Active site structure and catalytic mechanisms of human peroxidases

Myeloperoxidase (MPO), eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase are heme-containing oxidoreductases (EC 1.7.1.11), which bind ligands and/or undergo a series of redox reactions. Though sharing functional and structural homology, reflecting their phylogenetic origin, differences are observed regarding their spectral features, substrate specificities, redox properties, and kinetics of interconversion of the relevant redox intermediates ferric and ferrous peroxidase, compound I, compound II, and compound III. Depending on substrate availability, these heme enzymes path through the halogenation cycle and/or the peroxidase cycle and/or act as poor (pseudo-)catalases. Based on the published crystal structures of free MPO and its complexes with cyanide, bromide and thiocyanate as well as on sequence analysis and modeling, we critically discuss structure-function relationships. This analysis highlights similarities and distinguishing features within the mammalian peroxidases and intents to provide the molecular and enzymatic basis to understand the prominent role of these heme enzymes in host defense against infection, hormone biosynthesis, and pathogenesis.     

The crystal structure of Klebsiella pneumoniae FeoA reveals a site for protein-protein interactions

In order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe²⁺) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate that KpFeoA can assume both “open” and “closed” conformations, controlled by binding at this Leu-rich ridge. We propose a model in which a “C-shaped” clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions and provide a framework for testing FeoA-FeoB interactions, which could be exploited for future antibiotic developments.     

X-ray structure of human aromatase reveals an androgen-specific active site

Aromatase is a unique cytochrome P450 that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens for the synthesis of estrogens. All human estrogens are synthesized via this enzymatic aromatization pathway. Aromatase inhibitors thus constitute a frontline therapy for estrogen-dependent breast cancer. Despite decades of intense investigation, this enzyme of the endoplasmic reticulum membrane has eluded all structure determination efforts. We have determined the crystal structure of the highly active aromatase purified from human placenta, in complex with its natural substrate androstenedione. The structure shows the binding mode of androstenedione in the catalytically active oxidized high-spin ferric state of the enzyme. Hydrogen bond-forming interactions and tight packing hydrophobic side chains that complement the puckering of the steroid backbone provide the molecular basis for the exclusive androgenic specificity of aromatase. Locations of catalytic residues and water molecules shed new light on the mechanism of the aromatization step. The structure also suggests a membrane integration model indicative of the passage of steroids through the lipid bilayer.     

Crystal structure of the catalytic domain of human atypical protein kinase C-iota reveals interaction mode of phosphorylation site in turn motif

Atypical protein kinases C (aPKCs) play critical roles in signaling pathways that control cell growth, differentiation and survival. Therefore, they constitute attractive targets for the development of novel therapeutics against cancer. The crystal structure of the catalytic domain of atypical PKCiota in complex with the bis(indolyl)maleimide inhibitor BIM1 has been determined at 3.0A resolution within the frame of the European Structural Proteomics Project SPINE. The overall structure exhibits the classical bilobal kinase fold and is in its fully activated form. Both phosphorylation sites (Thr403 in the activation loop, and Thr555 in the turn motif) are well defined in the structure and form intramolecular ionic contacts that make an important contribution in stabilizing the active conformation of the catalytic subunit. The phosphorylation site in the hydrophobic motif of atypical PKCs is replaced by the phosphorylation mimic glutamate and this is also clearly seen in the structure of PKCiota (residue 574). This structure determination for the first time provides the architecture of the turn motif phosphorylation site, which is characteristic for PKCs and PKB/AKT, and is completely different from that in PKA. The bound BIM1 inhibitor blocks the ATP-binding site and puts the kinase domain into an intermediate open conformation. The PKCiota-BIM1 complex is the first kinase domain crystal structure of any atypical PKC and constitutes the basis for rational drug design for selective PKCiota inhibitors.     

Functional interactions of catalytic site and transmembrane channel in the sarcoplasmic reticulum ATPase

Vesicular fragments of longitudinal sarcoplasmic reticulum were loaded with calcium by active transport, sedimented by centrifugation, and resuspended in neutral buffer and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Under these conditions, calcium efflux from the loaded vesicles occurred at rates varying from 100 to 700 nmol/mg/min, depending on the calcium load. If either Ca2+ (microM), Mg2+ (mM), K+ or Na+ (greater than 10 mM) were added to the resuspension medium, the rate of efflux was reduced. In the presence of Mg2+ and EGTA, a large inhibition of calcium efflux was produced by formation of phosphoenzyme intermediate with Pi. In this case, addition of ADP again started calcium efflux, coupled with ATP synthesis. The rates of uncoupled or coupled efflux were approximately the same. The observed calcium fluxes are attributed to a slow channel formed by ATPase transmembrane helices (MacLennan, D. H., Brandl, C. J., Korczak, B., and Green, N. M. (1985) Nature 316, 686-700) and are capable of long range interaction with the catalytic site. Coupling of transport and catalytic activities is thereby produced by phosphorylation and ligand binding. The channel includes negatively charged residues that are likely to influence calcium fluxes through cation binding. It is proposed that this channel is the mechanistic device for active transport of calcium across the sarcoplasmic reticulum membrane, and for its reversal.     

Highly multiplexed bioactivity screening reveals human and microbiota metabolome-GPCRome interactions

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Further characterization of the putative human isopeptidase T catalytic site

The human isopeptidase T (isoT) is a zinc-binding deubiquitinating enzyme involved in the disassembly of free K48-linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site-directed mutagenized. None of the mutants were able to cleave a peptide-linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48-linked isopeptide ubiquitin dimer, which is an isoT-specific substrate that mimics the K48-linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48-linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.     

A catalytic synthesis of dialkylamines from alkylamines using neopentyl-substituted PNP pincer-iridium complex

A combination of neopentyl-substituted PNP-iridium complex 2 and NaH could catalyze dimerization of alkylamines to form dialkylamines with the highest activity ever reported. Primary and secondary alkylamines were applicable to the present catalytic reaction. Several mechanistic studies suggested a plausible catalytic cycle. The high activity of catalyst 2 may come from the role of neopentyl groups to make a space around the metal center.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

Structure of the human multidrug resistance protein 1 nucleotide binding domain 1 bound to Mg2+/ATP reveals a non-productive catalytic site

Human multidrug resistance protein 1 (MRP1) is a membrane protein that belongs to the ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 contributes to chemotherapy failure by exporting a wide range of anti-cancer drugs when over expressed in the plasma membrane of cells. Here, we report the first high-resolution crystal structure of human MRP1-NBD1. Drug efflux requires energy resulting from hydrolysis of ATP by nucleotide binding domains (NBDs). Contrary to the prokaryotic NBDs, the extremely low intrinsic ATPase activity of isolated MRP1-NBDs allowed us to obtain the structure of wild-type NBD1 in complex with Mg2+/ATP. The structure shows that MRP1-NBD1 adopts a canonical fold, but reveals an unexpected non-productive conformation of the catalytic site, providing an explanation for the low intrinsic ATPase activity of NBD1 and new hypotheses on the cooperativity of ATPase activity between NBD1 and NBD2 upon heterodimer formation.     

Crystal structure of human PNP complexed with guanine

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. More recently, the 3-D structure of human PNP has been refined to 2.3A resolution, which allowed a redefinition of the residues involved in the substrate-binding sites and provided a more reliable model for structure-based design of inhibitors. This work reports crystallographic study of the complex of Human PNP:guanine (HsPNP:Gua) solved at 2.7A resolution using synchrotron radiation. Analysis of the structural differences among the HsPNP:Gua complex, PNP apoenzyme, and HsPNP:immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design.     

Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site

PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.     

Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site

We report crystal structures of the human enzyme phosphoenolpyruvate carboxykinase (PEPCK) with and without bound substrates. These structures are the first to be determined for a GTP-dependent PEPCK, and provide the first view of a novel GTP-binding site unique to the GTP-dependent PEPCK family. Three phenylalanine residues form the walls of the guanine-binding pocket on the enzyme's surface and, most surprisingly, one of the phenylalanine side-chains contributes to the enzyme's specificity for GTP. PEPCK catalyzes the rate-limiting step in the metabolic pathway that produces glucose from lactate and other precursors derived from the citric acid cycle. Because the gluconeogenic pathway contributes to the fasting hyperglycemia of type II diabetes, inhibitors of PEPCK may be useful in the treatment of diabetes.     

Phylogenetic divergence of CD47 interactions with human signal regulatory protein alpha reveals locus of species specificity. Implications for the binding site

Cell-cell interactions between ubiquitously expressed integrin-associated protein (CD47) and its counterreceptor signal regulatory protein (SIRPalpha) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRPalpha binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRPalpha as a probe show that neither human CD47 nor SIRPalpha requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRPalpha-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRPalpha. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRPalpha to about one-third that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.     

Solid-state NMR analysis of ligand--receptor interactions reveals an induced misfit in the binding site of isorhodopsin

Rhodopsin is the photosensitive protein of the rod photoreceptor in the vertebrate retina and is a paradigm for the superfamily of G-protein-coupled receptors (GPCRs). Natural rhodopsin contains an 11-cis-retinylidene chromophore. We have prepared the 9-cis analogue isorhodopsin in a natural membrane environment using uniformly (13)C-enriched 9-cis retinal. Subsequently, we have determined the complete (1)H and (13)C assignments with ultra-high field solid-state magic angle spinning NMR. The 9-cis substrate conforms to the opsin binding pocket in isorhodopsin in a manner very similar to that of the 11-cis form in rhodopsin, but the NMR data reveal an improper fit of the 9-cis chromophore in this binding site. We introduce the term "induced misfit" to describe this event. Downfield proton NMR ligation shifts (Deltasigma(lig)(H) > 1 ppm) are observed for the 16,17,19-H and nearby protons of the ionone ring and for the 9-methyl protons. They provide converging evidence for global, nonspecific steric interactions between the chromophore and protein, and contrast with the specific interactions over the entire ionone ring and its substituents detected for rhodopsin. The Deltasigma(lig)(C) pattern of the polyene chain confirms the positive charge delocalization in the polyene associated with the protonation of the Schiff base nitrogen. In line with the misalignment of the ionone ring, an additional and anomalous perturbation of the (13)C response is detected in the region of the 9-cis bond. This provides evidence for strain in the isomerization region of the polyene and supports the hypothesis that perturbation of the conjugation around the cis bond induced by the protein environment assists the selective photoisomerization.     

Glycosylation is crucial for a proper catalytic site organization in human glucocerebrosidase

Gaucher disease, an autosomal recessive disorder, is caused by a deficiency of glucocerebrosidase (GCase) enzyme, a peripheral membrane-associated glycoprotein that hydrolyses glucosylceramide in lysosomes. Glycosylation is essential for the development of a catalytically active enzyme, specifically in the first site, located at Asn19. However, both the molecular basis of the relevance of N-glycosylation over GCase activity and the effects of glycosylation over its structure and dynamics are still not fully understood. Thus, the present work evaluated GCase enzyme in increasing glycosylation content using triplicate unbiased molecular dynamics simulations. Accordingly, the N-linked glycan chains caused local conformational stabilization effects over the protein, as well as in regions flanking the enzyme catalytic dyad. In the case of the Asn19-linked glycan, it also occurred around region 438–444, where one of the most prevalent GCase mutations is found. Markedly, an increasing catalytic dyad organization was related to increasing glycosylation contents, offering the first atomic-level explanation for the experimental observation that GCase activity is controlled by glycosylation, especially at Asn19.     

The catalytic site residues and interfacial binding of human pancreatic lipase.

In this study, the essential serine residue and 2 other amino acids in human pancreatic triglyceride lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) were tested for their contribution to the enzyme's catalytic site or interfacial binding site. By site-specific mutagenesis of the cDNA for human pancreatic lipase, amino acid substitutions were made at Ser153, His264, and Asp177. The mutant cDNAs were expressed in transfected COS-1 cells. Both the medium and the cells were examined for the presence of pancreatic lipase by Western blot analysis. The activity of the expressed proteins against triolein and the interfacial binding was measured. Proteins with mutations in Ser153 were secreted by the cells and bound to interfaces but had no detectable activity. Changing His264 to a leucine or Asp177 to an asparagine also produced inactive lipase. Substituting glutamic acid for Asp177 produced an active protein. These results demonstrate that Ser153 is involved in the catalytic site of pancreatic lipase and is not crucial for interfacial binding. Moreover, the essential roles of His264 and Asp177 in catalysis were demonstrated. A Ser-His-Asp catalytic triad similar to that present in serine proteases is present in human pancreatic lipase.