Substrate Recognition Profile Research Articles

Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites

Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (Vderivative) and PA-0 (VPA-0), we demonstrated that the Vderivative/VPA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile.

Protein Conformational Space at the Edge of Allostery: Turning a Nonallosteric Malate Dehydrogenase into an "Allosterized" Enzyme Using Evolution-Guided Punctual Mutations.

We unveil the intimate relationship between protein dynamics and allostery by following the trajectories of model proteins in their conformational and sequence spaces. Starting from a nonallosteric hyperthermophilic malate dehydrogenase, we have tracked the role of protein dynamics in the evolution of the allosteric capacity. Based on a large phylogenetic analysis of the malate (MalDH) and lactate dehydrogenase (LDH) superfamily, we identified two amino acid positions that could have had a major role for the emergence of allostery in LDHs, which we targeted for investigation by site-directed mutagenesis. Wild-type MalDH and the single and double mutants were tested with respect to their substrate recognition profiles. The double mutant displayed a sigmoid-shaped profile typical of homotropic activation in LDH. By using molecular dynamics simulations, we showed that the mutations induce a drastic change in the protein sampling of its conformational landscape, making transiently T-like (inactive) conformers, typical of allosteric LDHs, accessible. Our data fit well with the seminal key concept linking protein dynamics and evolvability. We showed that the selection of a new phenotype can be achieved by a few key dynamics-enhancing mutations causing the enrichment of low-populated conformational substates.

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover.

The evolution of new protein functions is dependent upon inherent biophysical features of proteins. Whereas, it has been shown that changes in protein dynamics can occur in the course of directed molecular evolution trajectories and contribute to new function, it is not known whether varying protein dynamics modify the course of evolution. We investigate this question using three related ß-lactamases displaying dynamics that differ broadly at the slow timescale that corresponds to catalytic turnover yet have similar fast dynamics, thermal stability, catalytic, and substrate recognition profiles. Introduction of substitutions E104K and G238S, that are known to have a synergistic effect on function in the parent ß-lactamase, showed similar increases in catalytic efficiency toward cefotaxime in the related ß-lactamases. Molecular simulations using Protein Energy Landscape Exploration reveal that this results from stabilizing the catalytically-productive conformations, demonstrating the dominance of the synergistic effect of the E014K and G238S substitutions in vitro in contexts that vary in terms of sequence and dynamics. Furthermore, three rounds of directed molecular evolution demonstrated that known cefotaximase-enhancing mutations were accessible regardless of the differences in dynamics. Interestingly, specific sequence differences between the related ß-lactamases were shown to have a higher effect in evolutionary outcomes than did differences in dynamics. Overall, these ß-lactamase models show tolerance to protein dynamics at the timescale of catalytic turnover in the evolution of a new function.

Strategies Towards Protease Inhibitors for Emerging Flaviviruses.

Infections with flaviviruses are a continuing public health threat. In addition to vaccine development and vector control, the search for antiviral agents that alleviate symptoms in patients are of considerable interest. Among others, the flaviviral protease NS2B-NS3 is a promising drug target to inhibit viral replication. Flaviviral proteases share a high degree of structural similarity and substrate-recognition profile, which may facilitate a strategy towards development of pan-flaviviral protease inhibitors. However, the success of various drug discovery attempts during the last decade has been limited by the nature of the viral enzyme as well as a lack of robust structural templates. Small-molecular, structurally diverse protease inhibitors have been reported to reach affinities in the lower micromolar range. Peptide-based, substrate-derived compounds are often nanomolar inhibitors, however, with highly compromised drug-likeness. With some exceptions, the antiviral cellular activity of most of the reported compounds have been patchy and insufficient for further development. Recent progress has been made in the elucidation of inhibitor binding using different structural methods. This will hopefully lead to more rational attempts for the identification of various lead compounds that may be successful in cellular assays, animal models and ultimately translated to patients.

Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor

About 50% of the world’s arable land is strongly acidic (pH ≤ 5). The low pH solubilizes root-toxic ionic aluminium (Al3+) species from clay minerals, driving the evolution of counteractive adaptations in cultivated crops. The food crop Sorghum bicolor upregulates the membrane-embedded transporter protein SbMATE in its roots. SbMATE mediates efflux of the anionic form of the organic acid, citrate, into the soil rhizosphere, chelating Al3+ ions and thereby imparting Al-resistance based on excluding Al+3 from the growing root tip. Here, we use electrophysiological, radiolabeled, and fluorescence-based transport assays in two heterologous expression systems to establish a broad substrate recognition profile of SbMATE, showing the proton and/or sodium-driven transport of 14C-citrate anion, as well as the organic monovalent cation, ethidium, but not its divalent analog, propidium. We further complement our transport assays by measuring substrate binding to detergent-purified SbMATE protein. Finally, we use the purified membrane protein as an antigen to discover native conformation-binding and transport function-altering nanobodies using an animal-free, mRNA/cDNA display technology. Our results demonstrate the utility of using Pichia pastoris as an efficient eukaryotic host to express large quantities of functional plant transporter proteins. The nanobody discovery approach is applicable to other non-immunogenic plant proteins.

Molecular Rationale behind the Differential Substrate Specificity of Bacterial RND Multi-Drug Transporters

Resistance-Nodulation-cell Division (RND) transporters AcrB and AcrD of Escherichia coli expel a wide range of substrates out of the cell in conjunction with AcrA and TolC, contributing to the onset of bacterial multidrug resistance. Despite sharing an overall sequence identity of ~66% (similarity ~80%), these RND transporters feature distinct substrate specificity patterns whose underlying basis remains elusive. We performed exhaustive comparative analyses of the putative substrate binding pockets considering crystal structures, homology models and conformations extracted from multi-copy μs-long molecular dynamics simulations of both AcrB and AcrD. The impact of physicochemical and topographical properties (volume, shape, lipophilicity, electrostatic potential, hydration and distribution of multi-functional sites) within the pockets on their substrate specificities was quantitatively assessed. Differences in the lipophilic and electrostatic potentials among the pockets were identified. In particular, the deep pocket of AcrB showed the largest lipophilicity convincingly pointing out its possible role as a lipophilicity-based selectivity filter. Furthermore, we identified dynamic features (not inferable from sequence analysis or static structures) such as different flexibilities of specific protein loops that could potentially influence the substrate recognition and transport profile. Our findings can be valuable for drawing structure (dynamics)-activity relationship to be employed in drug design.

The malonyl‐CoA:ACP transacylase R117A variant catalyzes acyl‐transfer with a broad range of acyl‐CoA substrates

Prokaryotic fatty acid biosynthesis is catalyzed by multiple dissociated enzymes and represents a target for pathway engineering to generate novel biorenewable chemicals. We probed the active site of the E. coli malonyl-CoA:ACP transacylase (FabD) with site directed mutagenesis to increase the substrate recognition profile. One single substitution, R117A, was predicted to disrupt anionic interaction with the malonate enzyme intermediate as well as several hydrogen bonds and allow for the entry of nonpolar and branched CoA substrates. We developed a rapid novel discontinuous fluorescence assay and probed FabD plus FabD R117A activity by measuring specific activities. FabD R117A efficiently transfers acetyl- (120 nmoles min−1 mg−1), acetoacetyl-(60), propionyl-(52), butyryl-(11), isobutyryl-(4), succinyl-(16), and methylmalonyl- (3200) thioesters, unlike the wild-type FabD that prefers malonyl-CoA (4 mmoles min−1 mg−1) over methylmalonyl-CoA(0.27) and acetoacetyl-CoA (2 nmoles min−1 mg−1). KM values for FabD R117A vary slightly when compared to the wild type enzyme with malonyl-CoA indicating catalytic rates are decreased. In total, these data suggest the FabD R117A variant represents a starting point for catalyst design to synthesize novel biorenewable molecules. Support or Funding InformationNational Science Foundation under Award No. EEC-0813570 Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology at Iowa State University

A Novel highly thermostable branched-chain amino acid aminotransferase from the crenarchaeon Vulcanisaeta moutnovskia.

A new fold-type IV branched-chain amino acid aminotransferase VMUT0738 from the hyperthermophilic Crenarchaeon Vulcanisaeta moutnovskia was successfully expressed in Escherichia coli. Purified VMUT0738 showed activity toward numerous aliphatic and aromatic l-amino acids and 2-oxo acids at optimal pH 8.0. Distinguishing features of the VMUT0738 compared with typical BCAT are the absence of activity toward acidic substrates, high activity toward basic ones, and low but detectable activity toward the (R)-enantiomer of α-methylbenzylamine (0.0076U/mg) The activity of VMUT0738 increases with a rise in the temperature from 60°C to 90°C. VMUT0738 showed high thermostability (after 24h incubation at 70°C the enzyme lost only 27% of the initial activity) and the resistance to organic solvents. The sequence alignment revealed two motifs (V/I)xLDxR and PFG(K/H)YL characteristic of BCATs from species of the related genera Vulcanisaeta, Pyrobaculum and Thermoproteus that might be responsible for the unique substrate recognition profile of the enzyme.

The Extended Cleavage Specificity of Human Thrombin

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1′-Ser/Ala/Gly/Thr, P2′-not acidic and P3′-Arg. Our analysis also identifies an important role for a P3′-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3′-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200–400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1–30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.

In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile

The need to identify anti-Flaviviridae agents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.

The extended substrate recognition profile of the dog mast cell chymase reveals similarities and differences to the human chymase

Human chymase (HC) constitutes a major granule protease in one of the two human mast cell (MC) types. The main biological role of this haematopoietic serine protease is probably not yet known, although it has been implicated in a large number of functions. Dogs, like humans, have only one chymase. This enzyme is closely related to its human homologue, and the MC subtypes of human and dog appear to be similar as well. Therefore, the functions of the dog chymase (DC) may closely reflect the functions of the HC. Moreover, dogs may serve as good models for studies of human MC functions and MC-related diseases. To reveal functional similarities and differences between the DC and HC, we have determined the extended cleavage specificity of the DC by substrate phage display. This method allows the simultaneous permutation of primed and unprimed substrate positions. The DC was found to have very similar preferences to its human counterpart for substrate positions P1, P3, P4 and P3', whereas their preferences differ at positions P2, P1' and P2'. Therefore, the HC and DC may have co-evolved with a substrate where positions P1, P3, P4 and P3' are conserved between dogs and humans, whereas positions P2 and P1' are not and P2'differs to a minor extent. The differences observed between these two enzymes suggest that results obtained from dog models cannot be directly extrapolated to human clinical settings but need to be evaluated carefully concerning potential differences in substrate preferences.

A P-type ATPase importer that discriminates between essential and toxic transition metals

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts.

The extended substrate specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile

The human chymase (HC) is a major granule constituent of mast cells (MCs) residing in the connective tissue and the sub-mucosa. Although many potential substrates have been described for this important MC enzyme, its full range of in vivo substrates has most likely not yet been identified. A major step toward a better understanding of the function of the HC is therefore to determine its extended cleavage specificity. Using a phage-displayed random nonapeptide library, we show that the HC has a rather stringent substrate recognition profile. Only aromatic amino acids (aa) are accepted in position P1, with a strong preference for Tyr and Phe over Trp. Aliphatic aa are preferred in positions P2 to P4 N-terminal of the cleaved bond. In the P1' position C-terminal of the cleaved bond, Ser is clearly over-represented and acidic aa Asp and Glu are strongly preferred in the P2' position. In P3', the small aliphatic aa Ala, Val and Gly were frequently observed. The consensus sequence, from P4 to P3': Gly/Leu/Val-Val/Ala/Leu-Ala/Val/Leu-Tyr/Phe-Ser-Asp/Glu-Ala/Val/Gly, provides an instrument for the identification of novel in vivo substrates for the HC. Interestingly, a very similar cleavage specificity was recently reported for the major chymase in mouse connective tissue mast cells (CTMCs), the beta-chymase mouse mast cell protease-4, suggesting functional homology between these two enzymes. This indicates that a rather stringent chymotryptic substrate recognition profile has been evolutionary conserved for the dominant CTMC chymase in mammals.

Characterization and kinetic analysis of enzyme-substrate recognition by three recombinant lactococcal PepVs

The dipeptidases (PepVs) from three typical lactococcal strains, Lactococcus lactis subsp. lactis (L9), L. lactis subsp. cremoris (L6) and L. lactis subsp. hordniae (hT) were cloned and characterized. The metal-binding, catalytic, and substrate-binding sites are highly conserved among of them. A computer-generated three-dimensional model suggested that the amino acid differences between these PepVs were mostly located away from the active center. L9 PepV does not hydrolyze dipeptides bearing Pro or d-amino acid at the C-terminal amino acid. Unlike PepV from Lactobacillus delbrueckii, L9 PepV does not cleave β-Asp-His, and has little ability to cleave dipeptides containing a β-alanine. In addition, L9 PepV has a much higher k cat for dipeptides with an N-terminal Ala but a significantly higher K m when the N-terminal amino acid is Gly. The substrate recognition profile of PepV is further discussed on the basis of the kinetic analysis and the structural model.

Promiscuity in multidrug recognition and transport: the bacterial MFS Mdr transporters

Multidrug (Mdr) transport is an obstacle to the successful treatment of cancer and infectious diseases, and it is mediated by Mdr transporters that recognize and export an unusually broad spectrum of chemically dissimilar toxic compounds. Therefore, in addition to its clinical significance, the Mdr transport phenomenon presents intriguing and challenging mechanistic queries. Recent studies of secondary Mdr transporters of the major facilitator superfamily (MFS) have revealed that they are promiscuous not only regarding their substrate recognition profile, but also with respect to matters of energy utilization, electrical and chemical flexibility in the Mdr recognition pocket, and surprisingly, also in their physiological functions.

Elucidation of the Substrate Specificity of the C1s Protease of the Classical Complement Pathway

The complement system is a central component of host defense but can also contribute to the inflammation seen in pathological conditions. The C1s protease of the first complement component, the C1 complex, initiates the pathway. In this study we have elucidated the full specificity of the enzyme for the first time using a randomized phage display library. It was found that, aside from the crucial P(1) position, the S(3) and S(2) subsites (in that order) played the greatest role in determining specificity. C1s prefers Leu or Val at P(3) and Gly or Ala residues at P(2). Apart from the S(2)' position, which showed specificity for Leu, prime subsites did not greatly affect specificity. It was evident, however, that together they significantly contributed to the efficiency of cleavage of a peptide. A peptide substrate based on the top sequence obtained in the phage display validated these results and produced the best kinetics of any C1s substrate to date. The results allow an understanding of the active site specificity of the C1s protease for the first time and provide a basis for the development of specific inhibitors aimed at controlling inflammation associated with complement activation in adverse pathological situations.

Identification and characterisation of Arabidopsis glycosyltransferases capable of glucosylating coniferyl aldehyde and sinapyl aldehyde

This study describes the substrate recognition profile of UGT72E1, an UDP–glucose:glycosyltransferase of Arabidopsis thaliana that is the third member of a branch of glycosyltransferases, capable of conjugating lignin monomers and related metabolites. The data show that UGT72E1, in contrast to the two closely related UGTs 72E2 and 72E3, is specific for sinapyl and coniferyl aldehydes. The biochemical properties of UGT72E1 are characterised, and are compared with that of UGT72E2, which is capable of glycosylating the aldehydes as well as coniferyl and sinapyl alcohols.

A unique substrate recognition profile for matrix metalloproteinase-2.

The catalytic domains of the matrix metalloproteinases (MMPs) are structurally homologous, raising questions as to the degree of distinction, or overlap, in substrate recognition. The primary objective of the present study was to define the substrate recognition profile of MMP-2, a protease that was historically referred to as gelatinase A. By cleaving a phage peptide library with recombinant MMP-2, four distinct sets of substrates were identified. The first set is structurally related to substrates previously reported for other MMPs. These substrates contain the PXX/X(Hy) consensus motif (where X(Hy) is a hydrophobic residue) and are not generally selective for MMP-2 over the other MMPs tested. Two other groups of substrates were selected from the phage library with similar frequency. Substrates in group II contain the L/IXX/X(Hy) consensus motif. Substrates in group III contain a consensus motif with a sequence of X(Hy)SX/L, and the fourth set of substrates contain the HXX/X(Hy) sequence. Substrates in Group II, III, and IV were found to be 8- to almost 200-fold more selective for MMP-2 over MMP-9. To gain an understanding of the structural basis for substrate selectivity, individual residues within substrates were mutated, revealing that the P(2) residue is a key element in conferring selectivity. These findings indicate that MMP-2 and MMP-9 exhibit different substrate recognition profiles and point to the P(2) subsite as a primary determinant in substrate distinction.

PH-Enhanced Cytopathic Effects of Clostridium sordellii Lethal Toxin

Clostridium sordellii lethal toxin (TcsL) is a large clostridial toxin (LCT) that glucosylates Ras, Rac, and Ral. TcsL differs from other LCTs because it modifies Ras, which does not cycle from cytosol to membrane. By using a suite of inhibitors, steps in cell entry by TcsL were dissected, and entry appears to be dependent on endosomal acidification. However, in contrast to TcdB, TcsL was substantially slower in its time course of entry. TcsL cytopathic effects (CPE) were blocked by bafilomycin A1 and neutralized by antiserum up to 2 h following treatment of cells with the toxin. The slow time course of intoxication and relatively high cytopathic dose were alleviated by exposing TcsL to acid pH, resulting in a time course similar to that of TcdB. The optimal pH range for activation was 4.0 to 5.0, which increased the rate of intoxication over 5-fold, lowered the minimal intoxicating dose by over 100-fold, and allowed complete substrate modification within 2 h, as shown by differential glucosylation. Fluorescence analysis of TcsL with 2-(p-toluidinyl) naphthalene-6-sulfonic acid as a probe suggested the acid pH stimulated a hydrophobic transition in the protein, a likely prelude to membrane insertion. Finally, acid entry by TcsL caused TcdB-like morphological changes in CHO cells, which suggesting that acid activation may impact substrate recognition profiles for TcsL.

A Substrate Phage Enzyme-Linked Immunosorbent Assay to Profile Panels of Proteases

It is estimated that proteases comprise nearly 2% of the human genome. Given that the primary structure of all known proteases will soon be available, an important challenge is to define the structure-activity relationships that govern substrate hydrolysis. Ideally this would be accomplished on a genome-wide scale. To this end, we have developed a one-pot phage selection system that yields the substrate recognition profile of multiple proteases from a single round of selection. The system meets five key criteria: (i) multiple proteases can be analyzed simultaneously, (ii) prior knowledge of substrate preference is not required, (iii) information regarding substrate preferences on both side of the scissile bond is obtained, (iv) the system yields selective substrates that distinguish closely related proteases, and (v) semiquantitative information on substrate hydrolysis is obtained, allowing for the assignment of initial rank-order preferences. As an illustration, a phage selection with a mixture of thrombin and factor Xa (serine proteases) along with matrix-metalloproteinase-9 and atrolysin C (metalloproteinases) was performed. Peptide substrates were identified that (i) have high k cat/ K m ratios, (ii) are selective for individual proteases, and (iii) match the sequences of known physiological substrates.