Active Site Cleft Research Articles

A model of zymogen factor XII: insights into protease activation.

In plasma, the zymogens factor XII (FXII) and prekallikrein reciprocally convert each other to the proteases FXIIa and plasma kallikrein (PKa). PKa cleaves high-molecular-weight kininogen (HK) to release bradykinin, which contributes to regulation of blood vessel tone and permeability. Plasma FXII is normally in a "closed" conformation that limits activation by PKa. When FXII binds to a surface during contact activation it assumes an "open" conformation that increases the rate of activation by PKa. Mutations in FXII that disrupt the closed conformation have been identified in patients with conditions associated with excessive bradykinin formation. Using FXII structures from the AlphaFold database, we generated models for the closed form of human FXII that we tested with site-directed mutagenesis. The models predict multiple interactions between the fibronectin type 2 (FN2), kringle, and catalytic domains involving highly conserved amino acids that restrict access to the FXII activation cleavage sites. Based on the model, we expressed FXII with single-amino acid substitutions and studied their effects on FXII activation by PKa. Replacements for Arg36 in the FN2 domain; Glu225, Asp253, or Trp268 in the kringle domain; or Lys346 near the activation cleavage site were activated >10-fold faster by PKa than wild-type FXII. Adding these proteins to plasma resulted in rapid HK cleavage due to markedly enhanced reciprocal activation with prekallikrein. The results support a model that explains the behavior of FXII in solution. Conformational changes involving the identified amino acids likely occur when FXII binds to a surface to facilitate activation.

Terminal spin labeling of xylotriose strongly affects interactions in the active site of xylanase BcX.

Paramagnetic probes provide long-range distance information and report on minor conformations of biomacromolecules. However, it is important to realize that any probe can affect the system of interest. Here, we report on the effects of attaching a small nitroxide spin label [TEMPO, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl] to xylotriose, a substrate of the enzyme xylanase from Bacillus circulans (BcX). BcX has a long and narrow active site cleft accommodating six xylose units and a secondary binding site on its surface. The aim of the study was to probe the interactions of thesubstrate with the enzyme using paramagnetic relaxation enhancements (PREs). Binding of the substrate to the surface exposed secondary binding site resulted in strong and localized PREs, indicative of well-defined binding. The xylotriose with diamagnetic control tag was still able to bind the active site cleft, though the rate of exchange was reduced relative to that of untagged xylotriose. The substrate with the paramagnetic TEMPO was not able to bind inside the active site cleft. Also, additional interactions on another surface location showed differences between the paramagnetic substrate and the diamagnetic control, despite the minimal chemical differences between TEMPO modified xylotriose and its reduced, diamagnetic counterpart. Our findings underscore the sensitivity of BcX substrate binding to minor substrate modifications. This study serves as a reminder that any probe, including the attachment of a small paramagnetic group, can affect the behavior of the system under investigation. Even the chemical difference between a paramagnetic tag and its diamagnetic control can result in differences in the molecular interactions.

Binding mode between peptidyl-tRNA hydrolase and the peptidyl-A76 moiety of the substrate.

Peptidyl-tRNA hydrolase (Pth) hydrolyzes the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of aborted translation, to prevent cell death by recycling tRNA. Numerous studies have attempted to elucidate the substrate recognition mechanism of Pth. However, the binding mode of the peptidyl-A76 (3'-terminal adenosine of tRNA) moiety of the substrate to Pth, especially the A76 moiety, still remains unclear. Here, we present the crystal structure of Thermus thermophilus Pth (TtPth) in complex with adenosine 5'-monophosphate (AMP), a mimic of A76. In addition, we show the crystal structure of TtPth in which the active site cleft interacts with the C-terminal three amino acid residues of a crystallographically-related neighboring TtPth molecule. Superimposition of these two crystal structures reveals that the C-terminal carboxyl group of the neighboring TtPth molecule and the 3'-hydroxyl group of AMP are located in positions favorable for ester bond formation, and we present a TtPth•peptidyl-A76 complex model. The complex model agrees with many previous NMR and kinetic studies, and our site-directed mutagenesis studies support its validity. Based on these facts, we conclude that the complex model properly represents the interaction between Pth and the substrate in the reaction. Furthermore, structural comparisons suggest that the substrate recognition mode is conserved among bacterial Pths. This study provides insights into the molecular mechanism of the reaction and useful information to design new drugs targeting Pth.

Binding of small molecules at the P-stalk site of ricin A subunit trigger conformational changes that extend into the active site.

Binding of small molecules at the P-stalk site of ricin A subunit trigger conformational changes that extend into the active site.

CryoEM and crystal structure analyses reveal the indirect role played by Trp89 in glutamate dehydrogenase enzymatic reactions.

Glutamate dehydrogenase from Thermococcus profundus is a homo-hexameric enzyme that catalyzes the reversible deamination of glutamate to 2-oxoglutarate in the presence of a cofactor. In each subunit, a large active-site cleft is formed between the two functional domains, one of which displays motion to open and close the cleft. Trp89 in the cleft displays two sidechain conformers in the open cleft and a single conformer in the closed cleft. To reveal the role of the Trp89 sidechain in the domain motion, we mutated Trp89 to phenylalanine. Despite the Trp89 sidechain being located away from the reaction center, the catalytic constant decreased to 1/38-fold of that of the wild-type without a fatal reduction of the affinities to the cofactor and ligand molecules. To understand the molecular mechanism underlying this reduction, we determined the crystal structure in the unliganded state and the metastable conformations appearing in the steady stage of the reaction using cryo-electron microscopy (cryoEM). The four identified metastable conformations were similar to the three conformations observed in the wild-type, but their populations were different from those of the wild-type. In addition, a conformation with a completely closed active-site cleft necessary for the reaction to proceed was quite rare. The crystal structure and the four metastable conformations suggested that the reduction in the catalytic constant could be attributed to changes in the interactions between Gln13 and the 89th side chains, preventing the closing domain motion.

Temperature-Dependent Structural Changes of the Active Site and Substrate-Binding Cleft in Hen Egg White Lysozyme

Lysozyme plays a crucial role in the natural immune system, protecting against invading bacteria or viruses. The room-temperature (RT) structure of lysozymes is important for understanding accurate structural information compared to the crystal structure determined at cryogenic temperature. Several RT structures of lysozymes are determined by serial crystallography, but their temperature-dependent structural properties are not fully elucidated. To better understand the temperature-dependent structural change, the RT and cryogenic temperature structures of hen egg white lysozyme (HEWL) were determined by serial synchrotron crystallography (SSX) and macromolecular crystallography (MX), respectively. Structural comparisons of HEWLRT and HEWLCryo showed that the positions of the loops above the substrate-binding cleft of HEWL differed. The width of the substrate-binding cleft between the α- and β-domains of HEWLRT was wider than that of HEWLCryo. The distance between the two catalytic residues Glu53 and Asp70 and their interaction with neighbor residues and water molecules showed the distant between HEWLRT and HEWLCryo. Due to temperature, the subtle movements of the active site and substrate-binding cleft of HEWL led to different docking results for N-acetylglucosamine and N,N′,N″-triacetylchitotriose. These results will provide useful information to more accurately understand the molecular function of HEWL and insights into the temperature effects for ligand design.

1,4,5-Trisubstituted-carboxylated 1,2,3-triazoles: an unconventional class of ribonuclease A inhibitors.

The "catalytic triad" present at the active site of ribonuclease A (RNase A) is responsible for the cleavage of the 5'-phosphodiester bond; amino acid residues His12, Lys41 and His119 constituting this triad provide a positively charged environment at the physiological pH. Based on docking studies, 1,4,5-trisubstituted-carboxylated 1,2,3-triazoles (1,4,5-TTs) were identified as a new class of RNase A inhibitors. Therefore, two different groups of 1,4,5-TTs, functionalized with carboxylic acid groups, were synthesized by reacting pre functionalized butyne-1,4-diol derivatives with several aryl/alkyl azides under solvent and catalyst free conditions. Inhibitory properties of the new molecules with heteroatom linked carboxylic acid "CH2XCH2CO2H" (X = S, O) functionalities were investigated by performing qualitative and quantitative biophysical studies. All the "CH2S" and "CH2O" linked acid derivatives (6a-e, 6f'-g' and 6h, and 8a-e, 8f'-g' and 8h) exhibited significant competitive inhibition with inhibition constant values (Ki) ranging from 9 to 34 μM determined by steady state enzyme kinetics. Uracil based bisthioglycolic acid (6h) and carboxylic acid based bisoxyacetic acid (8g') derivatives were found to be the most promising inhibitors with Ki values of 9.9 ± 0.7 and 15.6 ± 0.6 μM, respectively. Additional molecular docking studies revealed that a sufficient number of hydrogen bonding interactions were generated from various functional groups of inhibitors and the amino acid residues present at important subsites of RNase A. The study also established that the free rotating "CH2X" arms of 1,4,5-TTs provided a unique shape to accommodate the molecule within the active site cleft. A fairly good idea about the structure activity relationship (SAR) was obtained by correlating experimentally determined Ki values and the corresponding docking poses. This study reports an unconventional class of non-sugar, non-nucleosidic 1,4,5-TT based competitive inhibitors of RNase A.

Two medicinal molecules Hydralazine and Isoniazid have the potential to control wheat crown rot by combing with a N-acetyltransferase FDB2

Two medicinal molecules Hydralazine and Isoniazid have the potential to control wheat crown rot by combing with a N-acetyltransferase FDB2

Bimodal substrate binding in the active site of the glycosidase BcX.

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site.

Flavonoid-Quinoxaline Hybrid Compounds as Cathepsin Inhibitors Against Fascioliasis.

Fasciola hepatica is a parasitic trematode that infects livestock animals and humans, causing significant health and economic burdens worldwide. The extensive use of anthelmintic drugs has led to the emergence of resistant parasite strains, posing a threat to treatment success. The complex life cycle of the liver fluke, coupled with limited funding and research interest, have hindered progress in drug discovery. Our group has been working in drug development against this parasite using cathepsin proteases as molecular targets, finding promising compound candidates with in vitro and in vivo efficacy. Here, we evaluated hybrid molecules that combine two chemotypes, chalcones and quinoxaline 1,4-di- N-oxides, previously found to inhibit F. hepatica cathepsin Ls and tested their in vitro activity with the isolated targets and the parasites in culture. These molecules proved to be good cathepsin inhibitors and to kill the juvenile parasites at micromolar concentrations. Also, we performed molecular docking studies to analyze the compounds-cathepsins interface, finding that the best inhibitors interact at the active site cleft and contact the catalytic dyad and residues belonging to the substrate binding pockets. We conclude that the hybrid compounds constitute promising scaffolds for the further development of new fasciolicidal compounds.

Structure-guided discovery of ancestral CRISPR-Cas13 ribonucleases.

The RNA-guided ribonuclease CRISPR-Cas13 enables adaptive immunity in bacteria and programmable RNA manipulation in heterologous systems. Cas13s share limited sequence similarity, hindering discovery of related or ancestral systems. To address this, we developed an automated structural-search pipeline to identify an ancestral clade of Cas13 (Cas13an) and further trace Cas13 origins to defense-associated ribonucleases. Despite being one-third the size of other Cas13s, Cas13an mediates robust programmable RNA depletion and defense against diverse bacteriophages. However, unlike its larger counterparts, Cas13an uses a single active site for both CRISPR RNA processing and RNA-guided cleavage, revealing that the ancestral nuclease domain has two modes of activity. Discovery of Cas13an deepens our understanding of CRISPR-Cas evolution and expands opportunities for precision RNA editing, showcasing the promise of structure-guided genome mining.

Implication of Molecular Constraints Facilitating the Functional Evolution of Pseudomonas aeruginosa KPR2 into a Versatile α-Keto-Acid Reductase.

Theoretical concepts linking the structure, function, and evolution of a protein, while often intuitive, necessitate validation through investigations in real-world systems. Our study empirically explores the evolutionary implications of multiple gene copies in an organism by shedding light on the structure-function modulations observed in Pseudomonas aeruginosa's second copy of ketopantoate reductase (PaKPR2). We demonstrated with two apo structures that the typical active site cleft of the protein transforms into a two-sided pocket where a molecular gate made up of two residues controls the substrate entry site, resulting in its inactivity toward the natural substrate ketopantoate. Strikingly, this structural modification made the protein active against several important α-keto-acid substrates with varied efficiency. Structural constraints at the binding site for this altered functional trait were analyzed with two binary complexes that show the conserved residue microenvironment faces restricted movements due to domain closure. Finally, its mechanistic highlights gathered from a ternary complex structure help in delineating the molecular perspectives behind its kinetic cooperativity toward these broad range of substrates. Detailed structural characteristics of the protein presented here also identified four key amino acid residues responsible for its versatile α-keto-acid reductase activity, which can be further modified to improve its functional properties through protein engineering.

Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

Structural characterization of lytic transglycosylase MltD of Pseudomonas aeruginosa, a target for the natural product bulgecin A

Structural characterization of lytic transglycosylase MltD of Pseudomonas aeruginosa, a target for the natural product bulgecin A

Biosynthesis of Cytidine Diphosphate-6-d-Glucitol for the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in chickens and is the leading cause of human diarrheal disease worldwide. The various serotypes of C.jejuni produce structurally distinct capsular polysaccharides (CPSs) on the exterior surfaces of the cell wall. The capsular polysaccharide from C.jejuni serotype HS:5 is composed of a repeating sequence of d-glycero-d-manno-heptose and d-glucitol-6-phosphate. We previously defined the pathway for the production of d-glycero-d-manno-heptose in C.jejuni. Here, we elucidate the biosynthetic pathway for the assembly of cytidine diphosphate (CDP)-6-d-glucitol by the combined action of two previously uncharacterized enzymes. The first enzyme catalyzes the formation of CDP-6-d-fructose from cytidine triphosphate (CTP) and d-fructose-6-phosphate. The second enzyme reduces CDP-6-d-fructose with NADPH to generate CDP-6-d-glucitol. Using sequence similarity network (SSN) and genome neighborhood network (GNN) analyses, we predict that these pairs of proteins are responsible for the biosynthesis of CDP-6-d-glucitol and/or CDP-d-mannitol in the lipopolysaccharides (LPSs) and capsular polysaccharides in more than 200 other organisms. In addition, high resolution X-ray structures of the second enzyme are reported, which provide novel insight into the manner in which an open-chain nucleotide-linked sugar is harbored in an active site cleft.

Propagation of conformational instability in FK506-binding protein FKBP12

Propagation of conformational instability in FK506-binding protein FKBP12

First Validated Model of Zymogen Factor XII Provides Insights into Protein Assembly and Activation

First Validated Model of Zymogen Factor XII Provides Insights into Protein Assembly and Activation

Electrostatic interactions of enzymes in non-aqueous conditions: insights from molecular dynamics simulations

Electrostatic interactions of enzymes and their effects on enzyme activity and stability are poorly understood in non-aqueous conditions. Here, we investigate the contribution of the electrostatic interactions on the stability and activity of enzymes in the non-aqueous environment using molecular dynamics simulations. Lipase was selected as active and lysozyme as inactive model enzymes in non-aqueous media. Hexane was used as a common non-aqueous solvent model. In agreement with the previous experiments, simulations show that lysozyme has more structural instabilities than lipase in hexane. The number of hydrogen bonds and salt bridges of both enzymes is dramatically increased in hexane. In contrast to the other opinions, we show that the increase of the electrostatic interactions in non-aqueous media is not so favorable for enzymatic function and stability. In this condition, the newly formed hydrogen bonds and salt bridges can partially denature the local structure of the enzymes. For lysozyme, the changes in electrostatic interactions occur in all domains including the active site cleft, which leads to enzyme inactivation and destabilization. Interestingly, most of the changes in electrostatic interactions of lipase occur far from the active site regions. Therefore, the active site entrance regions remain functional in hexane. The results of this study reveal how the changes in electrostatic interactions can affect enzyme stability and activity in non-aqueous conditions. Moreover, we show for the first time how some enzymes, such as lipase, remain active in a non-aqueous environment.

Crystal Structure of Inhibitor-Bound GII.4 Sydney 2012 Norovirus 3C-Like Protease.

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.

Targeting Aspergillus allergen oryzin with a chemical probe at atomic precision

We report the molecular basis of Aspergillus fumigatus oryzin, allergen Asp f 13, or alkaline proteinase ALP1, containing the sequence motif His–Asp–Ser of the subtilisin family, structure, and function at atomic detail. Given the resolution of the data (1.06 Å), we use fragment molecular replacement with ideal polyalanine α-helices to determine the first crystal structure of oryzin. We probe the catalytic serine through formation of an irreversible bond to a small molecule compound, specifically labeling it, describing the amino acid residues performing the catalytic function. Defined by a self-processed pro-peptide, the active site architecture shapes up pocket-like subsites that bind to and unveil the S1′–S4′ substrate binding preferences. We use molecular modeling to dock a model of the pro-peptide in the S1–S4 region and to dock collagen along the active site cleft. Opposite to the face harboring the catalytic serine, the enzyme binds to a calcium ion in a binding site created by backbone flipping. We use thermal unfolding to show that this metal ion provides structural stability. With no known host inhibitor identified thus far, this structure may hasten the progress of developing new therapeutic agents for diseases caused by pathogenic fungi.