Protein structure and structure/function relationships

Scientific publication - Protein structure and structure/function relationships

 

Camilla Christiansen, Maher Abou Hachem, Esben Friis, Martin J. Baumann, Mikkel A. Glaring, Anders Viksø-Nielsen, Bent W, Sigurskjold, Birte Svensson and Andreas Blennow

"Exploring New Plant Carbohydrate Binding Modules (CBMs) from Glucan, Water Dikinase."

In: Starch Recent Progress in Biopolymer and Enzyme Technology. Tomasik, P., Bertoft, E. and Blennow A. (Editors). Polish Society of Food Technologists*, Chapter 7, pp. 85-102, ISBN 978-83-902699-7-X (2008)

 
Abstract
Starch binding domains (SBDs) are distinct starch-recognizing modules frequently present in starch active enzymes. SBDs are assigned in carbohydrate binding module (CBM) families based on primary structure similarities and conserved structural folds. SBDs from CBM family 20 are encountered in archaea, bacteria, and eukaryota and occur together with a variety of catalytic domains in α-amylases, β-amylases, cyclodextrin glucosyl transferases, glucoamylases, pullulanases, glucan, water dikinases (GWDs), and other enzymes. Typically, SBDs facilitate binding of extracellular hydrolytic enzymes to starch granules and they are considered as important motifs for developing enzymes with improved and new activities for effi cient degradation of starch. Members of the recently discovered family CBM45 are exclusively present in plastidial α-amylases, the role of which is still unknown and in GWDs implicated in starch phosphorylation. Interestingly, the plastidial GWD from Arabidopsis thaliana GWD3 has an SBD belonging to CBM20. The GWD3 SBD was cloned and produced for further characterisation and comparison of binding properties with the well characterized CBM20 of glucoamylase (GA) from Aspergillus niger. Binding to the low molecular weight starch mimic β-cyclodextrin and other soluble ligands was measured using surface plasmon resonance analysis, and confocal laser scanning microscopy was used to visualise binding to starch granules. The Arabidopsis thaliana GWD3-SBD displayed 70 fold lower affi nity to β-cyclodextrin than the GA-SBD. The low affi nity of these intracellular plant CBMs as compared to most other starch binding domains including the A. niger CBM20 suggests reversible or regulated binding of the plant SBD-containing enzymes. This is the fi rst report of an isolated plant CBM20 member and it highlights the presence of functional differences within CBM20.
  

Micheelsen, P.O., Vévodová, J., De Maria, L., Østergaard, P.R., Friis, E.P., Wilson, K., Skjøt, M.

"Structural and Mutational Analyses of the Interaction between the Barley α-Amylase/Subtilisin Inhibitor and the Subtilisin Savinase Reveal a Novel Mode of Inhibition"

Journal of Molecular Biology, . Article in Press. (2008)


Abstract
Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley α-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone. © 2008 Elsevier Ltd. All rights reserved.
 

Skálová, T., Dohnálek, J., Østergaard, L.H., Østergaard, P.R., Kolenko, P., Dušková, J., Hašek, J.

"Crystallization and preliminary X-ray diffraction analysis of the small laccase from Streptomyces coelicolor"

Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 63 (12), pp. 1077-1079. (2007)

 
Abstract
The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 Å. The crystals belong to either space group P41212 or P43212. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases will be determined from the anomalous signal of the natively present copper ions. © International Union of Crystallography 2007.
  

Gloster, T.M., Ibatullin, F.M., Macauley, K., Eklöf, J.M., Roberts, S., Turkenburg, J.P., Bjørnvad, M.E., Jørgensen, P.L., Danielsen, S., Johansen, K.S., Borchert, T.V., Wilson, K.S., Brumer, H., Davies, G.J.

"Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12"

Journal of Biological Chemistry, 282 (26), pp. 19177-19189. (2007)


Abstract
The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a β-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (β/α)8 and β-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the β-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (β/α)8 GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Greenbaum, J.A., Andersen, P.H., Blythe, M., Bui, H.-H., Cachau, R.E., Crowe, J., Davies, M., Kolaskar, A.S., Lund, O., Morrison, S., Mumey, B., Ofran, Y., Pellequer, J.-L., Pinilla, C., Ponomarenko, J.V., Raghava, G.P.S., Van Regenmortel, M.H.V., Roggen, E.L., Sette, A., Schlessinger, A., Sollner, J., Zand, M., Peters, B.

"Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools"

Journal of Molecular Recognition, 20 (2), pp. 75-82. (2007)


Abstract
A B-cell epitope is the three-dimensional structure within an antigen that can be bound to the variable region of an antibody. The prediction of B-cell epitopes is highly desirable for various immunological applications, but has presented a set of unique challenges to the bioinformatics and immunology communities. Improving the accuracy of B-cell epitope prediction methods depends on a community consensus on the data and metrics utilized to develop and evaluate such tools. A workshop, sponsored by the National Institute of Allergy and Infectious Disease (NIAID), was recently held in Washington, DC to discuss the current state of the B-cell epitope prediction field. Many of the currently available tools were surveyed and a set of recommendations was devised to facilitate improvements in the currently existing tools and to expedite future tool development. An underlying theme of the recommendations put forth by the panel is increased collaboration among research groups. By developing common datasets, standardized data formats, and the means with which to consolidate information, we hope to greatly enhance the development of B-cell epitope prediction tools. Copyright © 2007 John Wiley & Sons, Ltd.
 

Mirza, O., Skov, L.K., Sprogøe, D., Van Den Broek, L.A.M., Beldman, G., Kastrup, J.S., Gajhede, M.

"Structural rearrangements of sucrose phosphorylase from Bifidobacterium adolescentis during sucrose conversion"

Journal of Biological Chemistry, 281 (46), pp. 35576-35584. (2006)

 
Abstract
The reaction mechanism of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) was studied by site-directed mutagenesis and x-ray crystallography. An inactive mutant of BiSP (E232Q) was co-crystallized with sucrose. The structure revealed a substrate-binding mode comparable with that seen in other related sucrose-acting enzymes. Wild-type BiSP was also crystallized in the presence of sucrose. In the dimeric structure, a covalent glucosyl intermediate was formed in one molecule of the BiSP dimer, and after hydrolysis of the glucosyl intermediate, a β-D-glucose product complex was formed in the other molecule. Although the overall structure of the BiSP-glucosyl intermediate complex is similar to that of the BiSP(E232Q)-sucrose complex, the glucose complex discloses major differences in loop conformations. Two loops (residues 336-344 and 132-137) in the proximity of the active site move up to 16 and 4 Å, respectively. On the basis of these findings, we have suggested a reaction cycle that takes into account the large movements in the active-site entrance loops. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
 

Lyhne-Iversen, L., Hobley, T.J., Kaasgaard, S.G., Harris, P.

"Structure of Bacillus halmapalus α-amylase crystallized with and without the substrate analogue acarbose and maltose"

"Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 62 (9), pp. 849-854. (2006)


Abstract
Recombinant Bacillus halmapalus α-amylase (BHA) was studied in two different crystal forms. The first crystal form was obtained by crystallization of BHA at room temperature in the presence of acarbose and maltose; data were collected at cryogenic temperature to a resolution of 1.9 Å. It was found that the crystal belonged to space group P21212 1, with unit-cell parameters a = 47.0, b = 73.5, c = 151.1 Å. A maltose molecule was observed and found to bind to BHA and previous reports of the binding of a nonasaccharide were confirmed. The second crystal form was obtained by pH-induced crystallization of BHA in a MES-HEPES-boric acid buffer (MHB buffer) at 303 K; the solubility of BHA in MHB has a retrograde temperature dependency and crystallization of BHA was only possible by raising the temperature to at least 298 K. Data were collected at cryogenic temperature to a resolution of 2.0 Å. The crystal belonged to space group P2 12121, with unit-cell parameters a = 38.6, b = 59.0, c = 209.8 Å. The structure was solved using molecular replacement. The maltose-binding site is described and the two structures are compared. No significant changes were seen in the structure upon binding of the substrates. © 2006 International Union of Crystallography All rights reserved.
 

Von Ossowski, I., Eaton, J.T., Czjzek, M., Perkins, S.J., Frandsen, T.P., Schülein, M., Panine, P., Henrissat, B., Receveur-Bréchot, V.

"Protein disorder: Conformational distribution of the flexible linker in a chimeric double cellulase"

Biophysical Journal, 88 (4), pp. 2823-2832. (2005)


Abstract
The structural properties of the linker peptide connecting the cellulose-binding module to the catalytic module in bimodular cellulases have been investigated by small-angle x-ray scattering. Since the linker and the cellulose-binding module are relatively small and cannot be readily detected separately, the conformation of the linker was studied by means of an artificial fusion protein, Cel6BA, in which an 88-residue linker connects the large catalytic modules of the cellulases Cel6A and Cel6B from Humicola insolens. Our data showed that Cel6BA is very elongated with a maximum dimension of 178 A, but could not be described by a single conformation. Modeling of a series of Cel6BA conformers with interdomain separations ranging between 10 Å and 130 Å showed that good Guinier and P(r) profile fits were obtained by a weighted average of the scattering curves of all the models where the linker follows a nonrandom distribution, with a preference for the more compact conformers. These structural properties are likely to be essential for the function of the linker as a molecular spring between the two functional modules. Small-angle x-ray scattering therefore provides a unique tool to quantitatively analyze the conformational disorder typical of proteins described as natively unfolded. © 2005 by the Biophysical Society.

Davies, G.J., Marek Brzozowski, A., Dauter, Z., Rasmussen, M.D., Borchert, T.V., Wilson, K.S.

"Structure of a Bacillus halmapalus family 13 α-amylase, BHA, in complex with an acarbose-derived nonasaccharide at 2.1 Å resolution"Acta Crystallographica Section D: Biological Crystallography, 61 (2), pp. 190-193. (2005)


Abstract
The enzymatic digestion of starch by α-amylases is one of the key biotechnological reactions of recent times. In the search for industrial biocatalysts, the family GH13 α-amylase BHA from Bacillus halmapalus has been cloned and expressed. The three-dimensional structure at 2.1 Å resolution has been determined in complex with the (pseudo)tetrasaccharide inhibitor acarbose. Acarbose is found bound as a nonasaccharide transglycosylation product spanning the -6 to +3 subsites. Careful inspection of electron density suggests that the bound ligand could not have been formed through successive transglycosylations of acarbose and must also have featured maltose or maltooligosaccharides as an acceptor. © 2005 International Union of Crystallography - all rights reserved.
  

Ryttersgaard, C., Le Nours, J., Lo Leggio, L., Jørgensen, C.T., Christensen, L.L.H., Bjørnvad, M., Larsen, S.

"The structure of endo-β-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products"

Journal of Molecular Biology, 341 (1), pp. 107-117. (2004)

 
Abstract
The β-1,4-galactanase from Bacillus licheniformis (BLGAL) is a plant cell-wall-degrading enzyme involved in the hydrolysis of β-1,4-galactan in the hairy regions of pectin. The crystal structure of BLGAL was determined by molecular replacement both alone and in complex with the products galactobiose and galactotriose, catching a first crystallographic glimpse of fragments of β-1,4-galactan. As expected for an enzyme belonging to GH-53, the BLGAL structure reveals a (βα)8-barrel architecture. However, BLGAL βα-loops 2, 7 and 8 are long in contrast to the corresponding loops in structures of fungal galactanases determined previously. The structure of BLGAL additionally shows a calcium ion linking the long βα-loops 7 and 8, which replaces a disulphide bridge in the fungal galactanases. Compared to the substrate-binding subsites predicted for Aspergillus aculeatus galactanase (AAGAL), two additional subsites for substrate binding are found in BLGAL, -3 and -4. A comparison of the pattern of galactan and galactooligosaccharides degradation by AAGAL and BLGAL shows that, although both are most active on substrates with a high degree of polymerization, AAGAL can degrade galactotriose and galactotetraose efficiently, whereas BLGAL prefers longer oligosaccharides and cannot hydrolyze galactotriose to any appreciable extent. This difference in substrate preference can be explained structurally by the presence of the extra subsites -3 and -4 in BLGAL. © 2004 Elsevier Ltd. All rights reserved.

McAuley, K.E., Svendsen, A., Patkar, S.A., Wilson, K.S.

"Structure of a feruloyl esterase from Aspergillus niger"

Acta Crystallographica Section D: Biological Crystallography, 60 (5), pp. 878-887. (2004)

 
Abstract
The crystallographic structure of feruloyl esterase from Aspergillus niger has been determined to a resolution of 1.5 Å by molecular replacement. The protein has an α/β-hydrolase structure with a Ser-His-Asp catalytic triad; the overall fold of the protein is very similar to that of the fungal upases. The structure of the enzyme-product complex was determined to a resolution of 1.08 Å and reveals dual conformations for the serine and histidine residues at the active site. © 2004 International Union of Crystallography. Printed in Denmark - all rights reserved.
 
 

J. Le Nours; C. Ryttersgaard; L.L. Leggio; P.R. Østergaard; T.V. Borchert; L.L.H. Christensen; S. Larsen.

"Structure of two fungal beta-1,4-galactanases: Searching for the basis for temperature and pH optimum."

Protein Science, 12, 1195-1204 (2003)

 
Abstract
beta-1, 4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal beta-1, 4- galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the Gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing similar to56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 Angstrom and 2.14 Angstrom resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 Angstrom resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the a-helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/H91D mutant of MTGAL showed that th e difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.

 

W. Receveur; M. Czjzek; M. Schulein; P. Panine; B. Henrissat.

"Dimension, shape, and conformational flexibility of a two domain fungal cellulase in solution probed by small angle X- ray scattering."

J. Biol. Chem., 277(43), 40887-40892 (2002)


Abstract

Cellulase Ce145 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Ce145 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain. The glycosylation of the linker is the key factor defining its extended conformation, and a five proline stretch mutation on the linker was found to confer a higher rigidity to the enzyme. Our study shows that the respective positioning of the catalytic module and the CBM onto the insoluble substrate is most likely influenced by the linker structure and flexibility. Our results are consistent with a model where cellulases can move on the surface of cellulose with a caterpillar-like displacement with free energy restrictions.

J. E. Nielsen; T. V. Borchert; G. Vriend.

"The determinants of alpha-amylase pH activity profiles."

Protein engineering, 14, 505-512 (2001)

 
Abstract
The glycosyl hydrolases present a large family of enzymes that are of great significance for industry. Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions. Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involved stability engineering, but lately there has also been considerable interest in engineering their pH-activity profiles. We mutated four neutral residues (N190, F290, N326 and Q360) in the chimeric Bacillus Ba2 alpha -amylase to both charged and neutral amino acids. The results show that the pH-activity profile of the Ba2 alpha -amylase can be changed by inserting charged residues close to the active site. The changes in the pH-activity profile for these neutral --> charged mutations do not, however, correlate with the predictions from calculations of the pK(a) values of the active site residues. More surprisingly, the neutral --> neutral mutations change the pH-activity profile as much as the neutral --> charged mutations. From these results, it is concluded that factors other than electrostatics, presumably the dynamic aspects of the active site, are important for the shape of the pH-activity profiles of the alpha -amylases.

 

E. Sabini; K.S: Wilson; S. Danielsen; M. Schulein; G.J. Davies.

"Oligosaccharide binding to family 11 xylanases: both covalent intermediate and mutant product complexes display B-2,B-5 conformations at the active centre."

Acta Crystallographica Section D-Biological Crystallography, 57, 1344-1347 (2001)


Abstract
The glycoside hydrolase sequence-based classification reveals two families of enzymes which hydrolyse the beta -1, 4-linked backbone of xylan, xylanases, termed families GH-10 and GH- 11. Family GH-11 xylanases are intriguing in that catalysis is performed via a covalent intermediate adopting an unusual B-2, B-5 (boat) conformation, a conformation which also fulfils the stereochemical constraints of the oxocarbenium ion-like transition state. Here, the 1.9 Angstrom structure of a nucleophile, E94A, mutant of the Xyn11 from Bacillus agaradhaerens in complex with xylotriose is presented. Intriguingly, this complex also adopts the B-2, B-5 conformation in the -1 subsite, with the vacant space provided by the Glu --> Ala mutation allowing the sugar to adopt the alpha -configuration at C1. The structure of the covalent 2-deoxy-2-fluoroxylobiosyl-enzyme intermediate has been extended to atomic (1.1 Angstrom) resolution.

K. Zhu; A. Jutila; E.K.J. Tuominen; S.A. Patkar; A. Svendsen; P.K.J. Kinnunen.

"Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability."

Biochim. Biophys. Acta - Protein Structure and Molecular Enzymology, 1547, 329-338 (2001)


Abstract
Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared. Differential scanning calorimetry revealed unfolding of HLL at T-d = 74.4 degreesC whereas for W89L and W89m this endotherm was decreased to 68.6 and 62 degreesC, respectively, demonstrating significant contribution of the above Trp residues to the structural stability of HLL. Fluorescence emission spectra revealed the average microenvironment of Trps of wt HLL and W89L to become more hydrophilic at elevated temperatures whereas the opposite was true for W89m. These changes in steady-state emission were sharp, with midpoints (T-m) at approx. 70.5, 61.0, and 65.5 degreesC for wt HLL, W89L, and W89m, respectively. Both steady-state and time resolved fluorescence spectroscopy further indicated that upon increasing temperature, the local movements of tryptophan(s) in these lipases were first attenuated. However. faster mobilities became evident when the unfolding temperatures (T-m) were exceeded, and the lipases became less compact as indicated by the increased hydrodynamic radii. Even at high temperatures (up to 85 degreesC) a significant extent of tertiary and secondary structure was revealed by circular dichroism. Activity measurements are in agreement with increased amplitudes of conformational fluctuations of HLL. with temperature. Our results also indicate that the thermal unfolding of these lipases is not a two-state process but involves intermediate states. Interestingly, a heating and cooling cycle enhanced the activity of the lipases, suggesting the protein to be trapped in an intermediate, higher energy state. The present data show that the mutations. especially W89L in the lid, contribute significantly to the stability, structure and activity of HLL.

L. Beier; A. Svendsen; C. Andersen; T. P. Frandsen; T. V. Borchert; J.R. Cherry.

"Conversion of the maltogenic alpha-amylase into a CGT'ase."

Protein Engineering ,13,  509-513 (2000)


Abstract
Novamyl is a thermostable five-domain maltogenic alpha- amylase that shows sequence and structural homology with the cyclodextrin glycosyltransferases (CGTases), Comparing X-ray crystal structures of Novamyl and CGTases, two major differences in the active site cleft were observed: Novamyl contains a loop insertion consisting of five residues (residues 191-195) and the location of an aromatic residue known to be essential to obtain an efficient cyclization reaction. To convert Novamyl into a cyclodextrin (CD)producing enzyme, the loop was deleted and two substitutions, F188L and T189Y, were introduced. Unlike the parent Novamyl, the obtained variant is able to produce beta- CD and showed an overall conversion of starch to CD of 9%, compared with CGTases which are able to convert up to 40%. The lower conversion compared with the CGTase is probably due to additional differences in the active site cleft and in the starch-binding E domain. A variant with only the five- residue loop deleted was not able to form beta-CD.
 

A.M. Brzozowski; H. Savage; C.S. Verma; J.P.D.M. Lawson; A. Svendsen; S.A. Patkar.

"Structural origins of the interfacial activation on Thermomyces (Humicola) lanuginosa lipase."

Biochemistry, 39,15071-15082 (2000)


Abstract

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TIL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha (0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TIL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.