Interdomain Region Research Articles

Tertiary Contacts in αa-Lactalbumin at pH 7 and pH 2: A Molecular Dynamics Study

Molecular dynamics simulations of α-lactalbumin were performed under conditions of neutral pH and low pH in order to study the acid-induced molten globule state. Through the use of experimental techniques such as NMR and CD spectroscopy, molten globules have been characterized as being compact intermediates with secondary structure similar to that of the native protein but with tertiary structure that is disordered. The detailed structure of the molten globule state is unknown, however. Through the use of computer simulations we can study the structural changes which occur upon lowering pH. The simulations presented here differ from previous unfolding simulations in two important ways: the electrostatic interactions are treated more accurately than ever before, and artificially high temperatures are not used to force the protein to unfold. Simulations of 880 psec each were run at pH 7 (control simulation) and pH 2. We concentrate on the interesting changes in the tertiary interactions within the protein with lowering of pH. In particular, there is a loss of native tertiary contacts in the beta domain and interdomain region, and a large decrease in interdomain hydrogen bonds.

Melt texturing of NdBa Cu O Nd Ba Cu O superconductor in short time

We have melt processed NdBa2Cu3O -Nd4Ba2Cu2O10 (NdBCO) superconducting composites with various rates of cooling through their peritectic formation temperature. The microstructural studies reveal that the sample processed even with C/h rate of cooling is built up with aligned microstructure which is necessary for superior transport current and magnetic properties. In the case of YBa2Cu3O , C/h rate of cooling is necessary to get highly textured microstructure. Magnetic flux profile measurements reveal that even though the intradomain region is well textured and had good transport and magnetic properties, the interdomain region is relatively weak in the fast cooled sample in comparison with slow cooled ones but is much better than that in polycrystalline samples. The results suggest that, the NdBCO superconductor can be melt processed in short time with much better microstructural features in comparison with YBCO system.

Genes required for replication of the 15.5-kilobase RNA genome of a plant closterovirus.

A full-length cDNA clone of beet yellows closterovirus (BYV) was engineered and used to map functions involved in the replication of the viral RNA genome and subgenomic RNA formation. Among 10 open reading frames (ORFs) present in BYV, ORFs 1a and 1b suffice for RNA replication and transcription. The proteins encoded in these ORFs harbor putative methyltransferase, RNA helicase, and RNA polymerase domains common to Sindbis virus-like viruses and a large interdomain region that is unique to closteroviruses. The papain-like leader proteinase (L-Pro) encoded in the 5'-proximal region of ORF 1a was found to have a dual function in genome amplification. First, the autocatalytic cleavage between L-Pro and the remainder of the ORF 1a product was essential for replication of RNA. Second, an additional L-Pro function that was separable from proteolytic activity was required for efficient RNA accumulation. The deletion of a large, approximately 5.6-kb, 3'-terminal region coding for a 6-kDa hydrophobic protein, an HSP70 homolog, a 64-kDa protein, minor and major capsid proteins, a 20-kDa protein, and a 21-kDa protein (p21) resulted in replication-competent RNA. However, examination of mutants with replacements of start codons in each of these seven 3'-terminal ORFs revealed that p21 functions as an enhancer of genome amplification. The intriguing analogies between the genome organization and replicational requirements of plant closteroviruses and animal coronavirus-like viruses are discussed.

Role of Allosteric:Zinc Interdomain Region of the Regulatory Subunit in the Allosteric Regulation of Aspartate Transcarbamoylase fromEscherichia coli

The hydrophobic interface between the allosteric and the zinc domains of the regulatory subunit of aspartate transcarbamoylase has previously been implicated in the heterotropic ATP activation of the enzyme. The present work shows that this interface also affects CTP and CTP–UTP inhibition and proposes a structural explanation for the effects. Mutant enzymes derived from nonselective mutagenesis of residues r101–r106 (residues that contribute part of the interface) displayed a variety of homotropic and heterotropic effects. The cooperative behavior of the enzymes was affected, as indicated by reduced aspartateS0.5values and apparent Hill coefficient values for V106L, V106L/N105S, and I103F/R102C. In addition, both ATP activation and CTP inhibition were significantly reduced and CTP+UTP synergistic inhibition was decreased in these mutants. The D104G mutant enzyme was subject to inhibition by CTP andCTP+UTP, but was not activated by ATP. Finally, the I103T mutant enzyme had an increasedS0.5value of 11.5 mM and displayed altered effector responses: ATP acted as an inhibitor, and the CTP+UTP synergistic inhibition was reduced. Most of these allosteric variations can be explained in terms of perturbations to the “tongue and groove” hydrophobic interface between the allosteric and the zinc domains and a consequent impact on a second interface (“reg1:cat4”) between regulatory and catalytic subunits.

The biochemical and molecular spectrum of ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTCase) deficiency, the most common inherited urea cycle disorder, is transmitted as an X-linked trait. The clinical phenotype in affected males as well as heterozygous females shows a spectrum of severity ranging from neonatal hyperammonaemic coma to asymptomatic adults. The ornithine transcarbamylase enzyme is a trimer with three active sites per holoenzyme molecule, each of which is composed of an interdomain region of one polypeptide and a polar domain of the adjacent polypeptide. The OTC gene is located on the short arm of the X-chromosome and one of the two alleles undergoes inactivation in female cells. Approximately 140 mutations have been found in families affected with OTCase deficiency, most having their own 'private' mutation. Large deletions of one exon or more are seen in approximately 7% of patients, small deletions or insertions are seen in about 9%, and the remaining mutations are single base substitutions. Approximately 15% of mutations affect RNA splicing sites. The recurrent mutations are distributed equally among CpG dinucleotide hot spots. Generally, mutations causing neonatal disease affect amino acid residues that are 'buried' in the interior of the enzyme, especially around the active site, while those associated with late onset and milder phenotypes tend to be located on the surface of the protein. Very few mutations have been found in the sequence of the leader peptide, proportionally much fewer than in the sequence of the mature enzyme. Only few of the mutations have been expressed in bacteria or mammalian cells for the study of their deleterious mechanisms. Examples of expressed mutations include R277W and R277Q associated with late-onset disease, which markedly increase the Km for ornithine, shift the pH optimum to more alkaline and decrease the thermal stability of the purified mutant enzyme. R141Q (neonatal disease) disrupts the active site, whereas the purified R40H mutant has normal catalytic function and this mutation is likely to affect posttranslational processing such as mitochondrial targeting. It appears that most new mutations occur in male sperm and are then passed on to a transmitting heterozygous female. Uncommonly, mild mutations are transmitted by asymptomatic males to their daughters, subsequently resulting in clinical disease of males in future generations. The causes for variable expressivity of these mutations are currently unknown but are likely to involve a combination of environmental and genetic modifiers.

Mutations and Deletions within the S8–S9 Interdomain Region Abolish Complementation of N- and C-Terminal Domains of Ca2+-Activated K+(BK) Channels

A full length α-subunit of the Ca2+-activated K+(BK) channel with an inactivating mutation in the C-terminus can complement a functional C-terminal fragment. We analyzed deletions and amino acid changes within the S8–S9 interdomain region for their ability to allow complementation. Cys612and His616that are located in a region that contains two overlapping signature sequences, a immunoglobulin signature sequence and a heme binding domain, are essential for a functional channel. These two amino acid residues are also essential for complementation. The deletion of the PEST sequence does not affect the function of the BK channel; however, without the PEST sequence, complementation by a functional C-terminal fragments is no longer possible. The ability to complement a functional channel is restricted to the C-terminal fragment and requires that the complete α-subunit or the larger N-terminal fragment contains both, the immunoglobulin signature sequence the PEST sequence.

Cellulose binding domains and linker sequences potentiate the activity of hemicellulases against complex substrates

To evaluate the role of the CBDs and linker sequences in Pseudomonas xylanase A (XYLA) and arabinofuranosidase C (XYLC), the catalytic activity of derivatives of these enzymes, lacking either the linker sequences or CBDs, was assessed. Removal of the CBDs or linker sequences did not affect the activity of either XYLA or XYLC against soluble arabinoxylan, while derivatives of XYLA, in which either the CBD or interdomain regions had been deleted, exhibited decreased activity against the xylan component of cellulose/hemicellulose complexes. Although a truncated derivative of XYLC (XYLC″'), lacking its CBD, was less active than the full-length enzyme against plant cell wall material containing highly substituted arabinoxylan, XYLC‴ was more active than XYLC on complex substrates where the degree of substitution of arabinoxylan was very low. These data indicate that CBDs and linker sequences play an important role in the activity of hemicellulases against plant cell walls and other cellulose/hemicellulose complexes.

Correlated mutations contain information about protein-protein interaction

Many proteins have evolved to form specific molecular complexes and the specificity of this interaction is essential for their function. The network of the necessary inter-residue contacts must consequently constrain the protein sequences to some extent. In other words, the sequence of an interacting protein must reflect the consequence of this process of adaptation. It is reasonable to assume that the sequence changes accumulated during the evolution of one of the interacting proteins must be compensated by changes in the other.Here we apply a method for detecting correlated changes in multiple sequence alignments to a set of interacting protein domains and show that positions where changes occur in a correlated fashion in the two interacting molecules tend to be close to the protein-protein interfaces. This leads to the possibility of developing a method for predicting contacting pairs of residues from the sequence alone. Such a method would not need the knowledge of the structure of the interacting proteins, and hence would be both radically different and more widely applicable than traditional docking methods.We indeed demonstrate here that the information about correlated sequence changes is sufficient to single out the right inter-domain docking solution amongst many wrong alternatives of two-domain proteins. The same approach is also used here in one case (haemoglobin) where we attempt to predict the interface of two different proteins rather than two protein domains. Finally, we report here a prediction about the inter-domain contact regions of the heat- shock protein Hsc70 based only on sequence information.

Change in surface morphology of diamond films deposited by DC plasma glow discharge CVD

The influence of the deposition conditions on the morphology of two diamond samples prepared by DC plasma glow discharge CVD has been investigated. A structural change has been observed from a 〈110〉 textured morphology, to the morphology of randomly oriented grains with a heavily disturbed crystalline structure, by decreasing the substrate temperature from 1100 to 950°C. Features of the different morphologies have been studied by SEM and related to the results of Raman spectroscopy and X-ray diffraction. The growth side of the first sample presents cubo-ochtaedral grains with low mutual misorientation while the growth side of the second sample is characterised by ball-shaped structures due to an high density of renucleation on the growing planes, separated by deep interdomain regions. The size of the grains forming the rounded structures decreases gradually towards the bottom of these regions. The poorer crystalline quality of this sample is related to its Raman spectrum, showing an higher luminescence and the presence of graphite-like phases all over the growth surface. Moreover an higher density of defects is evidenced by the higher broadening of the Raman diamond line and of the Bragg diffraction peaks with respect to the textured sample. Analogous results have been reported for a microwave CVD reactor. In this work we confirm the hypothesis that this structural change does not depend on the particular CVD method which are likely to provide the same film quality and morphology at different plasma conditions if the activation of the reactant gases and the surface mobility are in the same range of values.

Calcium Determines the Shape of Fibrillin

Velocity sedimentation experiments using authentic fibrillin-1 demonstrated sedimentation coefficients of s20,w0 = 5.1 +/- 0.1 in the Ca2+ form and s20,w0 = 6.2 +/- 0.1 in the Ca2+-free form. Calculations based on these results and the corresponding molecular mass predicted a shortening of fibrillin by approximately 25% and an increase in width of approximately 13-17% upon removal of Ca2+. These observations were confirmed by analysis of Ca2+-loaded and Ca2+-free rotary shadowed fibrillin molecules. Analysis of recombinant fibrillin-1 subdomain rF17, consisting primarily of an array of 12 Ca2+-binding epidermal growth factor (cbEGF)-like repeats, by analytical ultracentrifugation and rotary shadowing further confirmed Ca2+-dependent structural changes in the tertiary structure of fibrillin-1. Based on these results, the contribution of a single cbEGF-like repeat to the length of tandem arrays is predicted to be approximately 3 nm in the Ca2+ form. Ca2+-free forms demonstrated a decrease of 20-30% in length, indicating significant structural changes of these motifs when they occur in tandem. Circular dichroism measurements of rF17 in the presence and absence of Ca2+ indicated secondary structural changes within and adjacent to the interdomain regions that connect cbEGF-like repeats. The results presented here suggest a flexible structure for the Ca2+-free form of fibrillin which becomes stabilized, more extended, and rigid in the Ca2+ form.

Heteronuclear NMR studies of E. coli translation initiation factor IF3. evidence that the inter-domain region is disordered in solution

Initiation factor IF3 from Escherichia coli plays a critical role in the selection of the correct initiation codon. This protein is composed of two domains, connected by a lysine-rich hydrophilic linker. The conformation of native IF3 was investigated by heteronuclear NMR spectroscopy. The two domains are independent and show little or no interaction. Heteronuclear relaxation studies of a sample selectively labelled on lysine residues demonstrates that the inter-domain linker is highly flexible, exhibiting increased 15N T2 values and negative 1H{15N} nuclear Overhause effects over a length of at least eight residues. Analysis of the rotational correlation times further shows that the motions of the two domains are most likely uncorrelated. The inter-domain linker thus displays almost totally unrestricted motions. Accordingly, the amide protons in the central region are shown to be in fast exchange with water. Such a high degree of flexibility of the inter-domain linker might be required for IF3 domains to interact with distant regions of the ribosome.

Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates.

Xylanase A (XYLA) and arabinofuranosidase C (XYLC) from Pseudomonas fluorescens subsp. cellulosa are modular enzymes consisting of discrete cellulose-binding domains (CBDs) and catalytic domains joined by serine-rich linker sequences. To evaluate the role of the CBDs and interdomain regions, the capacity of full-length and truncated derivatives of the two enzymes, lacking either the linker sequences or CBDs, to hydrolyse a range of substrates, and bind to cellulose, was determined. Removal of the CBDs did not affect either the activity of XYLA or XYLC against soluble arabinoxylan. Similarly, deletion of the linker sequences did not alter the affinity of the enzymes for cellulose or their activity against soluble substrates, even when bound to cellulose via the CBDs. Truncated derivatives of XYLA lacking either the linker sequences or the CBD were less active against xylan contained in cellulose-hemicellulose complexes, compared with the full-length xylanase. Similarly, removal of the CBD from XYLC diminished the activity of the enzyme (XYLC''') against plant-cell-wall material containing highly substituted arabinoxylan. The role of CBDs and linker sequences in the catalytic activity of hemicellulases against the plant cell wall is discussed.

A functional chimeric modular polyketide synthase generated via domain replacement

Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules. To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated. These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.

In vivo analysis reveals that the interdomain region of the yeast proliferating cell nuclear antigen is important for DNA replication and DNA repair.

To identify the regions of the proliferating cell nuclear antigen (PCNA) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs-) and 31 methyl methanesulfonate-sensitive (Mmss) mutations of the PCNA gene (POL30) in Saccharomyces cerevisiae. Unlike the Mmss mutations, the Cs- mutations are strikingly clustered in the interdomain region of the three-dimensional PCNA monomer structure. At the restrictive temperature, the Cs- pol30 mutants undergo a RAD9-dependent arrest as large-budded cells with a 2c DNA content. Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through S phase at the restrictive temperature. DNA repair defects are revealed by the observation that Cs- pol30 mutants are very sensitive to the alkylating agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation. Finally, analysis of the chromosomal DNA in pol30 cells by velocity sedimentation gradients shows that pol30 cells accumulate single-stranded DNA breaks at the restrictive temperature. Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo.

Topology of the secondary structure elements of ribosomal protein L7/L12 from E. coli in solution

Topology of the secondary structure elements of ribosomal protein L7/L12 has been studied. The sequential assignment was obtained for main and side chain resonances. This allows the overall secondary structure to be described. The results of high resolution NMR studies show that dimer of the ribosomal protein L7/L12 from Escherichia coli has a parallel (head-to-head) orientation of subunits, and N-terminal domain (NTD, residues Ser1-Ser33) has no contracts with the C-terminal domain (CTD, residues Lys51-Lys120). The NMR data for CTD are in line with crystallographic structure. The flexible interdomain (hinge) region (residues Ala34-Glu50) has an unordered structure, the Pro44 forming both cis and trans peptide bonds. Due to the conformational exchange the intensities of the peaks from the NTD are low. The conformation of the NTD, which is responsible for the formation of the L7/L12 dimer, is α-helical hairpin. The NTD dimer forms an antiparallel four-α-helix bundle.

Multiple domains contribute to the distinct inactivation properties of human heart and skeletal muscle Na+ channels.

Voltage-gated Na+ channels are essential for the normal electrical excitability of neuronal and striated muscle membranes. Distinct isoforms of the Na+ channel alpha-subunit have been identified by molecular cloning, and their functional attributes have been defined by heterologous expression coupled with electrophysiological recording. Two closely related Na+ channel alpha-subunit isoforms, hH1 (human heart) and hSkM1 (human skeletal muscle), exhibit differences in their inactivation properties and in their response to the coexpressed beta 1-subunit. To localize regions that contribute to inactivation and to beta 1-subunit response, we have exploited these functional differences by studying chimeric channels composed of segments from both hH1 and hSkM1. Chimeras in which one or more of the cytoplasmic interdomain regions (ID1-2, ID2-3, and ID3-4) were exchanged between hH1 and hSkM1 exhibit inactivation properties identical with the background channel isoform, suggesting that these regions are not sufficient to cause gating differences. In contrast, inactivation properties of chimeras composed of approximately equal halves of the two channel isoforms were intermediate between hH1 and hSkM1. Furthermore, the response to the coexpressed beta 1-subunit was dependent on structures located in the carboxy-terminal half of the alpha-subunit, although domains D3, D4, and the carboxy terminal are not singularly responsible for this effect. These data indicate that inactivation differences between hH1 and hSkM1 are determined by multiple alpha-subunit domains.

Three chitinase genes ( chiA, chiC and chiD) comprise the chitinase system of Bacillus circulans WL-12

This study was aimed at clarifying the origin and mechanism of processing of multiple chitinase detected in the culture supernatant of Bacillus circulans WL-12 grown in the presence of chitin. Re-examination of N-terminal amino acid sequences of chitinase B1 and B2 led us to propose that these chitinases are derived from chitinase D. Lysyl endopeptidase digestion of both chitinase B1 (39 kDa) and B2 (38 kDa) generated seven common peptide fragments which correspond to different portions of the catalytic domain of chitinase D (newly designated as chitinase D1). Peptide mapping of chitinase D1, B1 and B2 using CNBr digestion indicated that chitinase B1 and B2 are truncated forms of chitinase D1 formed by cleavage of its binding domain and fibronectin type III-like domain. Therefore, it is concluded that chitinase B1 and B2 are derived from chitinase D1 and correspond to the catalytic domain of this chitinase. Limited proteolysis of chitinase D1 with different endoproteases under non-denaturing conditions generated active molecules corresponding to chitinase B1 and B2, providing evidence that chitinase B1 and B2 are produced by proteolytic cleavage at interdomain region of chitinase D1. Results of binding experiments of chitinase D1, B2 and the proteolysed form of chitinase D1 to regenerated chitin demonstrated that the N-terminal portion of chitinase D1, lacking in chitinase B1 and B2, is important for binding activity. These overall findings suggested that chitinases of this bacterium detected to date in its culture supernatant are encoded by only three genes ( chiA, chiC and chiD). All of these genes have already been cloned, sequenced and analyzed.

Probing intradomain and interdomain conformational changes during equilibrium unfolding of phosphoglycerate kinase: fluorescence and circular dichroism study of tryptophan mutants.

Phosphoglycerate kinase is a monomeric protein composed of two globular domains of the alpha/beta type. Extensive domain-domain interactions involve three segments of the polypeptide chain that are distant from one another in the primary sequence: the N-terminus, the C-terminus, and a centrally located alpha-helix. In order to monitor spectroscopically the conformational changes that occur in the individual domains and at the interdomain interface during the unfolding process, we have constructed a series of single-tryptophan mutants. In addition to two previously described mutants, each with single tryptophans in the C-terminal domain (W308 and W333) [Szpikowska, B. K., Beechem, J. M., Sherman, M. A., & Mas, M. T. (1994) Biochemistry 33, 2217-2225], four new single-tryptophan mutants have been constructed: two with tryptophans located in the interdomain region (W194 and W399) and two with tryptophans in the N-terminal domain (W48 and W122). The equilibrium unfolding transitions induced by guanidine hydrochloride were monitored using far-UV CD, near-UV CD, steady-state, and time-resolved fluorescence. These studies reveal two unfolding transitions and suggest a sequential unfolding process for the mutants described in this paper. During the first transition (Cm approximately 0.5 M) the interdomain region and C-terminal domain unfold; the N-terminal domain remains relatively compact but lacks much of the tertiary structure that characterizes the native state. A hyperfluorescent intermediate is detected during this transition by tryptophan probes placed within the N-terminal domain. Complete unfolding of the N-terminal domain occurs during the second transition (Cm approximately 0.9 M).

Effects of C-Terminal Deletions on the Conformational State and Denaturation of Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK) contains two domains of approximately equal size, both of the alpha/beta type. An alpha-helix consisting of the middle section of the 415-amino acid polypeptide chain, and the N- and C-termini reside in the interdomain hinge region [Watson, H. C., et al. (1982) EMBO J. 1, 1635-1640]. The C-terminal end is an integral part of the N-terminal domain. The consequences of the deletion of fifteen and three C-terminal amino acids on the conformational state and on the guanidine hydrochloride-induced and thermal unfolding of PGK were investigated by using near- and far-UV CD, tryptophan fluorescence, 1-anilinonaphthalene-8-sulfonic acid binding, accessibility to chemical modification, and differential scanning calorimetry. The results of these studies indicate that the conformations of both domains and of the interdomain region were altered by these deletions. In the absence of the 15-amino acid C-terminal peptide [delta(401-415)], the N-terminal domain exhibits several characteristics of a molten globule state, whereas the C-terminal domain retains native-like, although distinctly different, tertiary structure. Deletion of three C-terminal amino acids [delta(413-415)] also globally affects PGK conformation, although to a much lesser extent. Both C-terminal deletions resulted in a significant decrease in protein stability, as demonstrated by their increased susceptibility to guanidine-induced and thermal denaturation. These results suggest that the formation of a native tertiary fold of PGK requires the presence of a complete polypeptide chain.

Structural Organisation of Human Bile-salt-activated Lipase Probed by Limited Proteolysis and Expression of a Recombinant Truncated Variant

Bile-salt-activated lipase belongs to the cholinesterase alpha/beta-hydrolase-fold family of proteins. Here, we have investigated the structural organisation of the human isoform by mapping tryptic cleavage sites using limited proteolysis and by expression studies using a recombinant truncated variant. Two accessible regions in the tertiary structure were identified. The first is defined by a tryptic cleavage at Lys429 and lies within the alpha/beta-hydrolase fold in bile-salt-activated lipase between a central beta-sheet and an active-site histidine residue, as deduced from sequence similarity across the cholinesterases and known structural properties. This region exhibits a proteolytic and topological similarity to the lid region in pancreatic lipase. The other accessible region in the tertiary structure is defined by a tryptic cleavage at Arg520 and occurs within a catalytically non-essential segment Leu519-Gln535, as identified by expression of a truncated variant which lacks the C-terminus starting from Leu519. This region is consistent with an interdomain region between the cholinesterase-related part of the protein structure and the unique proline-rich C-terminal repeats. Both protease-sensitive regions appear to occur at domain borders, and, therefore, are consistent with a multi-domain structure. The truncated variant was fully functional as a lipase and as a bile-salt-stimulated esterase. However, compared to the full-length enzyme, the truncated variant showed an increased susceptibility to limited proteolysis, suggesting that the C-terminal repeats may regulate proteolytic degradation of the protein.