Molecular modeling of the collagen-like tail of asymmetric acetylcholinesterase

Paola Deprez and Nibaldo C. Inestrosa1

Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, PO Box 114-D, Santiago, Chile


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The asymmetric form of acetylcholinesterase comprises three catalytic tetramers attached to ColQ, a collagen-like tail responsible for the anchorage of the enzyme to the synaptic basal lamina. ColQ is composed of an N-terminal domain which interacts with the catalytic subunits of the enzyme, a central collagen-like domain and a C-terminal globular domain. In particular, the collagen-like domain of ColQ contains two heparin-binding domains which interact with heparan sulfate proteoglycans in the basal lamina. A three-dimensional model of the collagen-like domain of the tail of asymmetric acetylcholinesterase was constructed. The model presents an undulated shape that results from the presence of a substitution and an insertion in the Gly-X-Y repeating pattern, as well as from low imino-acid regions. Moreover, this model permits the analysis of interactions between the heparin-binding domains of ColQ and heparin, and could also prove useful in the prediction of interaction domains with other putative basal lamina receptors.

Keywords: acetylcholinesterase/collagen/heparin binding/modeling/triple helix


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Acetylcholinesterase (AChE, EC 3.1.1.7) is responsible for the rapid hydrolysis of the neurotransmitter acetylcholine at cholinergic synapses, allowing for the precise control of neurotransmission and muscle contraction (Hall, 1973Go; Anglister et al., 1994Go). AChE presents an exceptional polymorphism, exhibiting both globular and asymmetric forms, that differs in their macromolecular structure and cellular localization (Inestrosa et al., 1982Go; Inestrosa and Perelman, 1989Go, 1990Go; Taylor, 1991Go; Massoulié et al., 1993Go).

In particular, asymmetric AChE consists of one to three catalytic tetramers linked to a collagen-like tail, denoted by ColQ (Massoulié et al., 1991Go). The N-terminal domain of this AChE tail is 117 residues long–including the signal peptide–and contains a proline-rich attachment domain, or PRAD, responsible for the recruitment of the catalytic subunits and their stabilization through disulfide bonds (MacPhee-Quigley et al., 1986Go; Bon et al., 1997Go). In addition, the predicted primary structure of ColQ contains a central collagen-like domain of 188 residues. The remaining 166 residues constitute the C-terminal domain which includes a proline-rich region and a cysteine-rich region (Krejci et al., 1991Go). This general organization as well as the amino acid sequence are highly conserved between Torpedo and mammalian ColQ (Krejci et al., 1997Go; Ohno et al., 1998Go).

The collagen-like domain of ColQ is characterized by Gly-X-Y repeats and a high proportion of the imino-acids proline and hydroxyproline, as well as the presence of hydroxylysine (Lwebuga-Mukasa et al., 1976Go; Rosenberry and Richardson, 1977Go; Anglister and Silman, 1978Go). These distinctive features allow the formation of a collagenous triple-helical structure, where each polypeptide chain adopts an extended left-handed polyproline II-like helix. The three helical chains are staggered by one residue with respect to each other, and are supercoiled in a right-handed manner (Brodsky and Shah, 1995Go; Brodsky and Ramshaw, 1997Go). In this conformation, which is stabilized by the high imino-acid content (Li et al., 1993Go; Bhatnagar and Gough, 1996Go), glycine residues are buried in the center of the superhelix whereas residues in the X and Y positions have their side chains pointing outwards with substantial exposure to solvent, making them available for interaction with other molecules. This structural characteristic allows ColQ as well as collagens to function as multidomain proteins (Brodsky and Shah, 1995Go; Prockop and Kivirikko, 1995Go).

In asymmetric AChE, the collagen-like tail is responsible for the anchorage of the enzyme to the basal lamina and for its specific localization at the neuromuscular junction (Lwebuga-Mukasa et al., 1976Go; Anglister and Silman, 1978Go). Although various molecules have been shown to bind the collagen-like tail of AChE (Emmerling and Lilien, 1981Go; Grassi et al., 1983Go; Vigny et al., 1983Go; Brandan and Inestrosa, 1984Go; Cohen and Barenholz, 1984Go; von Bernhardi and Inestrosa, 1990Go; Inestrosa et al., 1998Go), the exact identity of the tail receptor(s) in the basal lamina has yet to be elucidated. Nevertheless, there is strong evidence suggesting that heparan sulfate proteoglycans are responsible for the anchorage of asymmetric AChE to the basal lamina (Brandan et al., 1985Go; Rossi and Rotundo, 1996Go; Casanueva et al., 1998aGo). In this context, two heparin-binding domains (HBDs) that are present in the collagen-like tail of AChE could be responsible for this interaction, as shown by previous studies using synthetic peptides (Deprez and Inestrosa, 1995Go; Deprez et al., 1995Go; Inestrosa et al., 1998Go).

Because both the N-terminal and the C-terminal domains of ColQ present no significant homology with any other known protein, a three-dimensional model of the structure of the central collagen-like domain of the tail of asymmetric AChE was elaborated and is presented in this paper. By considering this model as an example of a complete collagen-like structure with a naturally occurring sequence, we were able to study the effect of low imino acid regions on the collagen-like conformation, as well as the effect of substitutions and insertions in the Gly-X-Y repeating pattern. In addition, this model is useful in the identification of the specific residues involved in the recognition of heparin, an interaction that is important in the anchorage of asymmetric AChE to the synaptic basal lamina.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Computational results were obtained using software programs from Biosym Technologies (San Diego, CA) run on a Silicon Graphics Indigo computer. Molecular dynamics (MD) and energy minimization (EM) calculations were done using the Discover® 2.97 program, using the CVFF 2.9 force field, and graphical displays were printed out from the InsightII® 95.0 molecular modeling system.

The model of the collagen-like domain of the asymmetric AChE tail was constructed using the structure of 1CLG (Brookhaven Protein Data Bank entry code) as template. 1CLG corresponds to the model structure for a synthetic collagen peptide with a (Gly-Pro-Pro)12 sequence (Chen et al., 1991Go). Because 1CLG is only 36 residues long and we needed a very long protein as template, we constructed a protein of 204 residues long, based on the regular nature of its collagen structure. Firstly, the heavy atoms of the last 12 residues of one 1CLG molecule were superimposed onto the first 12 residues of a second 1CLG molecule. In each chain, the peptide bond between the 12th and the 13th residue of the second molecule was broken, and a bond between the 36th residue of the first molecule and the 13th residue of the second was created, generating a new molecule 24 residues longer. This operation was repeated seven times. The root-mean-square deviation (r.m.s.d.) in aligned positions in each superimposition was 0.36 Å. Since some of the newly created bonds were about 0.1 Å longer than expected, the energy of the residues involved in such bonds was minimized, adjusting bond length without altering neither the torsion angles nor the hydrogen bonding pattern (data not shown). The final template molecule is shown in Figure 1AGo.



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Fig. 1. (A) Space-filling model of the template molecule (upper), where chain A is colored in blue, chain B in red and chain C in yellow, and of the structural model of the collagen-like domain of ColQ, from Torpedo AChE (lower). Both extremities, the Gly->Cys substitution region and the C-terminal insertion are colored in red, while collagen-like regions are colored in blue. (B) Ribbon model of the Gly->Cys substitution region, shown in red, with the rest of the molecule in blue. The side chains of Cys166 are also shown. (C) Ribbon model of the insertion region, colored in red, while the surrounding collagen-like regions are colored in blue.

 
The collagen-like domain of ColQ from Torpedo marmorata (Krejci et al., 1991Go) contains two interruptions in the Gly-X-Y repeats, a Gly->Cys substitution at position 166 and an insertion of 10 residues at position 283. This divides the collagen-like domain into three collagen-like zones: N-terminal (118–165), internal (169–282) and C-terminal (293–307) (Figure 1AGo). The coordinates of the backbone atoms of the constructed template molecule were assigned to the sequence of the collagen-like domain of ColQ, excepting the insertion zone where loops were generated (Homology User Guide, version 2.97, Biosym Technologies). Loop selection considered the lowest r.m.s.d. in the anchor points among loops without undesirable contacts between atoms. In order to include cysteine residues which have been proposed to bracket the central collagen-like structure (Krejci et al., 1991Go), the last nine residues of the N-terminal domain, and the first five residues of the C-terminal domain were also included. In this paper we will use the same sequence numbering given by Krejci et al. (1991), where the first residue of the model corresponds to residue 109 in the ColQ sequence.

The general optimization strategy consisted of minimizing the energy of the different zones of the molecule independently, whereby collagen-like regions were submitted to constraints which were progressively reduced, and submitting the unconstrained non-collagen-like zones to MD. The building and refinement of the model structure is detailed in the following steps:

The non-bonded interaction cut-off distance was fixed at 12 Å. All the minimization steps were done using a combination of steepest descent and conjugate gradients algorithms. The integration time step for the MD calculations was 1 fs, and snapshots of the protein structure were taken every picosecond. A distance-dependent dielectric constant was used to approximate the solvation effect. The {omega} torsion angles were forced to 180° using 80 kcal/rad2. At each EM step, the energy was minimized until the maximum derivative was less than 0.01 kcal.mol–1–1, and in the last step, less than 0.001 kcal.mol–1–1. The average absolute derivative of the global potential energy of the final model was 0.000032 ± 0.000037 kcal.mol–1–1.

PROCHECK v.2.1.4 was employed to check the stereochemical quality of the optimized structure (Laskowski et al., 1993Go). All main-chain as well as side-chain parameters for the whole structure were comparable to high resolution X-ray structures. PROCHECK reported only one non-glycine residue in the disallowed region of the Ramachandran plot, corresponding to 0.23% of all non-glycine residues detected. This residue correspond to Val292B located at the insertion zone, in the boundary with the C-terminal collagen-like zone.

An heparin molecule (1HPN, PDB code) was docked with both HBDs present in the model structure. 1HPN is a dodecasaccharide heparin comprising six repeating disaccharide units each composed of {alpha}-L-iduronic acid linked {alpha}1–4 to N-acetylglucosamine, the former being O-sulfated in position 2 and the latter in position 6, and N-sulfated in position 2 (Mulloy et al., 1993Go). Docking was performed by hand without further minimization, considering an approximate distance of 2.5 Å between the charged groups of heparin and the HBDs, and monitoring bumps between atoms and interaction energies. Thus, several fits with different zones of both HBDs were constructed and in all cases, a minimum of three basic residues was considered in the interaction with heparin.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Global structure

A major feature of the model structure is its undulated shape which is in contrast to the straight rod-like nature of the template (Figure 1AGo). This is consistent with negative staining micrographs of asymmetric AChE in which the collagen-like tail can be observed clearly (Dudai et al., 1973Go; Rieger et al., 1976Go). Interestingly, these undulations shorten the structure from 600 to 530 Å, the latter corresponding to the length of the collagen-like tail measured by electron microscopy (Dudai et al., 1973Go; Krejci et al., 1991Go). As observed in other systems, such perturbations are due to the presence of interruptions in the Gly-X-Y pattern as well as the presence of regions poor in imino acids (Vogel et al., 1988Go; Bella et al., 1994Go; Paterlini et al., 1994Go; Long et al., 1995Go). The undulated shape of the ColQ model is also in contrast with the three-dimensional model of bovine type I collagen, that maintains the straight rod-like and regular shape. This is an important requirement to be organized in microfibrils and is due to the lack of interruptions to the Gly-X-Y repeating pattern and its very high imino acid content (King et al., 1996Go).

The collagen-like domain of ColQ presents two interruptions in its Gly-X-Y repeating pattern. The first, a Gly->Cys substitution at position 166, generates a small kink which causes subtle local untwisting of the triple helix. As shown in Figure 1BGo, the side chain of these cysteine residues are pointing outwards, due to their inability to accommodate in the center of the triple helix. The same phenomenon has been observed in the X-ray diffraction structure of a (Pro-Hyp-Gly)10 peptide, with a Gly->Ala substitution (Bella et al., 1994Go), and in different mutants of type I collagen where Gly->Cys substitutions are implicated in osteogenesis imperfecta (Traub and Steinmann, 1986Go; Vogel et al., 1988Go). The second interruption corresponds to an insertion of 10 residues at position 283, which leads to an untwisting of the triple helix, including the region of the insertion, as well as the limiting triplets in both the internal and C-terminal collagen-like regions. This region could have a more flexible nature, since a bend in the straight triple helix is also generated in the insertion zone (Figure 1CGo).

Besides the perturbations to the collagen-like conformation created by interruptions to the Gly-X-Y repeating pattern, other shape variations are present in the collagen-like depending only on the identity of residues located in X and Y positions. This sequence-dependent local destabilization has been studied by Paterlini et al. (1994) in models of poly(GPP) structures containing a GAA triplet in the middle of the sequence, showing that the increased flexibility in the backbone of the GAA triplet allows the formation of a variety of low-energy conformations different from the rod-like shape.

Local analysis

The shape variations presented by the model of the collagen-like domain of ColQ were evaluated at a local level. One approach is to measure the r.m.s.d. between the backbone of the model and the template structure. In order to make local comparisons, each r.m.s.d. was calculated using a window of three residues and including the three chains (Figure 2Go). The highest r.m.s.d. value in the collagen-like domain of ColQ arises in the C-terminal insertion with a value of 4.14 Å. The Gly->Cys substitution also presents a high r.m.s.d. value, and as expected, so do both extremities that lack collagen-like sequences. In comparison, collagen-like zones presented lower r.m.s.d. mean values (Table IGo), with several local rises (Figure 2Go). In particular, the N-terminal collagen-like zone presented a high r.m.s.d. peak at residue 198 coinciding with a kink observed in the middle of the triple helical structure (Figure 1AGo).



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Fig. 2. R.m.s.d. values in angstroms between the backbone of the model and the template structures. Each r.m.s.d. was calculated using a window of three residues, for all the three chains, which was moved by one residue from the N-terminus to the C-terminus for each measurement. In the graph, the r.m.s.d. value of each window is assigned to the first residue of the window.

 

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Table I. Hydrogen-bonding pattern, Pro contribution and r.m.s.d. in the different zones of the ColQ model
 
An important structural feature of the collagen-like conformation is the presence of a hydrogen-bonding pattern between the backbone of adjacent chains, in which glycine amide groups from one chain hydrogen-bond to proline carbonyl groups in the X position of the next chain (Bella et al., 1994Go). In the present model, this pattern was also observed in the collagen-like regions even if the residue in the X position was not a proline. This is consistent with previous modeling studies using a 12 residue-long natural sequence from type I bovine skin collagen, where the presence of non-imino acids does not alter this pattern (Vitagliano et al., 1993Go). All the regions with high local r.m.s.d. values correlates with zones in which this hydrogen bonding pattern is interrupted. Considering that each triplet can form a maximum of three hydrogen bonds, the pattern of interruptions was analyzed according to the number of hydrogen bonds really formed, ranging from zero to three. Figure 3Go shows the occurrence of these hydrogen bonds in each triplet along the model structure. Also shown in Table IGo, the non-collagen-like zones contain no hydrogen bonds, and the r.m.s.d. mean value is very high (>2.4 Å).



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Fig. 3. Conservation of the hydrogen-bonding pattern in the model structure. The non-collagen-like zones are labeled with a dashed line above the amino acid sequence, below which the hydrogen-bonding pattern is shown. For each triplet, the residue in position X was considered for hydrogen-bonding. For a triplet number n, the line just under the amino acid sequence represents an hydrogen bond between the carbonyl group of the Xn residue of chain B, and the Glyn residue of chain A. The middle line corresponds to a hydrogen bond between the residue Xn from chain C and the Glyn+1 residue from chain B. Finally, the lower line represents a hydrogen bond between Xn from chain A and Glyn+1 from chain C.

 
For collagen-like regions, a high percentage of the hydrogen-bonding pattern is conserved, which is consistent with lower r.m.s.d. values. In these regions, some of the triplets that have lost hydrogen bonds are adjacent to the interruptions to the Gly-X-Y repeating pattern, but the majority of them depend only on the local specific sequence. The first phenomenon is specially important in the C-terminal collagen-like zone, comprising only five Gly-X-Y triplets, where there is only a single triplet with three hydrogen bonds (GER, 296–298). Interestingly, arginine in the Y position has shown to confer high stability, similar to hydroxyproline, in triple helical peptides (Yang et al., 1997Go; Ackerman et al., 1999Go). At the N-terminal collagen-like region, there is a single triplet (GEI, 142–144) that does not form a hydrogen bond, corresponding to the first rise in r.m.s.d. values seen in Figure 2Go. This is also true for the GQK triplet (199–201) in the internal collagen-like region, which coincides with a large rise in r.m.s.d. in this region and with the formation of a kink in the triple helix (Figure 1AGo). As for the interruptions, both triplets are surrounded by triplets that lack at least one of the three hydrogen bonds. Excepting for those unstable regions, all the imino acid containing triplets have the three expected hydrogen bonds (Figure 3Go and Table IGo).

Another way to analyze the divergence of the model from the template structure is to look at the torsion angles. Figure 4Go shows the Ramachandran plots for glycine and proline residues, and all other residues in various regions of the model. The polyproline II helix is characterized by torsion angles of about {phi} = –75° and {Psi} = 145°, and possesses conformational parameters similar to those generated by a polyglycine helix (Bhatnagar et al., 1997Go). Indeed, the majority of glycine residues are clustered in the polyproline II region of the plot (Figure 4AGo), although torsion angles are also presented in other regions. Proline residues, present only in the collagen-like zones of the model, are restricted to this zone of the Ramachandran plot (Figure 4BGo). Similarly, the majority of residues in the collagen-like zones are presented in the polyproline II region of the plot (Figures 4D, E and FGo). For the non-collagenous regions of the model structure, the dispersion of the torsion angle values is seen to increase (Figure 4CGo).



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Fig. 4. Ramachandran plots for (A) glycine residues and (B) proline residues, throughout the model structure. (C) to (F) represent Ramachandran plots divided into distinct regions of the model which consider all residues except glycines and prolines. Specifically, Ramachandran plots are shown of (C) all the non-collagen-like regions, (D) the N-terminal collagen-like zone, (E) the internal collagen-like zone and (F) the C-terminal collagen-like zone.

 
An interesting alteration arises with the triplet GVR (154–156), where the carbonyl group of valine (chain B) is not involved in the hydrogen-bonding pattern, which is maintained by the valines in the other two chains (Figure 3Go). In chain B, Val 155 is located in the lower left quadrant of the Ramachandran plot, in which the distance between {alpha}-carbons is shorter than in collagen. This is compensated by Arg156 located in the upper left corner of the plot, which is more extended than the polyproline II conformation, maintaining in this way the length of the triplet. In fact, the same phenomenon occurs in the internal collagen-like zone (Figure 4EGo) with the triplets GHR (244–246) and GKT (256–258). This suggest that these regions can present local conformational transitions that can be important for interacting with other molecules. A similar phenomenon was observed on the cell binding site of type I collagen that has been shown to present conformational tautomerism between polyproline II and strand–bend–strand conformations, maintaining the length of the corresponding triplet (Bhatnagar et al., 1997Go).

Heparin-binding domains

The only sites of interaction with other molecules that have been mapped in the collagen-like domain of ColQ are two HBDs that mediate the anchorage of asymmetric AChE in the basal lamina through interactions with heparan sulfate proteoglycans (Deprez and Inestrosa, 1995Go; Deprez et al., 1995Go). Each domain is composed of the core consensus sequence BBXB, where B represents a basic residue (Cardin and Weintraub, 1989Go), and other basic residues surrounding the core. The fact that only basic residues participate in heparin interaction has been confirmed by site-directed mutagenesis studies in ColQ (P.Deprez, E.Krejci, J.Massoulié and N.C.Inestrosa, unpublished results). Figure 5AGo shows the two HBDs in the model structure, each forming a broad basic belt around the triple helix. It is important to note that all other basic residues in the collagen-like domain are either paired with acidic residues, or are isolated residues that alone are insufficient to conform a HBD. In terms of stability, the hydrogen-bonding pattern is interrupted in both HBDs, and in particular, the triplets that contain the very unusual sequence GBB present only one hydrogen bond (Figure 3Go). This is likely to confer a local flexibility for accommodation of the ligand, in this case heparin.



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Fig. 5. Space-filling model of the collagen-like domain of ColQ showing the heparin-binding domains (A), where the basic residues are colored in blue, and the rest of the molecule is in orange. (B) and (C) show the N-terminal HBD docked by hand and without further minimization to a dodeca-saccharide heparin molecule (Mulloy et al., 1993Go), considering a distance of about 2.5 Å between the charged groups from basic residues and the sulfate groups from heparin. (C) is a lateral view and (D) is a transversal view. Heparin is colored in red with sulfate groups in yellow. All basic residues are colored in blue, and the interacting arginines present the terminal nitrogen atoms in green.

 
In order to elucidate the possible interactions between the ColQ HBDs and heparin, a dodecasaccharide heparin molecule (Mulloy et al., 1993Go) was docked with both HBDs present in the model structure. Figure 5B and CGo show one of the docking possibilities, where three arginines are interacting with sulfate groups of heparin. The distance between two consecutive interacting basic residues vary from 14 to 23 Å, consistent with the results of Margalit et al. (1993) who stated that the distance between two basic residues has to be about 20 Å in order to mediate an interaction with heparin in several globular proteins.

In this way, it is possible to obtain different arrangements between heparin and each HBD, suggesting that these domains can bind heparin in multiple ways. Within a HBD, the same group of basic residues can also bind heparin in different ways, for instance in a parallel and anti-parallel fashion. Interestingly, among all the possibilities observed, the N-terminal HBD always had a maximum of three residues interacting with heparin at the same time, while the C-terminal HBD generally involved more than three. An example of the latter involves residues Arg246A, Lys254C, Arg255B, Lys257A and Lys261A. This observation implies that the C-terminal HBD could bind heparin stronger than the N-terminal HBD, which is consistent with results obtained using synthetic peptides carrying either the HBD sequence that acquired a collagen-like conformation in solution (Inestrosa et al., 1998; P.Deprez, E.Doss-Pepe, B.Brodsky and N.C.Inestrosa, unpublished results).

It is important to consider the context of the triple helix, in which the three or more basic residues involved in heparin-binding are recruited from two or even three different chains, making this kind of interaction not comparable to that which arises with the same sequence in a monomeric state. It is interesting to note that a maximum of four heparin molecules could be docked simultaneously to each HBD. This could explain the fact that the collagen-like conformation binds heparin stronger than in the monomeric one, which could only bind one equivalent heparin molecule assuming it contains a typical HBD (Cardin and Weintraub, 1989Go). The last is consistent with results using peptides with both monomeric and trimeric conformations (P.Deprez, E.Doss-Pepe, B.Brodsky and N.C.Inestrosa, unpublished results). Another interesting point is that different arrangements are generated, leading to the interaction with either three or four heparin molecules depending on where the first heparin molecule is docked. This is consistent with the fact that both native and mutant asymmetric AChE containing one of the two HBDs, present different populations that bind heparin with different affinities when eluted with a NaCl gradient from an heparin–agarose column (P.Deprez, E.Krejci, J.Massoulié and N.C.Inestrosa, unpublished results). Hence, each population could correspond to enzymes that are interacting with a different number of heparin molecules and/or in different ways. Finally, heparin present acidic charge clusters on both sides of the molecule, and each side is able to interact simultaneously with two collagen-tail molecules, explaining the fact that heparin provokes the aggregation of asymmetric AChE at low salt concentrations through the collagen-like tails of this enzyme (Dudai et al., 1973Go; Bon et al., 1978Go; Lee and Taylor, 1982Go; Torres and Inestrosa, 1985Go).

Final remarks

It is possible to predict that regions that alter the regular and stable collagen-like conformation of ColQ are likely to constitute sites for interaction with other proteins, providing conformational flexibility and defining specificity, as seen in other systems. As for the HBDs present in ColQ, the more common examples correspond to low imino acid regions in the collagen-like molecule. This is the case, for instance, for the acetylated low-density lipoprotein and the tetraplex nucleic acid binding sites present on the macrophage scavenger receptor (Mielewczyk et al., 1996Go; Tanaka et al., 1996Go), as well as the cell binding site on the fibril forming type I collagen (Grab et al., 1996Go; Bhatnagar et al., 1997Go). Another example is provided by an antibody epitope on type III collagen characterized by the presence of a Gly-Gly-Y triplet, which has been shown to have a destabilizing effect on the local collagen-like structure (Shah et al., 1997Go). In the cases presented, it is interesting to note that the activity of the reactive sites depends on the trimeric conformation context even if a stable collagen-like conformation is not present locally. In non-fibrillar collagens and proteins with collagen-like domains such as asymmetric AChE, interruptions in the Gly-X-Y repeating pattern is a relatively common feature which has been shown to be important for function. In particular, the basement membrane type IV collagen contains several non-collagen-like regions that act as cellular recognition sites, some of which are conformational independent, while others need to be in a triple-helical environment, surrounded by collagen-like regions (Miles et al., 1994Go, 1995Go).

In this context, the collagen-like domain of ColQ presents two interruptions in the Gly-X-Y repeating pattern and several regions with perturbations in the collagen-like structure, all of which could constitute putative recognition sites. As expected for a protein with multidomain potential, the collagen-like tail of AChE can bind several molecules. In addition to heparan sulfate proteoglycans, known to have a physiological function (Brandan et al., 1985Go), other molecules have been shown to interact with ColQ in vitro, these include type I and type V collagens, laminin (Vigny et al., 1983Go), sphingomyelin liposomes (Cohen and Barenholz, 1984Go), and a collagenous protein of 140 kDa present in Torpedo extracellular matrix extracts (Casanueva et al., 1998bGo). Moreover, ColQ presents two collagenase-sensitive regions, one which is cleaved at 20°C and the other specifically at 37°C, which are found distal and proximal to the catalytic subunits, respectively (Anglister and Silman, 1978Go; Bon and Massoulié, 1978Go; Bon et al., 1979Go). By estimating the size of the cleaved ColQ segments, it is possible to speculate that the region sensitive to collagenase at 20°C is either in the C-terminal insertion or in the collagen-like zones bordering the insertion, which is consistent with the important untwisting present in this zone. On the other hand, the more resistant 37°C-sensitive site could occur near the GQK (199–201) triplet, which lacks hydrogen bonds between its backbone atoms (Figure 3Go), presents an important rise in the local r.m.s.d. value (Figure 2Go), and gives rise to a kink in the triple helical structure (Figure 1AGo). Interestingly, this region contains a -Pro-X-Gly-Pro-Y- sequence recognized by Clostridium collagenase (Harper and Kang, 1970Go; Katayama et al., 1978Go), specifically, the PMGPK sequence located between residues 191–195. This distribution for the collagenase recognition sites is consistent with the fact that asymmetric AChE treated with collagenase at 37°C is less sensitive to aggregation in low salt conditions than the enzyme cleaved at 20°C (Bon and Massoulié, 1978Go; Lee and Taylor, 1982Go), given that the former contains only the N-terminal HBD, and the latter contains both.

Using the same criteria, the region around the GEI (142–144) triplet which also lacks the characteristic hydrogen bonds (Figure 3Go), and the Gly->Cys substitution at position 166 could also constitute other recognition sites within ColQ. It is important to mention that since AChE is a secreted protein, most of its sulphydryl groups are expected to be disulfide bonded, particularly considering they are exposed to solvent. In fact, this occurs in the mutant type I collagen that causes lethal osteogenesis imperfecta (Traub and Steinmann, 1986Go). However, disulfide bond studies in ColQ show that no such inter-chain linkages occur in the collagen-like zone, although they do arise in the N- and C-terminal domains flanking the collagen-like domain (Rosenberry and Richardson, 1977Go; Lee and Taylor, 1982Go). One possible explanation is that the putative ligand for this site is bound very early in the secretory pathway, thus protecting sulphydryl groups from the reducing environment. Considering that this substitution is not conserved between Torpedo and mammalian ColQ sequences (Krejci et al., 1997Go), and that these substitutions frequently occur in other collagens, it is also possible that the specific clone of ColQ isolated and sequenced contained this single mutation (Krejci et al., 1991Go).

The model of the collagen-like domain of the tail of asymmetric AChE presented here show that both interruptions to the Gly-X-Y repeating pattern, as well as sequence-dependent low imino acid regions are determinant in the generation of shape variations to the rod-like shape of fibril-forming collagens. These low-stability regions can confer local flexibility important for interaction with other molecules. Future studies using this model will be directed to the prediction of other putative binding sites that are important for the anchorage at the basal lamina and the specific localization of asymmetric AChE at the neuromuscular junction.


    Acknowledgments
 
We thank Dr Barbara Brodsky for helpful comments on the manuscript. P.D. is a pre-doctoral fellow from CONICYT. This work was supported by FONDECYT No. 2970072 to P.D. and by a Presidential Chair in Science from the Chilean Government to N.C.I.


    Notes
 
1 To whom correspondence should be addressed; email: ninestr{at}genes.bio.puc.cl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Ackerman,M.S., Bhate,M., Shenoy,N., Beck,K., Ramshaw,J.A.M. and Brodsky,B. (1999) J. Biol. Chem., 274, 7668–7673.[Abstract/Free Full Text]

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Received March 9, 1999; revised October 15, 1999; accepted October 20, 1999.