Insights into substrate specificity and metal activation of mammalian tetrahedral aspartyl aminopeptidase

Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases.     

Insights into S100 target specificity examined by a new interaction between S100A11 and annexin A2

S100 proteins are a group of EF-hand calcium-signaling proteins, many of which interact with members of the calcium- and phospholipid-binding annexin family of proteins. This calcium-sensitive interaction enables two neighboring membrane surfaces, complexed to different annexin proteins, to be brought into close proximity for membrane reorganization, using the S100 protein as a bridging molecule. S100A11 and S100A10 are two members of the S100 family found to interact with the N-termini of annexins A1 and A2, respectively. Despite the high degree of structural similarity between these two complexes and the sequences of the peptides, earlier studies have shown that there is little or no cross-reactivity between these two S100s and the annexin peptides. In the current work the specificity and the affinity of the interaction of the N-terminal sequences of annexins A1 and A2 with Ca2+-S100A11 were investigated. Through the use of alanine-scanning peptide array experiments and NMR spectroscopy, an approximate 5-fold tighter interaction was identified between Ca2+-S100A11 and annexin A2 (approximately 3 microM) compared to annexin A1 (approximately 15 microM). Chemical shift mapping revealed that the binding site for annexin A2 on S100A11 was similar to that observed for the annexin A1 but with distinct differences involving the C-terminus of the annexin A2 peptide. In addition, kinetic measurements based on NMR titration data showed that annexin A2 binding to Ca2+-S100A11 occurs at a comparable rate (approximately 120 s(-1)) to that observed for membrane fusion processes such as endo- and exocytosis.     

Insights into the three-dimensional structure of crotalase: Implications for biological activity and substrate specificity

Crotalase is a serine protease from eastern diamondback rattlesnake (Crotalus adamanteus) venom. Crotalase has high amino-acid sequence similarity to three other members of the serine protease family, -thrombin, β-trypsin and kallikrein A. Their structural information was used to predict the folding of crotalase. The computational structural data were used to explain biochemical properties of this important enzyme.

The first computational model for the structure of crotalase is reported herein. The implications of the details of the structure for the biological activity are discussed.     

Insights into the substrate specificity of a novel snake venom serine peptidase by molecular modeling

The cDNA encoding BthaTL, a serine peptidase from the venom of the snake Bothrops alternatus, was cloned and sequenced. The deduced primary structure shows over 62% of identity with snake venom thrombin-like enzymes (SVTLEs), molecules with high substrate specificity toward different natural substrates. Indeed, a phylogenetic reconstruction by two different methods clustered this enzyme close to other SVTLEs. These enzymes generally affect the hemostatic system in several ways, and therefore are used as tools in pharmacology and clinical diagnosis. A three-dimensional model of BthaTL was built by homology modeling using TSV-PA (Trimeresurus stejnegeri venom plasminogen activator) crystal structure as template. BthaTL model showed that the typical catalytic triad conformation of serine peptidases was preserved. The calcium coordination ligands were absent or adopt an unfavorable conformation, preventing interactions with metals. On the other hand, the Asp97-Arg174 saline bridge of TSV-PA was not found and its specificity determinant Phe193 is replaced by a Gly in BthaTL. The substitution of essential residues in the neighborhoods of the catalytic site cleft of BthaTL indicates that these two proteins do not share the same enzymatic specificity, what means that BthaTL will probably not activate plasminogen. Such observations may be helpful in the understanding of the molecular mechanism for substrate specificity of these enzymes.     

Insights into the HER-2 receptor tyrosine kinase mechanism and substrate specificity using a transient kinetic analysis

The HER-2/erbB-2/c-neu proto-oncogene encodes for an EGF receptor-like protein which has been implicated in the pathogenesis of several human malignancies. Although much has been learned about the physiological significance of this receptor tyrosine kinase, its catalytic mechanism remains poorly understood. We have expressed, purified, and characterized two recombinant proteins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an optimal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degenerate peptide library. We have conducted a transient kinetic analysis of the HER-2 proteins (HCD and HKD) to illuminate mechanistic details of the HER-2 pathway. In particular, stopped-flow fluorescence studies with mant (N-methylanthraniloyl)-nucleotide derivatives provided direct measurements of the association and dissociation rate constants for these nucleotide interactions with the HER-2 recombinant proteins, thereby enabling the determination of nucleotide K(d) values. Moreover, the actual step of chemical catalysis was isolated using rapid chemical quench techniques and shown to occur approximately 3-fold faster than the steady-state rate which corresponds to product release. Evidence is also provided that suggests a conformational change that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was examined. Studies carried out with pre-phosphorylated recombinant HER-2 proteins suggest that while autophosphorylation is not a prerequisite for enzymatic activity, this protein modification actually directly affects the catalytic mechanism by enhancing the rate of ADP release and that of the rate-limiting step. While a pre-steady-state kinetic analysis has been carried out on the catalytic subunit of cAMP-dependent serine/threonine kinase, to our knowledge, this study represents the first reported transient kinetic investigation of a receptor tyrosine kinase. This work serves as a basis for comparison of these two important protein kinase families and in this report we highlight these similarities and differences.     

Insights into substrate recognition by the Escherichia coli Orf135 protein through its solution structure

Escherichia coli Orf135 hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-oxo-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP. Nucleotide pool sanitization by Orf135 is important since nucleotides are continually subjected to potential damage by reactive oxygen species produced during respiration. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, even though they recognize various substrates and possess a variety of substrate binding pockets. We investigated the tertiary structure of Orf135 and its interaction with a 2-hydroxy-dATP analog using NMR. We report on the solution structure of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrates.     

Insights into role of the hydrogen bond networks in substrate recognition by UDP-GalNAc 4-epimerases

UDP-hexose 4-epimerases are critical in galactose metabolism and often important in lipopolysaccharide biosynthesis as well. Three groups of these enzymes have been reported based on their substrate specificity towards non-acetylated substrates (group 1), dual specificity towards N-acetylated and non-acetylated substrates (group 2) and specificity towards N-acetylated substrates (group 3). We recently reported the structure of a novel UDP-GalNAc 4-epimerase called WbgU and based on the structure proposed a model of specific substrate recognition by UDP-GalNAc 4-epimerases. In this work, we present an analysis of the proposed model of substrate recognition using site-directed mutagenesis of WbgU and crystal structure of the His305Ala mutant. This investigation reveals that the wild-type activity of WbgU is retained in most single-point mutants targeting the active site. However, a graded loss in activity is observed in double-and triple-point mutants with the quadruple-point mutant being completely inactive corroborating the proposed rationale of substrate recognition. Furthermore, crystal structure of the His305Ala mutant shows that the structure is significantly similar to the wild-type WbgU, albeit a loss in the critical hydrogen bond network seated at His305 and ensuing minor conformational changes. It is inferred that the specific and non-specific interactions throughout the active site confer it sufficient elasticity to sustain wild-type activity for several of the single-point mutations.     

Molecular insight into substrate recognition by human cytosolic sialidase NEU2

Sialidases or neuramidases are glycoside hydrolases removing terminal sialic acid residues from sialo-glycoproteins and sialo-glycolipids. Viral neuraminidases (NAs) have been extensively characterized and represent an excellent target for antiviral therapy through the synthesis of a series of competitive inhibitors that block the release of newly formed viral particles from infected cells. The human cytosolic sialidase NEU2 is the only mammalian enzyme structurally characterized and represents a valuable model to study the specificity of novel NA inhibitory drugs. Moreover, the availability of NEU2 3D structure represents a pivotal step toward the characterization of the molecular basis of natural substrates recognition by the enzyme. In this perspective, we have carried out a study of molecular docking of NEU2 active site using natural substrates of increasing complexity. Moreover, selective mutations of the residues putatively involved into substrate(s) interaction/recognition have been performed, and the resulting mutant enzymes have been preliminary tested for their catalytic activity and substrate specificity. We found that Q270 is involved in the binding of the disaccharide α(2,3) sialyl-galactose, whereas K45 and Q112 bind the distal glucose of the trisaccharide α(2,3) sialyl-lactose, corresponding to the oligosaccharide moiety of GM3 ganglioside. In addition, E218, beside D46, is proved to be a key catalytic residue, being, together with Y334, the second member of the nucleophile pair required for the catalysis. Overall, our results point out the existence of a dynamic network of interactions that are possibly involved in the recognition of the glycans bearing sialic acid.     

Crystal structure of group A streptococcus Mac-1: insight into dimer-mediated specificity for recognition of human IgG

Group A Streptococcus secretes cysteine proteases named Mac-1 and Mac-2 that mediate host immune evasion by targeting both IgG and Fc receptors. Here, we report the crystal structures of Mac-1 and its catalytically inactive C94A mutant in two different crystal forms. Despite the lack of sequence homology, Mac-1 adopts the canonical papain fold. Alanine mutations at the active site confirmed the critical residues involved in a papain-like catalytic mechanism. Mac-1 forms a symmetric dimer in both crystal forms and displays the unique dimer interface among papain superfamily members. Mutations at the dimer interface resulted in a significant reduction in IgG binding and catalysis, suggesting that the dimer contributes to both IgG specificity and enzyme cooperativity. A tunnel observed at the dimer interface constitutes a target for designing potential Mac-1-specific antimicrobial agents. The structures also offer insight into the functional difference between Mac-1 and Mac-2.     

Comparative modeling and molecular docking of orphan human CYP4V2 protein with fatty acid substrates: Insights into substrate specificity

Cytochromes P450 (CYPs) are a super family of heme-containing enzymes well-known for their monooxgenase reaction. There are 57 CYP isoenzymes found in human which exhibit specific physiological functions. Thirteen members of this super family are classified as "orphan" CYP because of their unknown enzymatic functions. CYP4V2 is found to be a potential drug target for Bietti crystalline corneoretinal dystrophy (BCD). However, three-dimensional structure, the active site topology and substrate binding modes of CYP4V2 remain unclear. In this study, the three-dimensional model of CYP4V2 was constructed using the homology modeling method. Four possible fatty acid substrates namely, caprylic, lauric, myrisitc and palmitic acids were optimized and evaluated for drug likeness using Lipinski's rule of five. Further, these substrates were docked into active sites of CYP4V2 and several key residues responsible for substrate binding were identified. These findings will be helpful for the structure-based drug design and detailed characterization of the biological roles of CYP4V2.     

GroEL/S substrate specificity based on substrate unfolding propensity

Phage P22 wild-type (WT) coat protein does not require GroEL/S to fold but temperature-sensitive-folding (tsf) coat proteins need the chaperone complex for correct folding. WT coat protein and all variants absolutely require P22 scaffolding protein, an assembly chaperone, to assemble into precursor structures termed procapsids. Previously, we showed that a global suppressor (su) substitution, T1661, which rescues several tsf coat protein variants, functioned by inducing GroEL/S. This led to an increased formation of tsf:T1661 coat protein:GroEL complexes compared with the tsf parents. The increased concentration of complexes resulted in more assembly-competent coat proteins because of a shift in the chaperone-driven kinetic partitioning between aggregation-prone intermediates toward correct folding and assembly. We have now investigated the folding and assembly of coat protein variants that carry a different global su substitution, F170L. By monitoring levels of phage production in the presence of a dysfunctional GroEL we found that tsf:F170L proteins demonstrate a less stringent requirement for GroEL. Tsf:F170L proteins also did not cause induction of the chaperones. Circular dichroism and tryptophan fluorescence indicate that the native state of the tsf: F170L coat proteins is restored to WT-like values. In addition, native acrylamide gel electrophoresis shows a stabilized native state for tsf:F170L coat proteins. The F170L su substitution also increases procapsid production compared with their tsf parents. We propose that the F170L su substitution has a decreased requirement for the chaperones GroEL and GroES as a result of restoring the tsf coat proteins to a WT-like state. Our data also suggest that GroEL/S can be induced by increasing the population of unfolding intermediates.     

Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases

Modular polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as erythromycin and picromycin. Understanding PKSs at high resolution could present new opportunities for chemoenzymatic synthesis of complex molecules. The crystal structures of macrocycle-forming thioesterase (TE) domains from the picromycin synthase (PICS) and 6-deoxyerythronolide B synthase (DEBS) were determined to 1.8-3.0 A with an R(crys) of 19.2-24.4%, including three structures of PICS TE (crystallized at pH 7.6, 8.0, and 8.4) and a second crystal form of DEBS TE. As predicted by the previous work on DEBS TE [Tsai, S. C., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14808-14813], PICS TE contains an open substrate channel and a hydrophobic dimer interface. Notwithstanding their similarity, the dimer interfaces and substrate channels of DEBS TE and PICS TE reveal key differences. The structural basis for the divergent substrate specificities of DEBS TE and PICS TE is analyzed. The size of the substrate channel increases with increasing pH, presumably due to electrostatic repulsion in the channel at elevated pH. Together, these structures support previous predictions that macrocycle-forming thioesterases from PKSs share the same protein fold, an open substrate channel, a similar catalytic mechanism, and a hydrophobic dimer interface. They also provide a basis for the design of enzymes capable of catalyzing regioselective macrocyclization of natural or synthetic substrates. A series of high-resolution snapshots of a protein channel at different pHs is presented alongside analysis of channel residues, which could help in the redesign of the protein channel architecture.     

Achieving broad substrate specificity in damage recognition by binding accessible nondamaged DNA

The structure of the Rad4/Rad23 protein, shown in a recent issue of Nature (Min and Pavletich, 2007), reveals how structurally diverse lesions are recognized in eukaryotic nucleotide excision repair: by probing for accessible nondamaged DNA opposite the lesion.     

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4? resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes.     

Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis

Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4- and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.     

Endoglycosidase S Enables a Highly Simplified Clinical Chemistry Procedure for Direct Assessment of Serum IgG Undergalactosylation in Chronic Inflammatory Disease

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Highlights

• •Selective release of IgG Fc glycans in crude serum by endoglycosidase S.
• •CE-LIF-based measurements of IgG undergalactosylation levels (UGS).
• •UGS as a biomarker: Stratification of non-alcoholic steatohepatitis patients and controls.