Molecular mechanisms for antibody-mediated modulation of peptide-displaying enzyme sensors

The generation of molecular sensors based on peptide-displaying enzymes for the detection of antibodies or antigens represents an innovative field of protein engineering. The knowledge of the underlying molecular mechanisms of enzymatic modulation in such sensors would be of great importance for the rational construction and improvement of responsiveness of new peptide-enzyme molecules. Here we analyze the enzymatic characteristics of three different kinds of sensors based in engineered beta-galactosidase, alkaline phosphatase and beta-lactamase, to explore a common activation basis. We describe two different categories of enzyme sensors. In one of them, including only some modified beta-lactamases, the enzymatic activity is inhibited upon ligand binding and it seems to be caused by the steric coverage of the active site by the bound antibody. In a second group, embracing members of the three studied enzymes, the ability to be modulated upon effector binding depends on the ratio between the k(cat) of the engineered enzyme and the k(cat) of the intact enzyme. This proves a common mechanism for enzymatic modulation of enzyme biosensors that is probably caused by conformational effects induced by the bound antibody on the enzyme.     

Tailoring molecular sensing for peptide displaying engineered enzymes

Regulable enzymes displaying foreign peptides are valuable instruments for molecular targeting and fast analyte detection in homogeneous assays. Both the specificity and the intensity of the signal generated by the sensor are critical parameters that can be manipulated by trial-and-error protein engineering in the vicinity of the active site. An alternative approach is presented to enhance signal-background ratio in -galactosidase-based molecular sensors by an optimisation of the sensing conditions. The screening of the enzymatic response in a set of engineered enzymes has revealed an antibody-dependent increase in their specific activity up to 500% for the enzyme, HD72CA, that is reached with 0.25 pmol enzyme per reaction in presence of 1.75 mM substrate. This value, much higher than 200% enzyme activation achieved only by protein engineering, represents a step further in enhancing the enzyme's responsiveness. On the other hand, engineered -galactosidases are also highly dynamic without preliminary antibody incubation, rendering activation factors around 300% after global reaction times shorter than 15 min. Therefore, this enzymatic system has been revealed as extremely robust and suitable for efficient and fast molecular detection in the diagnosis of infectious diseases.     

The acceptor specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases

The in vitro and in vivo specificity of the family of peptide:N-acetylgalactosaminyltransferases (GalNAcT) is analyzed on the basis of the reactivity and/or inhibitory activity of peptides and protein segments. The transferases appear to be multi-substrate enzymes with extended active sites containing a least nine subsites that interact cooperatively with a linear segment of at least nine amino acid residues on the acceptor polypeptide. Functional acceptor sites are located on the surface of the protein and extended conformations (-strand conformation) are preferred. The acceptor specificity of GalNAc-T can be predicted from the primary structure of the acceptor peptide with an accuracy of 70 to 80%. The same GalNAc-T enzymes catalyze the glycosylation of both serine and threonine residues. The higher in vitro catalytic efficiency toward threonine versus serine is the result of enhanced binding as well as increased reaction velocity, both effects being the result of steric interactions between the active site of the enzyme and the methyl group of threonine. Results from substrate binding studies suggest that GalNAc-T catalyzed transfer proceeds via an ordered sequential mechanism.     

Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET)

The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand.     

Identification of the substrate-binding sites of 2'-5'-oligoadenylate synthetase

2'-5'-Oligoadenylate synthetases are interferon-induced enzymes that upon activation by double-stranded RNA polymerize ATP to 2'-5'-linked oligoadenylates. In our continuing effort to understand the mechanism of catalysis by these enzymes, we used photo affinity cross-linking and peptide mapping to identify the substrate-binding sites of the P69 isozyme of human 2'-5'-oligoadenylate synthetases. Radiolabeled azido 2'-5'-oligoadenylate dimers were enzymatically synthesized and used as ligands for cross-linking to the P69 protein by exposure to ultraviolet light. The radiolabeled protein was digested with trypsin, and two ligand-cross-linked peptides were purified by immobilized aluminum affinity chromatography followed by reverse phase high pressure liquid chromatography. The peptides were identified by mass spectrometry and peptide sequencing and were found to correspond to residues 420-425 and 539-547 of P69. To examine the functional importance of the cross-linking sites, specific residues in the two peptides were mutated. When residues in the two sites were mutated individually, ligand cross-linking was selectively eliminated at the mutated site, and the enzyme activity was lost almost completely. Using substrates that can serve either as a donor or as an acceptor but not both, we could identify one of the sites as the acceptor and the other as the donor site.     

Engineering homooligomeric proteins to detect weak intersite allosteric communication: aminotransferases, a case study

The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.     

Flexibility and mutagenic resiliency of glycosyltransferases

The human blood group A and B antigens are synthesized by two highly homologous enzymes, glycosyltransferase A (GTA) and glycosyltransferase B (GTB), respectively. These enzymes catalyze the transfer of either GalNAc or Gal from their corresponding UDP-donors to αFuc1–2βGal-R terminating acceptors. GTA and GTB differ at only four of 354 amino acids (R176G, G235S, L266M, G268A), which alter the donor specificity from UDP-GalNAc to UDP-Gal. Blood type O individuals synthesize truncated or non-functional enzymes. The cloning, crystallization and X-ray structure elucidations for GTA and GTB have revealed key residues responsible for donor discrimination and acceptor binding. Structural studies suggest that numerous conformational changes occur during the catalytic cycle. Over 300 ABO alleles are tabulated in the blood group antigen mutation database (BGMUT) that provides a framework for structure-function studies. Natural mutations are found in all regions of GTA and GTB from the active site, flexible loops, stem region and surfaces remote from the active site. Our characterizations of natural mutants near a flexible loop (V175M), on a remote surface site (P156L), in the metal binding motif (M212V) and near the acceptor binding site (L232P) demonstrate the resiliency of GTA and GTB to mutagenesis.     

Transpeptidation reactions of a specific substrate catalyzed by the Streptomyces R61 DD-peptidase: the structural basis of acyl acceptor specificity

Bacterial dd-peptidases, the targets of beta-lactam antibiotics, are believed to catalyze d-alanyl-d-alanine carboxypeptidase and transpeptidase reactions in vivo. To date, however, there have been few concerted attempts to explore the kinetic and thermodynamic specificities of the active sites of these enzymes. We have shown that the peptidoglycan-mimetic peptide, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, 1, is a very specific and reactive carboxypeptidase substrate of the Streptomyces R61 dd-peptidase [Anderson, J. W., and Pratt, R. F. (2000) Biochemistry 39, 12200-12209]. In the present paper, we explore the transpeptidation reactions of this substrate, where the enzyme catalyzes transfer of the glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl moiety to amines. These reactions are believed to occur through capture of an acyl-enzyme intermediate by amines rather than water. Experiments show that effective acyl acceptors require a carboxylate group and thus are amino acids and peptides. d(but not l)-amino acids, analogues of the leaving group of 1, are good acceptors. The effectiveness of d-alanine as an acceptor increases with pH, suggesting that the bound and reactive form of an amino acid acceptor is the free amine. Certain glycyl-l(but not d)-amino acids, such as glycyl-l-alanine and glycyl-l-phenylalanine, are also good acceptors. These molecules may resemble the N-terminus of the Streptomyces stem peptides that, presumably, are the acceptors in vivo. The acyl acceptor binding site therefore demonstrates a dual specificity. That d-alanyl-l-alanine shows little activity as an acceptor suggested that, on binding of acceptors to the enzyme, the carboxylate of d-amino acids does not overlap with the peptide carbonyl group of glycyl-l-amino acids. Molecular modeling of transpeptidation tetrahedral intermediates and products demonstrated the likely structural bases for the stereospecificity of the acceptors and the nature of the dual function acceptor binding site. For both groups of acceptors, the terminal carboxylate appeared to be anchored at the active site by interaction with Arg 285 and Thr 299.     

Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity

The enzymes from the alpha-amylase family all share a similar alpha-retaining catalytic mechanism but can have different reaction and product specificities. One family member, cyclodextrin glycosyltransferase (CGTase), has an uncommonly high transglycosylation activity and is able to form cyclodextrins. We have determined the 2.0 and 2.5 A X-ray structures of E257A/D229A CGTase in complex with maltoheptaose and maltohexaose. Both sugars are bound at the donor subsites of the active site and the acceptor subsites are empty. These structures mimic a reaction stage in which a covalent enzyme-sugar intermediate awaits binding of an acceptor molecule. Comparison of these structures with CGTase-substrate and CGTase-product complexes reveals three different conformational states for the CGTase active site that are characterized by different orientations of the centrally located residue Tyr 195. In the maltoheptaose and maltohexaose-complexed conformation, CGTase hinders binding of an acceptor sugar at subsite +1, which suggests an induced-fit mechanism that could explain the transglycosylation activity of CGTase. In addition, the maltoheptaose and maltohexaose complexes give insight into the cyclodextrin size specificity of CGTases, since they precede alpha-cyclodextrin (six glucoses) and beta-cyclodextrin (seven glucoses) formation, respectively. Both ligands show conformational differences at specific sugar binding subsites, suggesting that these determine cyclodextrin product size specificity, which is confirmed by site-directed mutagenesis experiments.     

Detection of molecular interactions by using a new peptide-displaying bacteriophage biosensor.

Foreign peptides fused to the carboxy terminus of P22 tailspike protein are solvent-exposed and highly antigenic when displayed on the surface of infectious virus particles. Binding of an anti-peptide specific Fab antibody fragment enhances the infectivity of chimeric bacteriophage particles in a titre-dependent fashion. Although the precise molecular basis of this enhanced infectivity remains unclear, experimental data and modelling approaches suggest that the antibody binding might restore conformational impairments in the assembled tail protein affecting its activity and performance during infection. These results suggest that in addition to free enzymes, peptide-displaying bacteriophages could be engineered as new biosensors to detect molecular interactions by using natural viral enzymes critical for cell infection.     

A detailed model of the active centre of Escherichia coli peptidyl transferase

On the basis of published data, a detailed model of the active centre of Escherichia coli peptidyl transferase is proposed. The major conclusions are as follows: A binding site is present at each of the acceptor (A′) and donor (P′) substrate binding sites of the enzyme for the 3′-terminal CpCpA of aminoacyl- and peptidyl-tRNA, respectively. In particular, the acceptor CpCpA binding site is composed of sites for the following groups: the terminal adenine, the first phosphoryl residue from the 3′-terminus, the 3′-penultimate cytosine, and the second 3′-CMP residue. In addition, two binding sites are present on each of the A′ and P′ sites, one for the basic and one for the hydrophobic aminoacyl R groups of both aminoacyl-tRNA and the carboxyl-terminal amino acid of peptidyl-tRNA. The role of these sites in the binding of inhibitors and substrates and in the mechanism of catalysis of peptide bond formation by peptidyl transferase is discussed.     

Multiple substrate binding sites in the ribozyme from Bacillus subtilis RNase P.

The ribozyme from Bacillus subtilis RNase P (P RNA) recognizes an RNA structure consisting of the acceptor stem and the T stem-loop of tRNA substrates. An in vitro selection experiment was carried out to obtain potential RNA substrates that may interact with the P RNA differently from the tRNA substrate. Using a P RNA-derived ribozyme that contains most, if not all, of the structural elements thought to be involved in active site formation of P RNA, but lacks the putative binding site for the T stem-loop of tRNA, a single RNA substrate was isolated after nine rounds of selection. This RNA is a competent substrate for the ribozyme used in selection as well as for the full-length P RNA. Biochemical characterization shows that this selected substrate interacts at a different site compared with the tRNA substrate. The selection experiment also identified a self-cleaving RNA seemingly different from other known ribozymes. These results indicate that a biological ribozyme can contain different binding sites for different RNA substrates. This alternate binding site model suggests a simple mechanism for evolving existing ribozymes to recognize RNA substrates of diverse structures.     

Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides

Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.     

Screening HIV‐1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors

We have analyzed the suitability of six antigenic peptides from several HIV‐1 structural proteins (namely gp41, gp120, p17, and p24), as anti‐HIV‐1 antibody receptors in an allosteric enzymatic biosensor. These peptides were inserted in a solvent‐exposed surface of Escherichia coli (E. coli) beta‐galactosidase by means of conventional recombinant DNA technology. The resulting enzymes were tested to allosterically respond to sera from HIV‐1‐infected individuals. Only stretches from gp41 and gp120 envelope proteins were able to transduce the molecular contact signal in the presence of immunoreactive sera. Intriguingly, the enzyme displaying the CD4 binding site segment KQFINMWQEVGKAMYAPP was activated by soluble CD4, suggesting that it produces conformational modifications on the allosteric enzyme as those occurring during antibody‐promoted induced fit. This fact is discussed in the context of the design of smart protein drugs and markers targeted to CD4⁺ cells. Copyright © 2009 John Wiley & Sons, Ltd.     

The active centres in penicillin-sensitive enzymes

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site no. 1. Interaction between the N-14 substituent of the bound molecule and binding site no. 2 induces changes in binding site no. 1. In turn, the catalytic site thus created increases the chemical reactivity of the beta-lactam amide bond. As the beta-lactam ring opens and acylates an enzyme serine residue, the interaction between the thiazolidine (or dihydrothiazine) ring and binding site no. 3 stabilizes the acyl-enzyme complex. Enzyme regeneration slowly proceeds either by direct elimination of the penicilloyl moiety or via C-5-C-6 splitting of the bound metabolite. The fragment arising from thiazolidine yields free N-formyl-D-penicillamine while the enzyme-linked N-acylglycyl fragment is immediately attacked by an exogenous nucleophile correctly positioned on the acceptor site. Similarly, the enzyme action on L-X-D-Ala-D-Ala terminated peptides is mediated via a binding site no. 1 that combines with D-Ala-D-Ala, a binding site no. 2 that interacts with the side chain of the preceding L-residue, an inducible catalytic site and an acceptor site. Enzymes are known that form a transitory L-X-D-Ala-enzyme complex where the acyl group is ester-linked to the same serine residue as that involved in the formation of the penicilloyl-enzyme complex (Waxman et al., this symposium). Other enzymes, however, may function as catalyst templates. Depending on the enzymes, the independence of the beta-lactam and L-X-D-Ala-D-Ala active centres is more or less pronounced.     

Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1)

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The “gate” is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.     

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.     

Phosphorylation of tyrosine 992, 1068, and 1086 is required for conformational change of the human epidermal growth factor receptor c-terminal tail

We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the beta-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr-->Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2-binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation.     

Restriction enzymes have limited access to DNA sequences in Drosophila chromosomes.

Sequence specific DNA binding proteins in eukaryotic cells must efficiently locate their binding sites in chromosomes. Restriction enzymes provide a simple model system with which to investigate the factors which influence this process. We have used P element mediated transformation to introduce a DNA fragment containing a set of characterized restriction sites into the Drosophila germline. Embryonic nuclei prepared from these transgenic animals were treated with restriction enzymes to probe the accessibility of the target restriction sites. The results show that the insert is within an accessible region of the chromosome and that restriction sites within the inserted sequence can be cut. However, the rate of cutting is biphasic. At each restriction site, a fraction of the chromosomes is cut rapidly after which the remainder is refractory. Similar levels of incomplete cutting are obtained when the same P element construct is examined at a different chromosomal location, when different sequence elements are introduced into the P element vector or when the experiment is carried out on nuclei from different embryonic stages. These results are discussed in terms of how sequence specific DNA binding proteins may locate their genomic targets in vivo.     

Conformational isomerism can limit antibody catalysis

Ligand binding to enzymes and antibodies is often accompanied by protein conformational changes. Although such structural adjustments may be conducive to enzyme catalysis, much less is known about their effect on reactions promoted by engineered catalytic antibodies. Crystallographic and pre-steady state kinetic analyses of antibody 34E4, which efficiently promotes the conversion of benzisoxazoles to salicylonitriles, show that the resting catalyst adopts two interconverting active-site conformations, only one of which is competent to bind substrate. In the predominant isomer, the indole side chain of Trp(L91) occupies the binding site and blocks ligand access. Slow conformational isomerization of this residue, on the same time scale as catalytic turnover, creates a deep and narrow binding site that can accommodate substrate and promote proton transfer using Glu(H50) as a carboxylate base. Although 34E4 is among the best catalysts for the deprotonation of benzisoxazoles, its efficiency appears to be significantly limited by this conformational plasticity of its active site. Future efforts to improve this antibody might profitably focus on stabilizing the active conformation of the catalyst. Analogous strategies may also be relevant to other engineered proteins that are limited by an unfavorable conformational pre-equilibrium.