Role of induced fit in enzyme specificity: a molecular forward/reverse switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.     

Promotion of Enzyme Flexibility by Dephosphorylation and Coupling to the Catalytic Mechanism of a Phosphohexomutase

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes an intramolecular phosphoryl transfer across its phosphosugar substrates, which are precursors in the synthesis of exoproducts involved in bacterial virulence. Previous structural studies of PMM/PGM have established a key role for conformational change in its multistep reaction, which requires a dramatic 180° reorientation of the intermediate within the active site. Here hydrogen-deuterium exchange by mass spectrometry and small angle x-ray scattering were used to probe the conformational flexibility of different forms of PMM/PGM in solution, including its active, phosphorylated state and the unphosphorylated state that occurs transiently during the catalytic cycle. In addition, the effects of ligand binding were assessed through use of a substrate analog. We found that both phosphorylation and binding of ligand produce significant effects on deuterium incorporation. Phosphorylation of the conserved catalytic serine has broad effects on residues in multiple domains and is supported by small angle x-ray scattering data showing that the unphosphorylated enzyme is less compact in solution. The effects of ligand binding are generally manifested near the active site cleft and at a domain interface that is a site of conformational change. These results suggest that dephosphorylation of the enzyme may play two critical functional roles: a direct role in the chemical step of phosphoryl transfer and secondly through propagation of structural flexibility. We propose a model whereby increased enzyme flexibility facilitates the reorientation of the reaction intermediate, coupling changes in structural dynamics with the unique catalytic mechanism of this enzyme.     

DNA binding induces active site conformational change in the human TREX2 3′-exonuclease

The TREX enzymes process DNA as the major 3′→5′ exonuclease activity in mammalian cells. TREX2 and TREX1 are members of the DnaQ family of exonucleases and utilize a two metal ion catalytic mechanism of hydrolysis. The structure of the dimeric TREX2 enzyme in complex with single-stranded DNA has revealed binding properties that are distinct from the TREX1 protein. The TREX2 protein undergoes a conformational change in the active site upon DNA binding including ordering of active site residues and a shift of an active site helix. Surprisingly, even when a single monomer binds DNA, both monomers in the dimer undergo the structural rearrangement. From this we have proposed a model for DNA binding and 3′ hydrolysis for the TREX2 dimer. The structure also shows how TREX proteins potentially interact with double-stranded DNA and suggest features that might be involved in strand denaturation to provide a single-stranded substrate for the active site.     

Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase

7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower beta-elimination reaction at the 3'-side of the resulting abasic site. Many features of OGG1 mechanism, including its low beta-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of beta-elimination rather than pre-excision dynamics of DNA and the enzyme.     

Crystal structure of the cysteine desulfurase DndA from Streptomyces lividans which is involved in DNA phosphorothioation

DNA phosphorothioation is widespread among prokaryotes, and might function to restrict gene transfer among different kinds of bacteria. There has been little investigation into the structural mechanism of the DNA phosphorothioation process. DndA is a cysteine desulfurase which is involved in the first step of DNA phosphorothioation. In this study, we determined the crystal structure of Streptomyces lividans DndA in complex with its covalently bound cofactor PLP, to a resolution of 2.4 ?. Our structure reveals the molecular mechanism that DndA employs to recognize its cofactor PLP, and suggests the potential binding site for the substrate L-cysteine on DndA. In contrast to previously determined structures of cysteine desulfurases, the catalytic cysteine of DndA was found to reside on a β strand. This catalytic cysteine is very far away from the presumable location of the substrate, suggesting that a conformational change of DndA is required during the catalysis process to bring the catalytic cysteine close to the substrate cysteine. Moreover, our in vitro enzymatic assay results suggested that this conformational change is unlikely to be a simple result of random thermal motion, since moving the catalytic cysteine two residues forward or backward in the primary sequence completely disabled the cysteine desulfurase activity of DndA.     

DNA recognition by the homing endonuclease PI-SceI involves a divalent metal ion cofactor-induced conformational change

PI-SceI, a homing endonuclease of the LAGLIDADG family, consists of two domains involved in DNA cleavage and protein splicing, respectively. Both domains cooperate in binding the recognition sequence. Comparison of the structures of PI-SceI in the absence and presence of substrate reveals major conformational changes in both the protein and DNA. Notably, in the protein-splicing domain the loop comprising residues 53-70 and adopts a "closed" conformation, thus enabling it to interact with the DNA. We have studied the dynamics of DNA binding and subsequent loop movement by fluorescence techniques. Six amino acids in loop53-70 were individually replaced by cysteine and modified by fluorescein. The interaction of the modified PI-SceI variants with the substrate, unlabeled or labeled with tetramethylrhodamine, was analyzed in equilibrium and stopped-flow experiments. A kinetic scheme was established describing the interaction between PI-SceI and DNA. It is noteworthy that the apparent hinge-flap motion of loop53-70 is only observed in the presence of a divalent metal ion cofactor. Substitution of the major Mg2+-binding ligands in PI-SceI, Asp-218 and Asp-326, by Asn or "nicking" PI-SceI with trypsin at Arg-277, which interferes with formation of an active enzyme.substrate complex, both prevent the conformational change of loop53-70. Deletion of the loop inactivates the enzyme. We conclude that loop53-70 is an important structural element that couples DNA recognition by the splicing domain with DNA cleavage by the catalytic domain and as such "communicates" with the Mg2+ binding sites at the catalytic centers.     

Prediction of a ligand-induced conformational change in the catalytic core of Cdc25A

The cell cycle control phosphatases Cdc25 are dual specificity phosphatases that dephosphorylate both phosphothreonine and phosphotyrosine residues on their substrate proteins. The determination of the apo-protein structure of Cdc25A revealed that this enzyme has a completely different fold compared to all other phosphatases crystallised to date. The conformation of the active site residues does not seem very suitable for catalysis in this unliganded structure. We have studied some structural features of the Cdc25A apo-structure and a modelled Cdc25A-ligand complex by molecular dynamics simulations. The simulations predict a conformational change in the peptide backbone of the complex, which is not observed in the apo-structure. This ligand-induced conformational change yields a structure that is similar to other protein tyrosine phosphatase-ligand complexes that have been crystallised. The change in conformation takes place in the position between a serine and a glutamic acid residue in the phosphate binding loop. We suggest that this type of conformational change is an important molecular switch in the catalytic process.     

The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy.

Aspartate transcarbamylase (EC 2.1.3.2) contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.     

Catalytically requisite conformational dynamics in the mRNA-capping enzyme probed by targeted molecular dynamics

The addition of a N7-methyl guanosine cap to the 5' end of nascent mRNA is carried out by the mRNA-capping enzyme, a two-domain protein that is a member of the nucleotidyltransferase superfamily. The mRNA-capping enzyme is composed of a catalytic nucleotidyltransferase domain and a noncatalytic oligonucleotide/oligosaccharide binding (OB) domain. Large-scale domain motion triggered by substrate binding mediates catalytically requisite conformational rearrangement of the GTP substrate prior to the chemical step. In this study, we employ targeted molecular dynamics (TMD) on the PBCV-1 capping enzyme to probe the global domain dynamics and internal dynamics of conserved residues during the conformational transformation from the open to the closed state. Analysis of the resulting trajectories along with structural and sequence homology to other members of the superfamily allows us to suggest a conserved mechanism of conformational rearrangements spanning all mRNA-capping enzymes and all ATP-dependent DNA ligases. Our results suggest that the OB domain moves quasi-statically toward the nucleotidyltransferase domain, pivoting about a short linker region. The approach of the OB domain brings a conserved RxDK sequence, an element of conserved motif VI, within proximity of the triphosphate of GTP, destabilizing the unreactive conformation and thereby allowing thermal fluctuations to partition the substrate toward the catalytically competent state.     

Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad

The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/β-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/β-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.     

Probing conformational plasticity of the activation domain of trypsin: the role of glycine hinges

Trypsin-like serine proteases play essential roles in diverse physiological processes such as hemostasis, apoptosis, signal transduction, reproduction, immune response, matrix remodeling, development, and differentiation. All of these proteases share an intriguing activation mechanism that involves the transition of an unfolded domain (activation domain) of the zymogen to a folded one in the active enzyme. During this conformational change, activation domain segments move around highly conserved glycine hinges. In the present study, hinge glycines were replaced by alanine residues via site directed mutagenesis. The effects of these mutations on the interconversion of the zymogen-like and active conformations as well as on catalytic activity were studied. Mutant trypsins showed zymogen-like structures to varying extents characterized by increased flexibility of some activation domain segments, a more accessible N-terminus and a deformed substrate binding site. Our results suggest that the trypsinogen to trypsin transition is hindered by the mutations, which results in a shift of the equilibrium between the inactive zymogen-like and active enzyme conformations toward the inactive state. Our data also showed, however, that the inactive conformations of the various mutants differ from each other. Binding of substrate analogues shifted the conformational equilibrium toward the active enzyme since inhibited forms of the trypsin mutants showed similar structural features as the wild-type enzyme. The catalytic activity of the mutants correlated with the proper conformation of the active site, which could be supported by varying conformations of the N-terminus and the autolysis loop. Transient kinetic measurements confirmed the existence of an inactive to active conformational transition occurring prior to substrate binding.     

Phosphorylation and mutations of Ser(16) in human phenylalanine hydroxylase. Kinetic and structural effects

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser(16) by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate l-Phe. So far there is no structural information on the flexible N-terminal tail (residues 1-18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser(16) induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg(13) as well as a repulsion by Glu(280) in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met(1)-Leu(15)) on phosphorylation is in agreement with the observed conformational change and increased accessibility of the substrate to the active site, as indicated by circular dichroism spectroscopy and the enzyme kinetic data for the full-length phosphorylated and nonphosphorylated human PAH. To further validate the model we have prepared and characterized mutants substituting Ser(16) with a negatively charged residue and found that S16E largely mimics the effects of phosphorylation of human PAH. Both the phosphorylated enzyme and the mutants with acidic side chains instead of Ser(16) revealed an increased resistance toward limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of alpha-helical structure. In agreement with the modeled structure, the formation of an Arg(13) to Ser(16) phosphate salt bridge and the conformational change of the N-terminal tail also explain the higher stability toward limited tryptic proteolysis of the phosphorylated enzyme. The results obtained with the mutant R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.     

Dimeric Crystal Structure of Rabbit l-Gulonate 3-Dehydrogenase/λ-Crystallin: Insights into the Catalytic Mechanism

l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD⁺-dependent enzyme in the uronate cycle but also as a taxon-specific λ-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as λ-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.     

Kinetic Evidence of a Noncatalytic Substrate Binding Site That Regulates Activity in Legionella pneumophilal-Serine Dehydratase

The l-serine dehydratase from Legionella pneumophila (lpLSD) has recently been shown to contain a domain (β domain) that has a high degree of structural homology with the ASB domain of d-3-phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis. Furthermore, this domain has been shown by sequence homology to be present in all bacterial l-serine dehydratases that utilize an Fe-S catalytic center. In PGDH, l-serine binds to the ACT domain to inhibit catalytic activity. However, substrate must be bound to the ASB domain for serine to exert its effect. As such, the ASB domain acts as a codomain for the action of l-serine. Pre-steady-state kinetic analysis of l-serine binding to lpLSD demonstrates that l-serine binds to a second noncatalytic site and produces a conformational change in the enzyme. The rate of this conformational change is too slow for its participation in the catalytic cycle but rather occurs prior to catalysis to produce an activated form of the enzyme. That the conformational change must occur prior to catalysis is shown by a lag in the production of product that exhibits essentially the same rate constant as the conformational change. The second, noncatalytic site for l-serine is likely to be the ASB domain (β domain) of lpLSD that functions in a manner similar to that in PGDH. A mechanism whose overall effect is to keep l-serine levels from accumulating to high levels while not completely depleting the l-serine pool in the bacterial cell is proposed.     

Mutation of a unique aspartate residue abolishes the catalytic activity but not substrate binding of the mouse N-methylpurine-DNA glycosylase (MPG)

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a variety of alkylated purine adducts. Although Asp was identified as the active site residue in various DNA glycosylases based on the crystal structure, Glu-125 in human MPG (Glu-145 in mouse MPG) was recently proposed to be the catalytic residue. Mutational analysis for all Asp residues in a truncated, fully active MPG protein showed that only Asp-152 (Asp-132 in the human protein), which is located near the active site, is essential for catalytic activity. However, the substrate binding was not affected in the inactive Glu-152, Asn-152, and Ala-152 mutants. Furthermore, mutation of Asp-152 did not significantly affect the intrinsic tryptophan fluorescence of the enzyme and the far UV CD spectra, although a small change in the near UV CD spectra of the mutants suggests localized conformational change in the aromatic residues. We propose that in addition to Glu-145 in mouse MPG, which functions as the activator of a water molecule for nucleophilic attack, Asp-152 plays an essential role either by donating a proton to the substrate base and, thus, facilitating its release or by stabilizing the steric configuration of the active site pocket.     

Mutations at the interdomain hinge region of the DadB alanine racemase: effects of length and conformational constraint of the linker sequence on catalytic efficiency

The pentapeptide "hinge" region of the DadB alanine racemase links two structural domains of the protein [Galakatos, N. G., & Walsh, C. T. (1987) Biochemistry 26, 8475]. The presence of substrate markedly reduces the rate of hinge-specific proteolysis of this racemase and induces a conformational change observed by circular dichroism. To evaluate the possible contribution of the proteolytically sensitive hinge residues (-Y253GGGY257-) on catalytic efficiency, site-directed mutations were generated to probe the effects of size and conformational rigidity of that region. A bacterial overproducing system for the dadB gene was constructed that expresses the enzyme as 4.5% of total soluble protein. On this construct, a four-part ligation allowed the engineering of two unique and proximal restriction sites required for cassette mutagenesis at the hinge region. For two of the eight mutants generated, expressed protein could not be detected (deletion of -GGGY-; termination codon at position 255). Deletion of one or two of the three Gly residues had no effect on catalytic efficiency. Insertion of a fourth Gly resulted in a 5-fold drop in Vmax/Km. For G254P, G255P, and G256P, Vmax/Km was 60%, 126%, and 26% of the native enzyme, respectively. In all cases, the Km's remained essentially constant, suggesting that the hinge region is not involved in substrate binding. The rate of hinge-specific proteolysis of the mutants was faster than that of wild-type DadB except for the G255P protein for which it was equivalent.     

色氨酸残基在内切葡聚糖酶分子中的作用

内切葡聚糖酶的化学修饰研究表明：色氨酸残基可能位于活性位点,与底物结合有关．荧光光谱测定指出该酶的荧光几乎都来自色氨酸残基,酶分子中色氨酸微环境对ｐＨ变化非常敏感,降低ｐＨ导致了酶分子构象发生了较大变化,配基结合使酶分子色氨酸微环境产生了改变,引发了与ｐＨ诱导不同的构象变化．     

Induced-fit Mechanism for Prolyl Endopeptidase

Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the β-propeller and α/β-hydrolase domains; addition of substrate to preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.     

Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.     

Dynamics and DNA substrate recognition by the catalytic domain of lambda integrase

Bacteriophage lambda integrase (lambda-Int) is the prototypical member of a large family of enzymes that catalyze site-specific DNA recombination via the formation of a Holliday junction intermediate. DNA strand cleavage by lambda-Int is mediated by nucleophilic attack on the scissile phosphate by a conserved tyrosine residue, forming an intermediate with the enzyme covalently attached to the 3'-end of the cleaved strand via a phosphotyrosine linkage. The crystal structure of the catalytic domain of lambda-Int (C170) obtained in the absence of DNA revealed the tyrosine nucleophile at the protein's C terminus to be located on a beta-hairpin far from the other conserved catalytic residues and adjacent to a disordered loop. This observation suggested that a conformational change in the C terminus of the protein was required to generate the active site in cis, or alternatively, that the active site could be completed in trans by donation of the tyrosine nucleophile from a neighboring molecule in the recombining synapse. We used NMR spectroscopy together with limited proteolysis to examine the dynamics of the lambda-Int catalytic domain in the presence and absence of DNA half-site substrates with the goal of characterizing the expected conformational change. Although the C terminus is indeed flexible in the absence of DNA, we find that conformational changes in the tyrosine-containing beta-hairpin are not coupled to DNA binding. To gain structural insights into C170/DNA complexes, we took advantage of mechanistic conservation with Cre and Flp recombinases to model C170 in half-site and tetrameric Holliday junction complexes. Although the models do not reveal the nature of the conformational change required for cis cleavage, they are consistent with much of the available experimental data and provide new insights into the how trans complementation could be accommodated.