Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering

Cellulase Cel45 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Cel45 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain. The glycosylation of the linker is the key factor defining its extended conformation, and a five proline stretch mutation on the linker was found to confer a higher rigidity to the enzyme. Our study shows that the respective positioning of the catalytic module and the CBM onto the insoluble substrate is most likely influenced by the linker structure and flexibility. Our results are consistent with a model where cellulases can move on the surface of cellulose with a caterpillar-like displacement with free energy restrictions. Cellulase Cel45 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Cel45 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain. The glycosylation of the linker is the key factor defining its extended conformation, and a five proline stretch mutation on the linker was found to confer a higher rigidity to the enzyme. Our study shows that the respective positioning of the catalytic module and the CBM onto the insoluble substrate is most likely influenced by the linker structure and flexibility. Our results are consistent with a model where cellulases can move on the surface of cellulose with a caterpillar-like displacement with free energy restrictions. The enzymatic hydrolysis of cellulose, the most abundant biopolymer on earth, is catalyzed by cellulases, which have considerable industrial and ecological importance. Most cellulases have a modular structure with a catalytic module and a cellulose-binding module (CBM) 1The abbreviations used are: CBM, cellulose-binding module; SAXS, small angle x-ray scattering; Cel45, H. insolens endoglucanase Cel45; R g, radius of gyration; Dmax, maximum dimension. usually joined by a glycosylated and presumably flexible linker peptide (1Gilkes N.R. Henrissat B. Kilburn D.G. Miller Jr., R.C. Warren R.A. Microbiol. Rev. 1991; 55: 303-315Google Scholar). Removal of the CBM results in a significantly reduced activity of the enzymes on crystalline cellulose probably because of a decreased binding capacity, but the activity on soluble cellulose oligomers is retained (2Gilkes N.R. Warren R.A. Miller Jr., R.C. Kilburn D.G. J. Biol. Chem. 1988; 263: 10401-10407Google Scholar, 3van Tilbeurgh H. Tomme P. Claeyssens M. Bhikhabhai R. Pettersson G. FEBS Lett. 1986; 204: 223-227Google Scholar, 4Tomme P. Van Tilbeurgh H. Pettersson G. Van Damme J. Vandekerckhove J. Knowles J. Teeri T. Claeyssens M. Eur. J. Biochem. 1988; 170: 575-581Google Scholar). Inversely, isolated CBMs retain most of the binding capacity on crystalline cellulose but are devoid of catalytic action (3van Tilbeurgh H. Tomme P. Claeyssens M. Bhikhabhai R. Pettersson G. FEBS Lett. 1986; 204: 223-227Google Scholar, 5Gilkes N.R. Jervis E. Henrissat B. Tekant B. Miller Jr., R.C. Warren R.A. Kilburn D.G. J. Biol. Chem. 1992; 267: 6743-6749Google Scholar). Enzymatic activity is sometimes affected when the interdomain linker has been shortened or completely deleted suggesting that it should be of sufficient length and/or flexibility to ensure an independent action of the two functional modules (6Shen H. Schmuck M. Pilz I. Gilkes N.R. Kilburn D.G. Miller R.C. Warren R.A.J. J. Biol. Chem. 1991; 266: 11335-11340Google Scholar, 7Srisodsuk M. Reinikainen T. Penttila M. Teeri T.T. J. Biol. Chem. 1993; 268: 20756-20761Google Scholar). The catalytic modules of cellulases have been classified into several distinct families of glycoside hydrolases on the basis of amino acid sequence similarities (8Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Google Scholar, 9Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Google Scholar) while, similarly, the CBMs form several families (10Tomme P. Warren R.A.J. Miller R.C.J. Kilburn D.G. Gilkes N.R. Saddler J.N. Penner M. Enzymatic Degradation of Insoluble Polysaccharides. 618. American Chemical Society, Washington, D. C.1995: 142-163Google Scholar, 11Boraston A.B. McLean B.W. Kormos J.M. Alam M. Gilkes N.R. Haynes C.A. Tomme P. Kilburn D.G. Warren R.A.J. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge1999: 202-211Google Scholar). Structural data on individual cellulase modules have been accumulating over the last few years, with three-dimensional structures solved in nine families of catalytic modules and seven families of CBMs (12Bourne Y. Henrissat B. Curr. Opin. Struct. Biol. 2001; 11: 593-600Google Scholar). There is no three-dimensional structure available for an intact cellulase whose catalytic module and CBM are separated by an interdomain linker longer than a dozen residues. The larger length, flexibility, and heterogenous glycosylation of fungal interdomain linkers are probably a major obstacle to protein crystallization. The only two domain cellulase known at the three-dimensional structural level is that of the bacterial endoglucanase E4 (Cel9A) of Thermobifida fusca (13Sakon J. Irwin D. Wilson D.B. Karplus P.A. Nat. Struct. Biol. 1997; 4: 810-818Google Scholar). However, in this particular protein, the interdomain linker is so short that the CBM is literally fused onto the catalytic module. So far, all the different experimental techniques used for structural elucidation failed to give precise information on the overall structure of full-length, multidomain cellulases bearing a long, glycosylated, and presumably flexible interdomain linker. At the end of 1980, Pilz and co-workers have reported pioneering studies using small angle x-ray scattering (SAXS), which provided the very first insight into the tertiary structure of two domain cellulases from Trichoderma reesei (14Abuja P.M. Schmuck M. Pilz I. Tomme P. Claeyssens M. Esterbauer H. Eur. Biophys. J. 1988; 15: 339-342Google Scholar, 15Abuja P.M. Pilz I. Claeyssens M. Tomme P. Biochem. Biophys. Res. Commun. 1988; 156: 180-185Google Scholar) and from Cellulomonas fimi (6Shen H. Schmuck M. Pilz I. Gilkes N.R. Kilburn D.G. Miller R.C. Warren R.A.J. J. Biol. Chem. 1991; 266: 11335-11340Google Scholar, 16Pilz I. Schwarz E. Kilburn D.G. Miller R.C. Warren R.A.J. Gilkes N.R. Biochem. J. 1990; 271: 277-280Google Scholar, 17Meinke A. Schmuck M. Gilkes N.R. Kilburn D.G. Miller R.C. Warren R.A.J. Glycobiology. 1992; 2: 321-326Google Scholar). However, these early results were necessarily imperfect because of the lack of known three-dimensional structure for the isolated cellulase modules and the equipment available then. The data collected on conventional sources in the 1980s were undermined by relatively poor statistics, despite the huge amounts of protein used for the experiments (several hundred milligrams for one set of experiment). Besides, the high protein concentrations used may have distorted the signal (18Zimmerman S.B. Minton A.P. Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 27-65Google Scholar, 19Receveur V. Durand D. Desmadril M. Calmettes P. FEBS Lett. 1998; 426: 57-61Google Scholar). Since then, only the works of Boisset et al. (20Boisset C. Borsali R. Schülein M. Henrissat B. FEBS Lett. 1995; 376: 49-52Google Scholar) have combined the structural information now available on isolated modules with dynamic light scattering experiments to establish a more precise estimate of the hydrodynamic dimensions of a full-length cellulase. However, it is difficult to obtain precise structural information on proteins from dynamic light scattering data as only rough models based on geometrical shape can be used. Moreover the choice of a model requires relying on preliminary assumptions. By contrast, SAXS is a method of choice to determine the average shape of a protein because it enables the direct measurement of the dimensions of a macromolecule in solution and the calculation of its envelope ab initio, without any starting hypothesis. Here, we report the first ab initio characterization of the two domain structure of cellulase Cel45 from the fungus Humicola insolens and of several of its variants by SAXS in order to shed light on the shape, dimension, and conformation of this modular protein. Cel45 has a catalytic module belonging to the glycoside hydrolase family 45 carrying a CBM of family 1 via a 36 amino acid long linker peptide (21Schülein M. Tikhomirov D. Schou C. Suominen P. Reinikainen T. Trichoderma reesei cellulases and other hydrolases. 8. Foundation for Biotechnical and Industrial Fermentation Research, Espoo1993: 109-116Google Scholar). The three-dimensional structure of the catalytic module has been determined (22Davies G.J. Dodson G.G. Hubbard R.E. Tolley S.P. Dauter Z. Wilson K.S. Hjort C. Mikkelsen J.M. Rasmussen G. Schülein M. Nature. 1993; 365: 362-364Google Scholar) and that of the CBM could be directly modeled from its 50% sequence identity to cellobiohydrolase I (Cel7A) from Trichoderma reesei, whose three-dimensional structure was solved by NMR (23Kraulis P.M. Clore M.G. Nilges M. Jones T.A. Pettersson G. Knowles J. Gronenborn A.M. Biochemistry. 1989; 28: 7241-7257Google Scholar). H. insolens Cel45 full-length and its isolated catalytic module (Cel45 core) were cloned and expressed in Aspergillus oryzae (24Rasmussen, G., Mikkelsen, J. M., Schülein, M., Patkar, S. A., Hagen, F., Hjort, C. M. 376: 49-52Google Scholar). Site-directed mutagenesis was done using the PCR method. The expression plasmids harboring the mutated genes were transformed into A. oryzae using selection on acetamide by cotransformation using the selection as already described H. E. S.B. M. 1988; Scholar). only the catalytic module and the linker was by a on the at a corresponding to amino acid a stretch of five proline in the interdomain linker Cel45 was by the for and to for a a linker Cel45 was by of the from to Cel45 and Cel45 which a CBM, were purified using as described (20Boisset C. Borsali R. Schülein M. Henrissat B. FEBS Lett. 1995; 376: 49-52Google Scholar). was purified using as described for the catalytic module (Cel45 core) (20Boisset C. Borsali R. Schülein M. Henrissat B. FEBS Lett. 1995; 376: 49-52Google Scholar). The proteins were in and a with a in order to each protein was a in order to from the and the was used for the SAXS The protein of each was by at using from the amino acid of each protein. and was to the as a for SAXS experiments at and SAXS experiments were on the on the of the and on at the the was and the were and The was with to the so that the for higher scattering could be to scattering from to and to The scattering is as where is the scattering to of were on the protein The protein solution was the so that no was the was and the was leading to scattering from to The was a x-ray and of 1 with between each were for each equipment was used to the protein and the solution at a and a solution was each into a from a no protein solution was longer than 1 was to for or effect was and individual could be was with a of at different protein concentrations from 1 to were for each protein to for scattering was measured or each protein using the corresponding solution and from the protein scattering and from the experiments were at The of of were from the A. angle scattering of R is the is the The radius of and are from the and the of the of at The the of the of a with a enables an calculation using the entire scattering is from the of The the maximum of the which is as the where were by the of the scattering using D. J. 1992; Scholar). of was and the choice of was based on should from should with that from the and the should when the of the scattering based on structures were using D. C. J. 1995; 28: Scholar). were to the experimental data in the of the isolated catalytic module by into the and the scattering by a a different than the The structural model for Cel45 was with The for the CBM were by with Biochem. 1996; Scholar) using the of the CBM from T. reesei cellobiohydrolase (23Kraulis P.M. Clore M.G. Nilges M. Jones T.A. Pettersson G. Knowles J. Gronenborn A.M. Biochemistry. 1989; 28: 7241-7257Google Scholar). The of Cel45 variants and were determined ab initio from the scattering using the Biophys. J. Scholar) and Biophys. J. 2001; Scholar). two of protein and a with or the experimental scattering by a the between the and the by and and can the scattering to a of a from the scattering in order to it to the G. Scholar) as the surface between the and the should be well a three-dimensional model from the entire scattering the protein as an of on the of with a more and higher models than the experimental data are of sufficient the cellulases are in the linker into this glycosylation we have for that a is to amino acid to its and its have the of and amino acid of the cellulase its structure and the average of in the protein by mass used this of for the protein in the of the several independent were with of and and with no and the of the solution was The isolated modules were in the envelope with to each other using A. C. Scholar). Cel45 as well as its isolated catalytic module and different variants in the interdomain linker have been studied by SAXS in order to estimate the of the linker on the conformation of this cellulase. Cel45 two amino of the linker were by two in a of five proline residues. short which a conformation in solution known as a B.W. J.M. M.G. Miller Biochemistry. 2001; was in order to the rigidity of the linker. Cel45 the catalytic module and the CBM are joined by a linker amino than the linker. in the CBM has been in a protein only the catalytic module and the full-length linker peptide. The of Cel45 and its variants has been by mass Schülein, were for all for the isolated catalytic to the of the linker. of the average for each protein with that from the amino acid an estimate of the overall glycosylation of the units for Cel45, Cel45 and and units for Cel45 to an average of about or in the linker. The glycosylation is likely to be by of in The average of Cel45 and its variants was by the of of shows the experimental SAXS in for all studied The the in the and no of can be The is used in the corresponding the proteins studied are probably and a of the was to be only to effect was for Cel45 for was to The of of Cel45 and of its variants are reported in The of of for the proteins bearing a linker are higher than be for a protein with the of amino S.B. V. Jones Biochemistry. Scholar). is when one the of of the isolated catalytic module amino with which of the catalytic module and the linker The exhibits an R of it is larger than the by only 36 amino and about By contrast, the CBM amino to the average of the protein the intact Cel45 an R of the radius of of Cel45 is but significantly larger than that of Cel45, a in average for the two proteins structures only by two amino and dimensions of Cel45 and from the from the three-dimensional from the three-dimensional from the from the three-dimensional in a were from the scattering of Cel45 and its The experimental and the of the CBM from its are in The of the isolated catalytic module has a which is of a protein. The experimental to the one from the structure with a maximum of 45 The of length Cel45 and of its variants a of an shape to the maximum for full-length Cel45, for Cel45 for and for Cel45 is consistent with the high of of measured for these with to The first is of the short the catalytic as from the on the catalytic The to the of the first that are larger the of the cellulase other than the catalytic module or the that the linker is The of the interdomain to the between the catalytic module and the linker the peptide and the as by the of The last extension of the in Cel45 and in its variants to the between the CBM and the catalytic module and that at of the The from all these are reported in I. The of Cel45 and of Cel45 have a and only by the of the the the of Cel45 and Cel45 are but are the on the that the of the two modules with to each other is in the two proteins or that the two proteins in the initio three-dimensional of Cel45 and its variants have been determined using the and is for a with the and which the However, the surface of a cellulase is very well because of the of the linker peptide and of the glycoside this of probably the we used this is for higher were with the two to the of the scattering on solution ab initio three-dimensional shape from scattering can to structures the data of the it significantly the of an The results with were using J. Scholar). that the are for each protein the used. The models provided a to the experimental data with for Cel45 for Cel45 and for Cel45 The and the are in The ab initio with a and a more or long and The is in the Cel45 its in the Cel45 with and in the is in Cel45 exhibits a in Cel45 and several in different in Cel45 are from one to with and for the two are in the of the linker in the models for Cel45 of Cel45 and full-length Cel45 to the isolated modules in these at of the The of the envelope the glycosylated linker peptide. The SAXS experiments on cellulase Cel45 and variants reported to determine the structural of a full-length cellulase and to obtain the first ab initio shape of a two domain cellulase. Our results the first models of cellulases that Pilz and co-workers (14Abuja P.M. Schmuck M. Pilz I. Tomme P. Claeyssens M. Esterbauer H. Eur. Biophys. J. 1988; 15: 339-342Google Scholar, 15Abuja P.M. Pilz I. Claeyssens M. Tomme P. Biochem. Biophys. Res. Commun. 1988; 156: 180-185Google Scholar, 16Pilz I. Schwarz E. Kilburn D.G. Miller R.C. Warren R.A.J. Gilkes N.R. Biochem. J. 1990; 271: 277-280Google Scholar) at a when the and structure of individual modules were we show that the shape of cellulase Cel45 is to a the dimensions of the linker peptide from experiments are more and consistent with the of in the linker. a of the maximum dimensions for and from T. reesei were probably and to for the of the and for the linkers of and By contrast, results are consistent with the of Boisset et al. (20Boisset C. Borsali R. Schülein M. Henrissat B. FEBS Lett. 1995; 376: 49-52Google Scholar) on H. insolens Cel45 using dynamic light scattering of the protein of is to that a scattering the average of all in the solution by the the a scattering may to the of different protein the structure exhibits flexibility. is well known that the radius of is the of all to the of the scattering of the in by scattering other the average conformation by all the protein in contrast, Dmax, from the to the maximum between two a all in is very to these two for the different The of the of and of the of Cel45 that the of the linker is very with to its from Cel45 to The is but has an shape to the and is the catalytic module. on the basis of one can that the of the CBM to the conformation of the linker of the cellulase in the of to the of the CBM from its modeled the linker is shortened by amino Cel45 the R and corresponding to a of in The of about two that the linker can a extended conformation of a structure by short that the linker is The are of a of all in Our results that the linker a with to its small of amino the catalytic module and the CBM have an the linker is at all and flexibility. that short the of the peptide. However, or may the to the glycosylation of the linker probably the of the heterogenous length and different each protein in the or on the peptide and of the that the linker can is that the R of Cel45 and of Cel45 are different are and have the of amino in the linker The maximum extension of the linker is the the average is in the of the that is a of for Cel45 The ab initio are different in the linker Cel45 and Cel45 the of the linker is a of the glycosylation the linker and of the it can the we can a of the linker to the catalytic module in Cel45 with a at higher no mutation affected the glycosylation in Cel45 with Cel45 this that the flexibility of the linker of Cel45 is the linker. Since Cel45 average more extended than the but with an maximum we that Cel45 more than Cel45 and that the five proline stretch in the linker a higher rigidity to the protein. The at which the catalytic module can from the of the CBM on cellulose is an to the of the isolated modules of of by crystalline cellulose, each is by with to the in a cellobiose Cel45 we have a of which results in a maximum between the centers of the constitutive modules of one can that from a binding of the CBM onto a cellulose Cel45 has a maximum of about may only a maximum of four of on a chain. be a as the by one catalytic domain be has been that CBMs are on the cellulose but that are and to on the substrate surface Haynes C.A. Kilburn D.G. J. Biol. Chem. 1997; Scholar). Our results this where the of CBMs on the cellulose surface enables an hydrolysis by the catalytic module Haynes C.A. Kilburn D.G. J. Biol. Chem. 1997; Scholar, A. S. P. A. Gilbert H.J. Biochem. J. 1998; Scholar). et al. M. Reinikainen T. Penttila M. Teeri T.T. J. Biol. Chem. 1993; 268: 20756-20761Google Scholar) have that a sufficient spatial between the two modules is for activity in the reesei that the linker the between the two modules with to the results are consistent with a model where cellulases can move on cellulose a caterpillar-like the CBM to a the catalytic module can several the by the flexibility of the linker. the linker the free energy of the cellulase be via the of the CBM the cellulose the free energy is likely to extension or of the the on the of the enzyme the end to the other or on the of the CBM at the or end of the cellulase. a model is consistent with the of Scholar) already that the two modules should in on the cellulose surface and that relatively long linker with flexibility are to The on the of the SAXS with protein and NMR for the study of the conformation of modular The reported the first precise model of a cellulase in solution small angle x-ray scattering data with the now available three-dimensional structures of each individual module. Our results the first information on the structural of the interdomain peptide with a and show that the linker is flexible and extended but exhibits structural The amino acid of the linker an as by results on Cel45 Our SAXS data show that the of a sequence to a higher rigidity of the linker. the flexibility of the linker is the of high glycosylation only may be to the linker from and to the protein, but it to the linker to an extended is likely that or linkers be more flexible than studies of the of the glycosylation on the structure and flexibility of the linker in cellulases are in the the of the of a two domain cellulase shed light on the of action of cellulases on the substrate surface and catalytic modules and the for of and we and for in using and for for with