Structure of Cellulolytic Enzymes - ACS Symposium Series (ACS

Chapter 23

Structure of Cellulolytic Enzymes 1

2

H. Esterbauer, M. Hayn, P. M. Abuja , and M. Claeyssens

Downloaded by CORNELL UNIV on May 16, 2017 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch023

Institute of Biochemistry, University of Graz, Schubertstrasse 1, A-8010 Graz, Austria The structures of two cellobiohydrolases (E.C.3.2.1.91)fromTrichoderma reesei (CBH I, CBH II) were determined by small angle X-ray scattering (SAXS) analysis. Both enzymes have a tadpole-like shape with an isotropic head and a longflexibletail. The CBH I molecule has a length of 18.0 nm, the head has a maximum diameter of 2.09 nm; CBH II has a length of 21.5 nm with a head of 2.10 nm. The SAXS data are discussed in relation to recent X-ray diffraction studies and other known structural properties (amino acid sequences, disulfide linkages, glycosylation, active center localization, cellulose-binding sites) of the enzymes.

Cellulases are highly specialized fungal or bacterial enzymes catalyzing the hydrolysis of beta-l,4-glycosidic bonds in crystalline and amorphous cellulose, soluble cellodextrins and some derivatives such as carboxymethyl cellulose, hydroxyethyl cellulose and chromophoric glycosides of cellodextrins (for review see réf. 1). Considerable progress has recently been made in the purification of these enzymes (for review see réf. 1), the determination of their substrate specificity and kinetic properties 2,3, gene analysis and cloning 4 as well as in their structural and functional characterization 5. All evidences available point to a two domain organization of these cellulolytic enzymes: a catalytic (hydrolytic) domain (core) and a separate cellulose binding domain. In most sequenced cellulases 80 to 90 % of all amino acids are located in the core part which thus contains the major mass of the cellulase protein. Such an organization has been demonstrated unambiguously for four cellulases (CBH I, CBH II, E G I and E G III) from Trichoderma reesei 5-7, and two enzymes (CenA and Cex) from Cellulomonasfimi8. It was tentatively deduced for several other cellulases e.g. those from Clostridium thermocellum 9. Analysis of a large number of genes coding for cellulases clearly showed that most of the cellulase proteins are in fact products of individual genes. For example, cbhl, cbh2, egll, egl3 were identified and sequenced from Trichoderma reesei (for review see 4); more than fifteen cellulase genes were found in Clostridium thermocellum and five of them were sequenced 9. The comparison of the amino acid sequences suggests that cellulases usually show no or only weak sequence homology in the catalytic domain. Exceptions are CBH I and E G I of T. reesei with 50% sequence homology in the head domain. Comparisons of secondary structure using more elaborate methods such as hydrophobic cluster analysis have established the existence of at least six structural cell1

Current address: Institute of Physical Chemistry, University of Graz, Heinrichstr. 28, A-8010 Graz, Austria Current address: Laboratory for Biochemistry, State University Ghent, K.L.Ledeganckstraat 35, B-9000 Ghent, Belgium 2

u097-6156/91/0460-0301$06.00/0 © 1991 American Chemical Society

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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ENZYMES IN BIOMASS CONVERSION

ulase families composed of enzymes from widely different organisms, but showing strong homologies within each family 10. Most cellulases also contain a 30 to 60 amino acid long sequence rich in proline and highly glycosylated threonine and serine residues. This region is frequently called block Β 4 or PT-box 8. Adjacent to block Β the so called "cellulose binding domain" (block A) is found. It contains about 30 to 100 amino acids. The sequence in this block A appears to be highly conserved in cellulases from the same organism. For example, in T. reesei the enzymes CBH I, CBH II, E G I and E G III show 70% sequence homology in block A consisting of 30 amino acids. Two cellu lases from C. thermocellum (EG A and E G B) show an identical reiterated sequence of 24 amino acids in block A which is preceded in both enzymes by a domain rich in proline, threonine and serine. The repeated sequence of 24 amino acids is also found in a third C. thermocellum cellulase (EG D) but here the proline-rich sequence is missing 9. Similarly in two cellulases from Cellulomonas fimi a highly conserved block A is apparent, adjacent again to a PT box 8. The contiguous AB regions, i.e. the tail domains, are located at either the carboxy- or at the amino terminus of the polypep tide chain. In the first case the sequence of the three blocks from the Ν - t o the C terminus is therefore "core-Β-A", in the latter case "A-Β-core". It seems reasonable to assume that this interchanged position of the Β and A part was mediated in evolu tion by gene shuffling. For Trichoderma reesei the cellulases most thoroughly investigated with respect to their structural properties are CBH I and CBH II and to a lesser extent the two endoglucanases (EG I and E G III) 11 (Table I). For most other cellulases structural characterizations are confined to the amino acid sequence as deduced from the base sequence of the respective genes and to sequence related investigations such as hydro phobic cluster analysis 10 or computer aided molecular modelling and homology TABLE I: Analysis performed on T. reesei enzymes Enzyme

amino acids

domain sequence

analysis performed

CBH I

497

core-ΒΑ

aa sequence in part from protein and in full from gene (cbhl), number and location of SS bridges, region of O-glycosylation, types of car bohydrate, papain cleavage site, hydrophobic cluster analysis, computer model of active site, 2D-NMR on a synthetic tail fragment, SAXS on whole CBH I, head domain and xylan/CBH I complex

CBH II

447

ABB'-core

aa sequence in part from protein and in full from gene (cbh2), number of SS bridges, region of O-glycosylation, types of carbohydrate, papain cleavage site, hydrophobic cluster analysis, SAXS on whole CBH II and head domain, three d i mensional structure of head by X-ray diffraction

EG I

437

core-ΒΑ

aa sequence in part from protein and in full from gene (egll), hydrophobic cluster analysis, SAXS not successful

EG III

397

AB-core

aa sequence in part from protein and in full from gene (egl3), high mannose content 47 mole/mole enzyme, hydrophobic cluster analysis


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comparison. One should however keep in mind that such investigations provide only approximations of the structural and functional organization. Only X-ray diffraction analysis of crystalline cellulases with resolution in the 0.1 - 0.2 nm range would allow precise determination of the tertiary conformation and the spatial position of the amino acid residues. The core domains of CBH I and CBH II have been crystallized 11,12 and very recently, the fully resolved three-dimensional structure of the CBH II core was published 13. EGD from Clostridium thermocellum cloned in E. coli has also been crystallized 14.


Small-Angle X-Ray Scattering Small-angle X-ray scattering (SAXS) has been used since quite some time for the determination of the structure of macromolecules in solution (for a review see 14)· Also for non-crystallizing proteins some structural details can be obtained and since measurements can be performed under more or less "natural" conditions in aqueous solutions and various influences on the shape of the molecule can be studied (e.g. effects of ligand binding). Colleagues from the Institute of Physical Chemistry at the University of Graz have a long standing tradition and experience in using this technique. In fact most of the pioneering work in theory and practice (Kratky camera) and the development of this method have been performed in this Institute under the leadership of Prof. Dr. O. Kratky (For review see 16). Several parameters, e. g. the radius of gyration (Rg) and the maximal diameter (Dmax), of the molecule are available directly from the scattering function 1(h) or from the so-called distance distribution function p(r) which may be obtained by Fourier transformation of the 1(h) function 17. The distance distribution function p(r) gives some preliminary information concerning the shape of the measured molecule. Compu ter-aided modelling to simulate the distance distribution and scattering functions with models made up of small spheres can be used to reveal further structural details by trial and error. Models for the shape of the molecule are generated in this way until their p(r) and 1(h) functions closely match the experimental data 16. The computed model which fits best to the experimental functions is taken as representative for the shape of the investigated molecule. The resolution of SAXS depends on the form of the molecule and is on average between 0.2 and 0.5 nm. The main difference from high resolution X-ray diffraction is that SAXS can resolve only details of the tertiary and quarternary structure of the molecule and thus give information about the molecular shape but not on the secon dary structure. The principle of the method is briefly as follows: A series of at least four con centrations (ranging from 5 - 5 0 mg/ml) are measured successively for several hours each. During the measurement of each SAXS curve the scattered radiation is recorded for a range of angles between nearly zero and 3 to 5 angular degrees with respect to the primary beam. The measurement can be performed at any temperature that is suited to the needs of the sample. After completion of the measurements the data set is extrapolated to zero concentration and Fourier transformed. The results are the desmeared scattering and distance distribution functions. The average amount of sample needed for a complete SAXS analysis is 30 to 50 mg of an at least 95% pure sample. Structure of CeUobiohydrolase I from T. reesei Cellobiohydrolase I (CBH I, 1,4-β-D-glucan-cellobiohydrolase, E.C. 3.2.1.91) is the main protein (ca. 60%) of the cellulase complex produced by T. reesei strains. CBH I hydrolyses crystalline cellulose, acid swollen cellulose and 4-methylumbelliferyl-cellodextrins by cleaving off the terminal cellobiose unit from the non reducing end of the chain. It operates with retention of configuration in the reaction products 19,20. The abundance of this enzyme and its stability has facilitated its purification to homogeneity



304


by various chromatographic methods in quantities up to several hundred milligrams, amounts sufficient for detailed biochemical and physicochemical studies. Large parts of its amino acid sequence had already been determined in 1984 21 and this had later been confirmed by sequencing the corresponding gene cbhl 22,23. CBH I consists of 497 amino acid residues: 33 Asp, 23 Asn, 57 Thr, 56 Ser, 23 Glu, 19 Gin, 28 Pro, 63 Gly, 29 Ala, 23 Val, 24 Cys, 6 Met, 11 He, 26 Leu, 24 Tyr, 15 Phe, 13 Lys, 5 His, 9 Arg, 9 Trp. The degree of glycosylation (5 to 10%) depends on the mutant and probably also on the culture conditions. Identified sugar residues include 16 mannose, 6 glucose and 5 galactose units per CBH I molecule 24. Upon SDS po lyacrylamide electrophoresis and native polyacrylamide electrophoresis homogeneity is proven and the molecular mass found by different methods is between 59 and 68 kDa 24. The CBH I, prepared from Trichoderma reesei MCG 77 and purified by chromatofocusing exhibited a molecular mass (SDS-PAGE) of 59±1.5 kDa and its isoelectric point was 3.6. After initial SAXS measurements were performed on this sample, results surprisingly pointed to an unusual shape. The scattering curve indeed suggested that the molecule has a rather large head and a flexible tail. It was first thought that this might be an artifact caused by impurities (e.g. peptides) in the preparation or some other unknown factors. CBH I was therefore prepared again from a batch of another fermentation, and no impurities could be detected by SDS-PAGE or FPLC on a Mono Q column or in the analytical ultracentrifuge. The SAXS measurements gave again nearly the same result as obtained with the first CBH I preparation (Figure 1) and we were therefore convinced that CBH I has an unusual "tadpole" shape. The model pub lished in 1986 24 together with the two distance distribution functions p(r) experimen tally measured for these two CBH I preparations and the p(r) curve calculated for the tadpole model is shown in Figure 1. The dimensions of the molecule are given in Table II. This publication 24 started a series of collaborative efforts which confirmed and significantly extended our knowledge about the three-dimensional shape of cellobiohydrolases. A CBH I prepared from the T. reesei strain VTT D-80133 by affinity chromatography 25 revealed by SAXS molecular dimensions which are very close to those previously measured (Figure 2). The model derived from the scattering curves again showed a tadpole structure with a total length of 18.0 nm. About two-thirds of this length is confined to the tail. This time more distinct structural details could be observed, e.g. the collar-like part in the tail probably representing the region of gly cosylation (block B). The presence of two structural domains in CBH I has been postulated previously based on the amino acid sequence and the location of disulfide bridges 26 and this was later confirmed by limited proteolytic studies 27. Treatment with papain cleaves the polypeptide chain of CBH I at about residue 430 leading to a small (10 kDa) C-ter minal glycoprotein which strongly binds to cellulose and a large 54 kDa N-terminal core protein which contains the active center. It could be shown by SAXS 25 that this core protein is the head domain of the CBH I molecule (Figure 3). The molecular dimensions of the cleaved head part isolated from the papain digest by affinity chro matography coincide with the dimensions of the head seen in the intact molecule (Table II). Such a polypeptide chain folding into two distinct domains could be correlated with the amino acid sequence. The N-terminus of the polypeptide chain is located at an as yet unknown site of the head part and starts with the sequence Pyr.Glu-Ser-AlaCys-Thr-, the C-terminal region is located at the end of the tail and has the se quence -Pro-Tyr-Tyr-Ser-Gln-Cys-Leu. From the total of 497 amino acids about 430 are located in the head part, where the chain is partially ordered in β-structures. Ten disulfide bridges give stability and rigidity to this part of the molecule 24. The bridges interconnect the cysteine residues 4-50, 19-25, 61-71(?), 67-72(?), 138-397, 172-209(7), 176-210(7), 230-256, 238-243, and 261-331. The exact position of some


Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991. r

[nm]

Figure 1. Model of CBH I deduced from SAXS measurements proposed 1986 (from ref. 24 with permission of the authors). The distance distribution function p(r) was determined for two different CBH I preparations ( ,—· ), the dotted line ( . . . · ) giving the p(r) function calculated for the model.

>



306


^

r

[nm]

Figure 2. Model of CBH I deduced from a second series of SAXS measurements (redrawn from réf. 25 with permission). The sequence of the domains from the N-terminus to the C-terminus is C - B A The arrow indicates the site papain cleavage.

r [nm] Figure 3. SAXS-based model for CBH I core protein (Reproduced with permission from ref. 25; Copyright Springer 1988). The core protein was prepared by limited papain proteolysis from the intact enzyme preparation shown in Fig. 2



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ESTERBAUER ET A L


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local bridges is yet unknown. It should be noted that two SS-bridges connect regions which are 70 (261 to 331) and 259 (138 to 397) amino acid residues distant from each other. Such non-local bridges could contribute significantly to the compactness of the head part of the molecule. After the amino acid at position 429 (Thr) the protein chain probably exits from the main body of the head and the sequence which follows is (430) Glv-Asn-Pro-Ser-Glu-Glv-Asn-Pro-Pro-Glv-Glv-Asn-Pro (442). In analogy to other structurally characterized proteins the Gly-Asn-Pro - boxes can be seen as a hinge region which makes a flexible connection between the head and the remaining tail part. That this region has a flexible connection to the head was already proposed in the SAXS study from the variability of the distance distribution function seen in the tail part of the molecule 21. The following sequence contains 22 amino acids (443 to 464) rich in threonine (8 residues) and serine (3 residues) which probably reflects the region of the Ο-glycosylated block B. The last stretch of the tail comprises 33 amino acids (465 to 497) and contains two SS bridges which span from residue 469 to 486 and from 480 to 496 26. This last part of the tail domain (block A) contains binding site(s) for cellulose. In the tadpole model the region of glycosylation is pro bably the bulgy, collar-like part of the tail. If so, the connecting piece to the head should be the hinge region and the part following the bulge should be the cellulose binding domain A In summary, the tail of CBH I consists of 67 amino acid residues, the first part (hinge region and glycosylated region, block B) contains 34 residues, the second part (cellulose binding region, block A) consists of 33 amino acid residues. The C-terminal polypeptide block A (residues 462-497) has also been synthe sized and its three dimensional structure in solution (90% H O/10%D O) was deter mined by two-dimensional NMR-spectroscopy with a 600 MHz instrument 28. Accor ding to this analysis the peptide has a wedge-like shape with dimensions 3 χ 1.8 χ 1.0 nm. This wedge probably represents the last part in the SAXS model which has a length of about 4 nm and a maximum diameter of 1.4 nm. The principle element of the secondary structure in this part is the 0-conformation. Three antiparallel short βsheets composed of residues 466 to 470 (β ), 485 to 489 (β ) and 493 to 496 (β ) were identified. The sites which could be responsible for binding to cellulose were not identified but the NMR data indicate that one face of the wedge is flat and hydrophilic (4 tyrosine residues) whereas the other face is hydrophobic. Both structures could po tentially interact with the cellulose chain on the surface of the crystallites. Some evi dence has been advanced for the fact that tyrosine residues play a part in the binding phenomenon 5. The core protein prepared by papain cleavage is enzymatically active, which proves that the active center, where the 0-1,4-glycosidic bonds are cleaved, is in the head domain of the protein. The amino acids of the active site have not yet been identified with certainty. From chemical modification experiments with more or less COOH-group specific reagents (carbodiimide, Woodward's reagent K) it seems now rather well estab lished that a carboxylate group, probably a glutamic acid residue, plays a crucial role in catalytic activity 29. Glutamic acid has also been identified as a part of the catalytic domain in several lysozymes, e.g. human milk, duck egg white, hen egg white or T4 phage lysozyme. In T4 phage lysozyme glutamic acid 11 and aspartic acid 20 are catalytically active residues and it has been proposed that in CBH I catalytically active residues are either glutamic acid 64 and aspartic acid 74 30 or glutamic acid 126 29 and aspartic acid 130 29. SAXS investigations have also been presented which suggest that CBH I can un dergo some kind of induced fit with a change of shape if it interacts with a ligand 32. The insoluble cellulose itself cannot be used for such studies since it would disturb the scattering experiment. Instead of cellulose a water soluble xylan (Rhoaymenia palmata) with an average molecular weight of 3000 to 5000 was used. This xylan interacts very strongly with the intact CBH I and its core, most probably by binding to the active (hydrolytic) center and, to a lesser extent, to the cellulose binding domain. SAXS measurements of CBH I in the presence of a twofold molar excess of xylan revealed 2

χ

2

2


3


308

that the binding of xylan to the head leads to a significant elongation of the particle, in particular in the tail domain (Table II). From computer aided simulation studies it was suggested that the xylan binding site (which eventually might be found to be the catalytic center of the enzyme) is located at the surface of the head near the tail part.

Table II: Molecular dimensions of T. reesei cellobiohydrolases and their core as determined by SAXS Rg = radius of gyration in nm ± 5%, D n.d.: not determined

m

= maximum distance in nm±5%, a

x

whole enzyme


Rg

D

core shape

Rg

D

tadpole

n.d.

max n.d.

shape

CBH I

a

4.20

max 18.0

CBH I

b

4.27

18.0

tadpole

2.09

6.5

ellipsoid

5.40

21.5

tadpole

2.10

6.0

ellipsoid

5.18

22.0

tadpole

2.06

5.8

ellipsoid

e

CBH II

d

CBH I/xylan

a) MCG 77, 24; b) QM 9414, (VTT-D-80133); 25 c) QM 9414 31; d) QM 9414 in presence of Rhodymenia palmata xylan (molar ratio 2.0 to 2.5) 32. Structure of Cellobiohydrolase II from T. reesei The second cellobiohydrolase (E.C. 3.2.1.91) excreted by T. reesei was termed CBH II. This enzyme also produces mainly cellobiose with minor amounts of glucose and cellotriose from crystalline or amorphous cellulose. Its specific activity as measured with Avicel is about twice as high as this of CBH I. CBH II operates with inversion of configuration as opposed to the reaction mechanism valid for CBH I 19,20. Immunologically, the CBH II protein is clearly distinct from that of CBH I 33 and it is proven that both enzymes are encoded by different genes, i.e. cbhl and cbh2 7. The functional role of these enzymes in cellulose hydrolysis is not yet clear but it appears that CBH II acts synergistically in a cooperative mode with CBH I (for review see 1). Recent evidence point to a possible "loose complex" formation between both enzymes occurring in solution prior to cellulose adsorption 34. The amino acid sequence has been partly determined on the purified protein and was fully deduced from the sequenced gene 7,35. According to this analysis the mature CBH II protein sequence is 447 amino acids long, the numbers of amino acid residues deduced from the cDNA sequence per CBH II molecule being as follows: 56 Ala, 13 Arg, 30 Asn, 21 Asp, 12 Cys, 21 Gin, 8 Glu, 39 Gly, 4 His, 15 He, 32 Leu, 10 Lys, 4 Met, 12 Phe, 31 Pro, 46 Ser, 34 Thr, 12 Try, 21 Tyr, 26 Val. At the N-terminus the chain begins with Pyr.Glu-Ala-Cys-Ser-Ser-Val-Thr.... and ends at the C - t e r minus with ...Thr-Asn-Ala-Asn-Pro-Ser-Phe-Leu. For the SAXS studies a CBH II sample was prepared by affinity chromatography from T. reesei QM 9414 to give the enzyme in a homogeneous form 27. In SDSPAGE the protein had a size of 58 kDa and the isoelectric point was 4.9. Glycosylation was estimated as 8 to 18 % 36. The molar absorptivity at 280 nm was 75 000 M cm . To obtain the core protein partial proteolytic hydrolysis with papain was per-



23. ESTERBAUERETAL.


309

formed, similarly as in the case of CBH I studies 27. The core exhibited a size of 45 kDa (SDS-PAGE) and an isoelectric point of 4.4. Figures 4 and 5 show the distance distribution functions measured by SAXS for the intact CBH II and its core protein together with the models which best fit these curves 31. The dimensions of the mole cule are given in Table II. Although the three-dimensional shapes of the CBH I and CBH II molecules appear to be very similar, some important differences are evident For example CBH II has a somewhat smaller head (by about 10%) but its tail is signifi cantly longer (by about 35%). In the CBH II molecule the tail part shows a long symmetrical part (light and dark grey in Figure 4), which probably represents the re gions of block Β and B'. In the amino acid sequence this region is rich in threonine and serine and it can be assumed that it is the region of O-glycosylation. In CBH II the glycosylation does not lead to the characteristic collar-like structure seen in CBH I. In contrast to CBH I the N-terminus of the protein chain of CBH II coincides with the tail, whereas the C-terminus represents the core. Based on the sequence and homologies with CBH I, the CBH II molecule is organized in four domains, A, B, B ' and the core, where ABB' represents the tail linked to the head part. A is the ter minal part consisting of 38 amino acids with 6 cysteine residues located at positions 3, 10, 20, 21, 27 and 37. These probably form three local SS bridges 7. The exact po sition of these bridges has not yet been determined. The domain A is the black part in the model of Figure 4. Following A is a sequence (block B) of 24 amino acids (39-62) rich in serine (9 residues) and threonine (5 residues). This sequence is fol lowed by block B' consisting of 26 amino acids (63-89) again rich in serine (6 resi dues) and threonine (6 residues). The amino acid sequences in block Β and B' are highly conserved. A distinct hinge region like that found in CBH I is not found in the B' block although this region is rich in proline (5 residues) but a typical hinge region with the Gly-Asn-Pro sequence (amino acid 90-92) does connect the tail part (i.e. B') with the main body of the protein. It can be assumed that the amino acids following this hinge region (Val 93 to Leu 447) are part of the head domain. The point of papain cleavage is at amino acid 82 27. The core part of the polypeptide chain is mainly folded in β-sheets (34 %) and to a lesser extent (15 %) arranged in alpha-helical structures 7. In contrast with CBH I the core of CBH II possesses only 2 disulfide bridges (176-235; 368-415) and four free sulfhydryl groups. Similarly to CBH I carboxyl functions are involved in the active center (Asp 175 and Glu 184) 28. Very recently 13 the three dimensional structure of the CBH II core was fully determined by X-ray diffraction. The polypeptide chain is folded in α-helices and β strands (α,β-protein with a central β-barrel built up by seven parallel strands. Six of the jS-strands are linked by α-helices. Near the C-terminus of the enzyme is a tunnel with dimensions well suited to take up a single cellulose chain. Two aspartic acid residues (175 and 221) are probably involved in the active center. Conclusions Cellulases apparently share a common structural organization which is characterized by a central core containing the catalytically active domain and a tail containing a highly glycosylated region (block B) and a cellulose binding domain (block A). Region A may serve as an anchor which strongly binds to cellulose and thereby loosens the rigid cellulose structure; this could facilitate the accessibility to the glycosidic bond of the active center in the core. The hydrolysis of β-1,4-glycosidic bonds probably proceeds by a general acid catalyzed mechanism, mediated by COOH groups of glutamic and aspartic acid residues, either through a double displacement mechanism (CBH I) or by single displacement (CBH II).


310


S nm

120

N

80

core

k- β


10

•

20

/5

W r / nm

Figure 4: SAXS-based model of CBH II (redrawn from réf. 31 with permission) Note that differently to CBH I the C-terminus of the chain is at the core domain and the N-terminus at the tail domain. The sequence of the domains from the N-terminus to the C-terminus is therefore A - B - B ' - C . B ' is a repeat of Β with strong sequence homologies. The arrow indicates the papain cleavage site.

300

200 Snm

100

-L

3

4

r / nm Figure 5: SAXS-based model for the CBH II core protein (reproduced with per mission from ref. 31; Copyright Academic Press 1988). The core protein was prepared by limited papain proteolysis from the intact mole cule shown in Fig. 4.


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