Mechanistic insights from the structures of HincII bound to cognate DNA cleaved from addition of Mg2+ and Mn2+

The three-dimensional X-ray crystal structures of HincII bound to cognate DNA containing GTCGAC and Mn(2+) or Mg(2+), at 2.50A and 2.95A resolution, respectively, are presented. In both structures, the DNA is found cleaved, and the positions of the active-site groups, cleaved phosphate group, and 3' oxygen atom of the leaving group are in very similar positions. Two highly occupied Mn(2+) positions are found in each active site of the four crystallographically independent subunit copies in the HincII/DNA/Mn(2+) structure. The manganese ion closest to the previously identified single Ca(2+) position of HincII is shifted 1.7A and has lost direct ligation to the active-site aspartate residue, Asp127. A Mn(2+)-ligated water molecule in a position analogous to that seen in the HincII/DNA/Ca(2+) structure, and proposed to be the attacking nucleophile, is beyond hydrogen bonding distance from the active-site lysine residue, Lys129, but remains within hydrogen bonding distance from the proRp oxygen atom of the phosphate group 3' to the scissile phosphate group. In addition, the position of the cleaved phosphate group is on the opposite side of the axis connecting the two metal ions relative to that found in the BamHI/product DNA/Mn(2+) structure. Mechanistic implications are discussed, and a model for the two-metal-ion mechanism of DNA cleavage by HincII is proposed.     

DNA-induced conformational changes in type II restriction endonucleases: the structure of unliganded HincII

The 2.1A crystal structure of the unliganded type II restriction endonuclease, HincII, is described. Although the asymmetric unit contains only a single monomer, crystal lattice contacts bring two monomers together to form a dimer very similar to that found in the DNA bound form. Comparison with the published DNA bound structure reveals that the DNA binding pocket is expanded in the unliganded structure. Comparison of the unliganded and DNA liganded structures reveals a simple rotation of subunits by 11 degrees each, or 22 degrees total, to a more closed structure around the bound DNA. Comparison of this conformational change to that observed in the other type II restriction endonucleases where DNA bound and unliganded forms have both been characterized, shows considerable variation in the types of conformational changes that can occur. The conformational changes in three can be described by a simple rotation of subunits, while in two others both rotation and translation of subunits relative to one another occurs. In addition, the endonucleases having subunits that undergo the greatest amount of rotation upon DNA binding are found to be those that distort the bound DNA the least, suggesting that DNA bending may be less facile in dimers possessing greater flexibility.     

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg²⁺ ions, EcoRV is able to cleave the DNA but not in presence of Ca²⁺, although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg²⁺(A)-EcoRV-DNA complex compared to Ca²⁺(S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca²⁺(S)-EcoRV-DNA compared to Mg²⁺(A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca²⁺(S) bound complex than in Mg²⁺(A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.     

Understanding the structural and dynamic consequences of DNA epigenetic modifications: Computational insights into cytosine methylation and hydroxymethylation

We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule''s dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding.     

Understanding the structural and dynamic consequences of DNA epigenetic modifications: Computational insights into cytosine methylation and hydroxymethylation

We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule's dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding.     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: recognition of pyrimidine-purine and purine-purine steps

The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.     

Five-stranded β-sheet sandwiched with two α-helices: A structural link between restriction endonucleases EcoRI and EcoRV

Examination of crystal structures of restriction endonucleases EcoRI and EcoRV complexes with their cognate DNA revealed a common structural element, which forms the core of both proteins. This element consists of a five-stranded β-sheet and two α-helices packed against it and could be described as α–β sandwich in which helices and β-strands lie in two stacked layers. While the spatial structure of this α–β sandwich is conserved in both enzymes, there are no detectable similarities between amino acid sequences except of a few residues involved in active site formation. Probably, other restriction endonucleases which have similar organization of the active site might possess similar structural element regardless of DNA sequence recognized and recognition elements in the enzyme used. © 1994 Wiley-Liss, Inc.     

Substrate mobility in a deeply buried active site: analysis of norcamphor bound to cytochrome P-450cam as determined by a 201-psec molecular dynamics simulation.

While cytochrome P-450cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5-exo-, 47% 6-exo-, and 8% 3-exo-hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754-3760, 1987). The present study describes a 201-psec molecular dynamics (MD) stimulation of norcamphorbound cytochrome P-450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the heme iron and the substrate of about 1.0 A; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphorbound crystal structure possesses mobility that correlates well with the spin-state equilibrium of this enzyme-substrate complex.     

Identification of selectivity-determining residues in cytochrome P450 monooxygenases: a systematic analysis of the substrate recognition site 5

The large and diverse family of cytochrome P450 monooxygenases (CYPs) was systematically analyzed to identify selectivity- and specificity-determining residues in the substrate recognition site 5, which is located in close vicinity to the heme center. A positively charged heme-interacting residue was identified in the structures of 29 monooxygenases and in 97.7% of the 6379 CYP sequences investigated here. This heme-interacting residue restricts the conformation of the substrate recognition site 5 and is preferentially located at position 10 or 11 after the conserved ExxR motif (in 94.4% of the sequences), in 3.3% of the sequences at position 9 or 12. As a result, a classification by the position of the heme-interacting residue allows to predict residues that are closest to the heme center and restrict its accessibility. In 98.4% of all CYP sequences a preferentially hydrophobic residue is located at position 5 after the ExxR motif that is predicted to point close to the heme center. Replacing this residue by hydrophobic residues of different size has been shown to change substrate specificity and regioselectivity for CYPs of different superfamilies. Twenty-seven percent of all CYPs are predicted to contain a second selectivity-determining residue at position 9 after the ExxR motif that can be identified by the pattern EXXR-X(7)-{P}-x-P-[HKR].     

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.     

Expression,purification, and crystallization of restriction endonuclease PvuII with DNA containing its recognition site

We have overexpressed the type II restriction endonuclease PvuII (R.PvuII) in E. coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site. The cocrystals are orthorhombic space group P2₁2₁2₁ with cell constants a = 95.8 Å, b = 86.3 Å, c = 48.5 Å, and diffract X-rays to at least 2.7 Å. There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit. © 1994 Wiley-Liss, Inc.     

Probing the active site of cellodextrin phosphorylase from Clostridium stercorarium: kinetic characterization, ligand docking, and site-directed mutagenesis

Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl β-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite +2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite +2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites -1 and +1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme-substrate interactions that determine the activity and specificity of cellodextrin phosphorylases.     

Photochemical crosslinking of bacteriophage T4 single-stranded DNA-binding protein (gp32) to oligo-p(dT)8: identification of phenylalanine-183 as the site of crosslinking

Using ultraviolet light, both the 33,000-dalton single-stranded DNA-binding protein from T4 bacteriophage (gp32) as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography. Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:p(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.     

Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease DraIII

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5′ half (CAT) and a canonical 3′ half (GTG). DraIII nicks the 3′ canonical half site at a faster rate than the 5′ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through sitedirected mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.     

The structural basis for substrate versatility of chloramphenicol acetyltransferase CATI

Novel antibiotics are needed to overcome the challenge of continually evolving bacterial resistance. This has led to a renewed interest in mechanistic studies of once popular antibiotics like chloramphenicol (CAM). Chloramphenicol acetyltransferases (CATs) are enzymes that covalently modify CAM, rendering it inactive against its target, the ribosome, and thereby causing resistance to CAM. Of the three major types of CAT (CAT(I-III)), the CAM-specific CAT(III) has been studied extensively. Much less is known about another clinically important type, CAT(I). In addition to inactivating CAM and unlike CAT(III), CAT(I) confers resistance to a structurally distinct antibiotic, fusidic acid. The origin of the broader substrate specificity of CAT(I) has not been fully elucidated. To understand the substrate binding features of CAT(I), its crystal structures in the unbound (apo) and CAM-bound forms were determined. The analysis of these and previously determined CAT(I)-FA and CAT(III)-CAM structures revealed interactions responsible for CAT(I) binding to its substrates and clarified the broader substrate preference of CAT(I) compared to that of CAT(III).     

Inositol phosphate-induced stabilization of inositol 1,3,4,5,6-pentakisphosphate 2-kinase and its role in substrate specificity

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) on their inositol rings to yield an array of signaling molecules. IPKs must possess the ability to recognize their physiological substrates from among a pool of over 30 cellular IPs that differ in numbers and positions of phosphates. Crystal structures from IPK subfamilies have revealed structural determinants for IP discrimination, which vary considerably between IPKs. However, recent structures of inositol 1,3,4,5,6‐pentakisphosphate 2‐kinase (IPK1) did not reveal how IPK1 selectively recognizes its physiological substrate, IP5, while excluding others. Here, we report that limited proteolysis has revealed the presence of multiple conformational states in the IPK1 catalytic cycle, with notable protection from protease only in the presence of IP. Further, a 3.1‐Å crystal structure of IPK1 bound to ADP in the absence of IP revealed decreased order in residues 110–140 within the N‐lobe of the kinase compared with structures in which IP is bound. Using this solution and crystallographic data, we propose a model for recognition of IP substrate by IPK1 wherein phosphate groups at the 4‐, 5‐, and 6‐positions are recognized initially by the C‐lobe with subsequent interaction of the 1‐position phosphate by Arg130 that stabilizes this residue and the N‐lobe. This model explains how IPK1 can be highly specific for a single IP substrate by linking its interactions with substrate phosphate groups to the stabilization of the N‐ and C‐lobes and kinase activation.     

Site-specific DNA-nicking mutants of the heterodimeric restriction endonuclease R.BbvCI

The restriction enzyme R.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence, thus: CCTCAGC/GCTGAGG-->CC--TCAGC/GC--TGAGG. We show that R.BbvCI comprises two different subunits, R(1) and R(2); that each subunit contains a catalytic site for DNA strand hydrolysis; and that these sites act independently and strand-specifically. In turn, each catalytic site was inactivated by mutagenesis to form dimeric enzymes in which only one site remained functional. The altered enzymes hydrolyzed just one strand of the recognition sequence, nicking the DNA rather than cleaving it. Enzymes in which the catalytic site in the R(1) subunit remained functional nicked the bottom strand of the sequence, producing CCTCAGC/GC--TGAGG, while those in which the catalytic site in the R(2) subunit remained functional nicked the top strand, producing CC--TCAGC/GCTGAGG. These DNA-nicking enzymes could prove useful for investigation of DNA repair, recombination, and replication, and for laboratory procedures that initiate from nicks, such as DNA degradation, synthesis, and amplification.     

Investigation of the chiral recognition ability of human carboxylesterase 1 using indomethacin esters

Human carboxylesterase 1 (hCES1) is an enzyme that plays an important role in hydrolysis of pharmaceuticals in the human liver. In this study, elucidation of the chiral recognition ability of hCES1 was attempted using indomethacin esters in which various chiral alcohols were introduced. Indomethacin was condensed with various chiral alcohols to synthesize indomethacin esters. The synthesized esters were hydrolyzed with a human liver microsome (HLM) solution and a human intestine microsome (HIM) solution. High hydrolytic rate and high stereoselectivity were confirmed in the hydrolysis reaction in the HLM solution but not in the HIM solution, and these indomethacin esters were thought to be hydrolyzed by hCES1. Next, these indomethacin esters were hydrolyzed in recombinant hCES1 solution and the hydrolysis rates of the esters were calculated. The stereoselectivity confirmed in HLM solution was also confirmed in the hCES1 solution. In the hydrolysis reaction of esters in which a phenyl group is bonded next to the ester, the V_max value of the (R) form was 10 times larger than that of the (S) form.     

How to use the MEROPS database and website to help understand peptidase specificity

The MEROPS website ( https://www.ebi.ac.uk/merops ) and database was established in 1996 to present the classification and nomenclature of proteolytic enzymes. This was expanded to include a classification of protein inhibitors of proteolytic enzymes in 2004. Each peptidase or inhibitor is assigned to a distinct identifier, based on its biochemical and biological properties, and homologous sequences are assembled into a family. Families in which the proteins share similar tertiary structures are assembled into a clan. The MEROPS classification is thus a hierarchy with at least three levels (protein‐species, family, and clan) showing the evolutionary relationship. Several other data collections have been assembled, which are accessed from all levels in the hierarchy. These include, sequence homologs, selective bibliographies, substrate cleavage sites, peptidase–inhibitor interactions, alignments, and phylogenetic trees. The substrate cleavage collection has been assembled from the literature and includes physiological, pathological, and nonphysiological cleavages in proteins, peptides, and synthetic substrates. In this article, we make recommendations about how best to analyze these data and show analyses to indicate peptidase binding site preferences and exclusions. We also identify peptidases where co‐operative binding occurs between adjacent binding sites.     

An update view on the substrate recognition mechanism of phosphodiesterases: a computational study of PDE10 and PDE4 bound with cyclic nucleotides

Early studies strongly implied that the specificity of cyclic nucleotide phosphodiesterases (PDEs) toward its endogenous substrates can be uniquely determined by the amido orientation of the invariant glutamine locating in the binding pocket of the enzyme. However, recently solved crystal structures of PDE4 (cAMP specific) and PDE10 (dual specific) in the presence of endogenous substrates have revealed that their invariant glutamine orientations are very similar despite exhibiting different substrate specificities proven physiologically. To understand this subtle specificity issue in the PDE family, here several experimentally inaccessible PDE-substrate complex models have been studied computationally, and the results are juxtaposed and compared in detail. Modeling results show that PDE10 in fact favors cAMP energetically but still can bind to cGMP owing to the robust hydrogen-bond network in the vicinity of the invariant glutamine side chain. PDE4 fails to accommodate cGMP is correlated to the weakening of this same hydrogen-bond network but not owing to any steric strain in the binding pocket. An Asn residue in the binding pocket of PDE4 has enhanced the specificity of the binding to cAMP sideway as observed in our computer simulation. Further to the previously studied syn- versus anti-conformational specificity of cAMP in PDE10, the unexpected substrate-binding mode in PDE10 versus PDE4 as reported here strongly suggested that there are remaining uncertainties in the substrate orientation and recognition mechanism in the PDE families. The molecular details of the binding pocket observed in this study provide hints for more optimal PDE4 and PDE10 inhibitor design.