

Allosteric regulation of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) controls the entry into aromatic amino acid biosynthesis in plants and microorganisms. DAH7PS has acquired a diverse range of allosteric machinery to enable this functionality. This review provides an overview of the current knowledge of the structural basis of allostery in this enzyme family and the evolutionary relationships between the different solutions to allosteric control of aromatic metabolite biosynthesis.

Because of their special organization, multifunctional enzymes play crucial roles in improving the performance of metabolic pathways. For example, the bacterium Prevotella nigrescens contains a distinctive bifunctional protein comprising a 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS), catalyzing the first reaction of the biosynthetic pathway of aromatic amino acids, and a chorismate mutase (CM), functioning at a branch of this pathway leading to the synthesis of tyrosine and phenylalanine. In this study, we characterized this P. nigrescens enzyme and found that its two catalytic activities exhibit substantial hetero-interdependence and that the separation of its two distinct catalytic domains results in a dramatic loss of both DAH7PS and CM activities. The protein displayed a unique dimeric assembly, with dimerization solely via the CM domain. Small angle X-ray scattering (SAXS)-based structural analysis of this protein indicated a DAH7PS-CM hetero-interaction between the DAH7PS and CM domains, unlike the homo-association between DAH7PS domains normally observed for other DAH7PS proteins. This hetero-interaction provides a structural basis for the functional interdependence between the two domains observed here. Moreover, we observed that DAH7PS is allosterically inhibited by prephenate, the product of the CM-catalyzed reaction. This allostery was accompanied by a striking conformational change as observed by SAXS, implying that altering the hetero-domain interaction underpins the allosteric inhibition. We conclude that for this C-terminal CM-linked DAH7PS, catalytic function and allosteric regulation appear to be delivered by a common mechanism, revealing a distinct and efficient evolutionary strategy to utilize the functional advantages of a bifunctional enzyme.

In coiled-coil (CC) protein structures α-helices wrap around one another to form rope-like assemblies. Most natural and designed CCs have two-four helices and cyclic (Cn) or dihedral (Dn) symmetry. Increasingly, CCs with five or more helices are being reported. A subset of these higher-order CCs is of interest as they have accessible central channels that can be functionalised; they are α-helical barrels. These extended cavities are surprising given the drive to maximise buried hydrophobic surfaces during protein folding and assembly in water. Here, we show that α-helical barrels can be maintained by the strategic placement of β-branched aliphatic residues lining the lumen. Otherwise, the structures collapse or adjust to give more-complex multi-helix assemblies without Cn or Dn symmetry. Nonetheless, the structural hallmark of CCs-namely, knobs-into-holes packing of side chains between helices-is maintained leading to classes of CCs hitherto unobserved in nature or accessed by design.

We describe de novo-designed α-helical barrels (αHBs) that bind and discriminate between lipophilic biologically active molecules. αHBs have five or more α-helices arranged around central hydrophobic channels the diameters of which scale with oligomer state. We show that pentameric, hexameric, and heptameric αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene (DPH) in the micromolar range and fluoresce. Displacement of the dye is used to report the binding of nonfluorescent molecules: palmitic acid and retinol bind to all three αHBs with submicromolar inhibitor constants; farnesol binds the hexamer and heptamer; but β-carotene binds only the heptamer. A co-crystal structure of the hexamer with farnesol reveals oriented binding in the center of the hydrophobic channel. Charged side chains engineered into the lumen of the heptamer facilitate binding of polar ligands: a glutamate variant binds a cationic variant of DPH, and introducing lysine allows binding of the biosynthetically important farnesol diphosphate.

In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.

In Pseudomonas aeruginosa ( Pae ), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d- arabino -heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α 2a and α 2b , relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type II A and type II B based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.

With the pharmaceutical industry striving for efficiency, cost-effectiveness, and sustainability, computational approaches such as molecular dynamics, quantum mechanics, and hybrid Quantum Mechanical/Molecular Mechanical methods are becoming increasingly important in drug discovery and development. Thanks to major software and hardware advances, these techniques enable don't need these words robust and accurate predictions at a fraction of the computational costs previously required. These approaches are particularly effective in supporting the design of new active pharmaceutical ingredients, shedding light on the molecular mechanisms that govern therapeutically relevant biological processes, or developing synthetic routes relying on biocatalysis.

Multifunctional proteins play a variety of roles in metabolism. Here, we examine the catalytic function of the combined 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM) from Geobacillus sp. DAH7PS operates at the start of the biosynthetic pathway for aromatic metabolites, whereas CM operates in a dedicated branch of the pathway for the biosynthesis of amino acids tyrosine and phenylalanine. In line with sequence predictions, the two catalytic functions are located in distinct domains, and these two activities can be separated and retain functionality. For the full-length protein, prephenate, the product of the CM reaction, acts as an allosteric inhibitor for the DAH7PS. The crystal structure of the full-length protein with prephenate bound and the accompanying small angle x-ray scattering data reveal the molecular mechanism of the allostery. Prephenate binding results in the tighter association between the dimeric CM domains and the tetrameric DAH7PS, occluding the active site and therefore disrupting DAH7PS function. Acquisition of a physical gating mechanism to control catalytic function through gene fusion appears to be a general mechanism for providing allostery for this enzyme.

Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

Allosteric regulation of enzyme activity plays important metabolic roles. Here we review the allostery of enzymes of amino-acid metabolism conferred by a discrete domain known as the ACT domain. This domain of 60-70 residues has a βαββαβ topology leading to a four-stranded β4β1β3β2 antiparallel sheet with two antiparallel helices on one face. Extensive sequence variation requires a combined sequence/structure/function analysis for identification of the ACT domain. Common features include highly varied modes of self-association of ACT domains, ligand binding at domain interfaces, and transmittal of allosteric signals through conformational changes and/or the manipulation of quaternary equilibria. A recent example illustrates the relatively facile adoption of this versatile module of allostery by gene fusion.

This chapter contains sections titled: Introduction Basic Principles of Chromatography Process Optimization to Reduce Eluent Consumption Use of a Green Solvent: Supercritical Carbon Dioxide Solvent Recycling Technologies Application Examples Conclusion: An Environmentally Friendly Solution for Each Separation Acknowledgment References