RESUMEN
The economics of affinity-tagging technologies, particularly at preparative scales, depends in part on the cost and efficiency of the bioprocessing step used to remove the affinity tag and obtain the final purified product (Lowe et al., J Biochem Biophys Methods. 2001;49:561-574). When CBM9, the family 9 cellulose binding module from Thermotoga maritima, serves as the affinity tag, the overall efficiency of tag removal is a function of the choice of processing enzyme and the local structure of the cleavage site, most notably the linker sequence flanking the bioprocessing recognition site on the tag side. A novel spectroscopic method is reported and used to rapidly and accurately measure CBM9 fusion-tag bioprocessing kinetics and their dependence on the choice of linker sequence. The assay monitors energy transfer between a lanthanide-based donor bound to the CBM9 tag and an acceptor fluorophore presented on the target protein or peptide. Enzyme-catalyzed cleavage of the fusion tag terminates this resonance energy transfer, resulting in a change in fluorescence intensity that can be monitored to quantify substrate concentration over time. The assay is simple, fast and accurate, providing k(cat)/K(M) values that contain standard errors of less than 3%. As a result, both substantial and subtle differences in bioprocessing kinetics can be measured and used to guide bioproduct design.
Asunto(s)
Proteínas Bacterianas/química , Celulosa/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Thermotoga maritima/metabolismoRESUMEN
A mechanically stable cellulose-based chromatography media was synthesized to permit inexpensive affinity purification of recombinant proteins containing the family 9 carbohydrate-binding module (CBM9) fused to either the N- or C-terminus of the target protein. A second-order response surface model was used to identify optimal concentrations of the primary reactants, epichlorohydrin and dimethyl sulfoxide (DMSO), required to cross-link the starting material, Perloza MT100, a compressible but inexpensive cellulose-based chromatography resin. This resulted in a mechanically stable cross-linked affinity chromatography media capable of operating at an order-of-magnitude higher linear velocity than permitted by unmodified MT100. Moments and Van Deemter analyses were used to show that rates of solute mass transfer within the column are largely unaffected by the cross-linking reaction, while the binding capacity decreased by 20% to 7.1 micromol of protein/g resin, a value superior to most commercial affinity chromatography media. In sharp contrast to MT100, the mechanical stability and purification performance of the cross-linked media are not diminished by scale-up or repeated column use.
Asunto(s)
Celulosa/metabolismo , Cromatografía de Afinidad/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Adsorción , Reactivos de Enlaces Cruzados/química , Equipo ReutilizadoRESUMEN
A novel two-zone model (TZM) is presented to describe the rate of solute uptake by the stationary phase of a sorption-type chromatography column. The TZM divides the porous stationary-phase particle into an inner protein-free core and an outer protein-containing zone where intraparticle transport is limited by pore diffusion and binding follows Langmuir theory. The TZM and the classic pore-diffusion model (PDM) of chromatography are applied to the prediction of stationary-phase uptake and elution bands within a cellulose-based affinity chromatography column designed to selectively purify proteins genetically labelled with a CBM9 (family 9 cellulose binding module) affinity tag. Under both linear and nonlinear loading conditions, the TZM closely matches rates of protein uptake within the stationary phase particles as measured by confocal laser scanning microscopy, while the PDM deviates from experiment in the linear-binding region. As a result, the TZM is shown to provide improved predictions of product breakthrough, including elution behavior from a bacterial lysate feed.
Asunto(s)
Celulosa/aislamiento & purificación , Cromatografía de Afinidad/métodos , Modelos Químicos , Proteínas/aislamiento & purificación , Adsorción , Dextranos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Porosidad , Proteínas Recombinantes de Fusión/aislamiento & purificación , Thermotoga maritima , Factores de TiempoRESUMEN
The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9) serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using the bioinformatics software MEROPS to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)(x)P and MEROPS-designed S(3)N(10) linkers were significantly more stable, indicating both strategies provide a useful approach to linker design. Factor X(a) processing of the fusion proteins depended strongly on linker chemistry, with poly(G) and S(3)N(10) linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the T(m) of GFP but did result in a destabilization, as seen by both a decrease in T(m) and DeltaH(cal), of CBM9. The degree of destabilization increased with decreasing length of the (PT)(x)P linker such that DeltaT(m) = -8.4 degrees C for the single P linker.
Asunto(s)
Marcadores de Afinidad/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/fisiología , Péptidos/química , Ingeniería de Proteínas/métodos , Marcadores de Afinidad/metabolismo , Reactivos de Enlaces Cruzados , Proteínas de Escherichia coli/genética , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/químicaRESUMEN
A carbohydrate-binding module (CBM) was fused to the N-termini of mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (EndoF1) and peptide N-glycosidase F (PNGaseF), two glycosidases from Chryseobacterium meningosepticum that are used to remove N-linked glycans from glycoproteins. The fusion proteins CBM-EndoF1 and CBM-PNGaseF also carry a hexahistidine tag for purification by immobilized metal affinity chromatography after production by Escherichia coli. CBM-EndoF1 is as effective as native EndoF1 at deglycosylating RNaseB; the glycans released by both enzymes are identical. Like native PNGaseF, CBM-PNGaseF is active on denatured but not on native RNaseB. Both fusion proteins are as active on RNaseB when immobilized on cellulose as they are in solution. They retain activity in the immobilized state for at least 1 month at 4 degrees C. The hexahistidine tag can be removed with thrombin, leaving the CBM as the only affinity tag. The CBM can be removed with factor Xa if required.
Asunto(s)
Enzimas Inmovilizadas , Glicoproteínas/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Cromatografía de Afinidad , Chryseobacterium/enzimología , Estabilidad de Enzimas , Escherichia coli/enzimología , Factor X/metabolismo , Glicosilación , Histidina/química , Oligopéptidos/química , Polisacáridos/metabolismo , Ribonucleasas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The extracellular matrix provides structural components that support the development of tissue morphology and the distribution of growth factors that modulate the overall cellular response to those growth factors. The ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells for tissue engineering applications. Cellulose binding domain (CBD) fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by surface-localized growth factors. We previously reported the synthesis and initial characterization of a fusion protein comprised of a CBD and murine stem cell factor (SCF) (Doheny et al. [1999] Biochem J 339:429-434). A significant advantage of the CBD fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of nonfactor-derived interactions between the cell and matrix. In this work, the long-term stability and bioactivity of SCF-CBD fusions adsorbed to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound SCF-CBD is shown to stimulate receptor polarization in the cell membrane and adherence to the cellulose matrix. In addition, cellulose-surface presentation of the SCF-CBD attenuates c-kit dephosphorylation kinetics, potentially modulating the overall response of the cell to the SCF signal.
Asunto(s)
Técnicas de Cultivo de Célula , Celulasa/química , Celulosa/química , Enzimas Inmovilizadas/química , Factor de Células Madre/farmacología , Animales , Membrana Celular/efectos de los fármacos , Células Cultivadas , Celulasa/genética , Estabilidad de Enzimas , Enzimas Inmovilizadas/genética , Ratones , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Factor de Células Madre/genética , Células Madre/efectos de los fármacos , Células Madre/enzimología , Ingeniería de TejidosRESUMEN
The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellulose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 +/- 3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein.
Asunto(s)
Cellulomonas/enzimología , Cromatografía de Afinidad/métodos , Escherichia coli/metabolismo , Lectinas/aislamiento & purificación , Modelos Químicos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Canadá , Cromatografía de Afinidad/economía , Simulación por Computador , Lectinas/biosíntesis , Lectinas/genética , Proteínas Recombinantes de Fusión/química , Proteína Estafilocócica A/biosíntesis , Proteína Estafilocócica A/química , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/aislamiento & purificaciónRESUMEN
A novel inexpensive affinity purification technology is described based on recombinant expression in Escherichia coli of the polypeptide or protein target fused through its N-terminus to TmXyn10ACBM9-2 (CBM9), the C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima. Measured association constants (K(a)) for adsorption of CBM9 to insoluble allomorphs of cellulose are between 2 x 10(5) and 8 x 10(6) M(-1). CBM9 also binds a range of soluble sugars, including glucose. As a result, a 1M glucose solution is effective in eluting CBM9 and CBM9-tagged fusion proteins from a very inexpensive commercially-available cellulose-based capture column. A processing site is encoded at the C-terminus of the tag to facilitate its rapid and quantitative removal by Factor X(a) to recover the desired target protein sequence following affinity purification. Fusion of the CBM9 affinity tag to the N-terminus of green fluorescent protein (GFP) from the jellyfish, Aquorin victoria, is shown to yield >200 mgl(-1) of expressed soluble fusion protein that can be affinity separated from clarified cell lysate to a purity of >95% at a yield of 86%.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Cromatografía de Afinidad/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Thermotoga maritima/enzimología , Xilosidasas/aislamiento & purificación , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Xilosidasas/química , Xilosidasas/metabolismoRESUMEN
Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 x 10(7) cells ml(-1) and a final saCBMFX concentration of 4 mg l(-1). The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day(-1). The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 x 10(7) Sf9 cells ml(-1) were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 x 10(7) cells ml(-1) the saCBMFX production rate was doubled to 6 mg l(-1) day(-1). The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l(-1) day(-1)) by an order of magnitude.
RESUMEN
Carbohydrate-binding modules (CBMs; previously called cellulose-binding domains) make excellent fusion partners for the immobilization or purification of polypeptides. However, their use in eukaryotic hosts has been limited by glycosylation, which interferes with the ability of the CBM to bind to cellulose. We have engineered the C-terminal carbohydrate-binding module from Cellulomonas fimi xylanase 10A such that it lacks N-glycosylation sites. This variant, called CBM2aNgly-, was produced and secreted by the methylotrophic yeast Pichia pastoris and found to be O-glycosylated. The O-linked glycans were composed entirely of mannose in a ratio of 1 mol of mannose to 4 mol of protein. The overall distribution of mannose on the O-glycosylated CBM mutant ranged from 1 to 9 mannose residues with the oligosaccharide sizes ranging from Man(1) to Man(4). MALDI-TOF (all matrix-assisted-laser-desorption time of flight) mass spectrometry (MS) was used to map the O-glycosylation to three regions of the polypeptide, each region having a maximum of 4 mannose residues attached to each. Glycans chemically released from CBM2aNgly- and analyzed by fluorophore-assisted carbohydrate electrophoresis were found to contain alpha-1,2-, alpha-1,3-, and alpha-1,6-linkages. Significantly, the O-glycosylation did not influence binding, making CBM2aNgly- a suitable fusion partner for polypeptides produced in P. pastoris and other eukaryotic hosts.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Cellulomonas/enzimología , Pichia/metabolismo , Xilosidasas/química , Xilosidasas/metabolismo , Secuencia de Aminoácidos , Celulosa/metabolismo , Glicosilación , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Pichia/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/genéticaRESUMEN
Carbohydrate-binding polypeptides, including carbohydrate-binding modules (CBMs) from polysaccharidases, and lectins, are widespread in nature. Whilst CBMs are classically considered distinct from lectins, in that they are found appended to polysaccharide-degrading enzymes, this distinction is blurring. The crystal structure of CsCBM6-3, a "sequence-family 6" CBM in a xylanase from Clostridium stercorarium, at 2.3 A reveals a similar, all beta-sheet fold to that from MvX56, a module found in a family 33 glycoside hydrolase sialidase from Micromonospora viridifaciens, and the lectin AAA from Anguilla anguilla. Sequence analysis leads to the classification of MvX56 and AAA into a family distinct from that containing CsCBM6-3. Whilst these polypeptides are similar in structure they have quite different carbohydrate-binding specificities. AAA is known to bind fucose; CsCBM6-3 binds cellulose, xylan and other beta-glucans. Here we demonstrate that MvX56 binds galactose, lactose and sialic acid. Crystal structures of CsCBM6-3 in complex with xylotriose, cellobiose, and laminaribiose, 2.0 A, 1.35 A, and 1.0 A resolution, respectively, reveal that the binding site of CsCBM6-3 resides on the same polypeptide face as for MvX56 and AAA. Subtle differences in the ligand-binding surface give rise to the different specificities and biological activities, further blurring the distinction between classical lectins and CBMs.
Asunto(s)
Fucosa/química , Lectinas/química , Carbohidratos/química , Celobiosa/química , Clostridium/enzimología , Cristalografía por Rayos X , ADN/metabolismo , Disacáridos/química , Electrones , Cinética , Ligandos , Modelos Químicos , Modelos Moleculares , Oligosacáridos/química , Polisacáridos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Termodinámica , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/químicaRESUMEN
Cellulase Cel5A from alkalophilic Bacillus sp. 1139 contains a family 17 carbohydrate-binding module (BspCBM17) and a family 28 CBM (BspCBM28) in tandem. The two modules have significantly similar amino acid sequences, but amino acid residues essential for binding are not conserved. BspCBM28 was obtained as a discrete polypeptide by engineering the cel5A gene. BspCBM17 could not be obtained as a discrete polypeptide, so a family 17 CBM from endoglucanase Cel5A of Clostridium cellulovorans, CcCBM17, was used to compare the binding characteristics of the two families of CBM. Both CcCBM17 and BspCBM28 recognized two classes of binding sites on amorphous cellulose: a high affinity site (K(a) approximately 1 x 10(6) M(-1)) and a low affinity site (K(a) approximately 2 x 10(4) M(-1)). They did not compete for binding to the high affinity sites, suggesting that they bound at different sites on the cellulose. A polypeptide, BspCBM17/CBM28, comprising the tandem CBMs from Cel5A, bound to amorphous cellulose with a significantly higher affinity than the sum of the affinities of CcCBM17 and BspCBM28, indicating cooperativity between the linked CBMs. Cel5A mutants were constructed that were defective in one or both of the CBMs. The mutants differed from the wild-type enzyme in the amounts and sizes of the soluble products produced from amorphous cellulose. This suggests that either the CBMs can modify the action of the catalytic module of Cel5A or that they target the enzyme to areas of the cellulose that differ in susceptibility to hydrolysis.
Asunto(s)
Celulosa/química , Celulosa/metabolismo , Secuencia de Aminoácidos , Bacillus/metabolismo , Unión Competitiva , Celulasa/química , Celulasa/metabolismo , Celulosa/genética , Clonación Molecular , Cristalización , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.
Asunto(s)
Reactores Biológicos , Metanol/metabolismo , Pichia/crecimiento & desarrollo , Pichia/metabolismo , Transferrina/biosíntesis , Células Cultivadas , Relación Dosis-Respuesta a Droga , Retroalimentación , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Metanol/farmacología , Pichia/efectos de los fármacos , Pichia/genética , Ingeniería de Proteínas/métodos , Control de Calidad , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Transferrina/genéticaRESUMEN
In this work, a new derivative of FX was engineered. It comprises a cellulose-binding module (CBM) fused to the N-terminus of the truncated light chain (E2FX) of FX and a hexahistidine tag (H6) fused to the C-terminus of the heavy chain. The sequence LTR at the site of cleavage of the activation peptide from the N-terminus of the heavy chain is changed to IEGR to render the derivative self-activating. However, N-linked glycans on the CBM of the derivative blocked its binding to cellulose and those on the activation peptide slowed its activation. Therefore, the sites of N-linked glycosylation on the CBM and on the activation peptide were eliminated by mutation. The final derivative can be produced in good yield by cultured mammalian cells. It is purified easily with Ni(2+)-agarose, it is self-activating, and it can be immobilized on cellulose. When immobilized on a column of cellulose beads, the activated derivative retains approximately 80% of its initial activity after 30 days of continuous hydrolysis of a fusion protein substrate. Under these conditions of operation, the effective substrate:enzyme ratio is >10(4).
Asunto(s)
Celulosa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Factor X/biosíntesis , Factor X/genética , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Células Cultivadas , Células Inmovilizadas , Clonación Molecular , Cricetinae , Estabilidad de Enzimas , Enzimas Inmovilizadas , Escherichia coli/clasificación , Factor X/química , Riñón/citología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Sefarosa , Especificidad de la EspecieRESUMEN
Competition isotherms are used to identify the set of cellulose substructures to which cellulose binding modules (CBMs) from families 2a, 3, 4, 9, and 17 bind. The experiments are based on coupling a unique fluorescent tag to each CBM in a manner that does not alter the natural binding properties of the CBM and therefore allows the surface and solution concentrations of each CBM to be monitored as a function of time and composition. Adsorption and surface exchange of like or competing CBMs are monitored using a range of cellulose preparations varying in both crystallinity and provenance. CBMs from families 2a, 3, 4, 9, and 17 are shown to recognize different physical forms of prepared cellulose. The demonstration of the very fine binding specificity of cellulose-specific CBMs implies that the polysaccharide targets of CBMs extend down to the resolution of cellulose microstructures.
Asunto(s)
Celulosa/química , Sitios de Unión , Unión Competitiva , Carbohidratos/química , Celulasa/metabolismo , Celulosa/metabolismo , Cristalización , Cinética , Espectrometría de Masas , Difracción de Rayos X , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/metabolismoRESUMEN
Enzymes active on complex carbohydrate polymers frequently have modular structures in which a catalytic domain is appended to one or more carbohydrate-binding modules (CBMs). Although CBMs have been classified into a number of families based upon sequence, many closely related CBMs are specific for different polysaccharides. In order to provide a structural rationale for the recognition of different polysaccharides by CBMs displaying a conserved fold, we have studied the thermodynamics of binding and three-dimensional structures of the related family 4 CBMs from Cellulomonas fimi Cel9B and Thermotoga maritima Lam16A in complex with their ligands, beta-1,4 and beta-1,3 linked gluco-oligosaccharides, respectively. These two CBMs use a structurally conserved constellation of aromatic and polar amino acid side-chains that interact with sugars in two of the five binding subsites. Differences in the length and conformation of loops in non-conserved regions create binding-site topographies that complement the known solution conformations of their respective ligands. Thermodynamics interpreted in the light of structural information highlights the differential role of water in the interaction of these CBMs with their respective oligosaccharide ligands.
Asunto(s)
Actinomycetales/química , Celulasa/química , Celulasa/metabolismo , Glucanos/metabolismo , Oligosacáridos/metabolismo , Thermotoga maritima/química , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Evolución Molecular , Glucanos/química , Concentración de Iones de Hidrógeno , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato , TermodinámicaRESUMEN
Carbohydrate-binding module (CBM) family 13 includes the "R-type" or "ricin superfamily" beta-trefoil lectins. The C-terminal CBM, CBM13, of xylanase 10A from Streptomyces lividans is a family 13 CBM that is not only structurally similar to the "R-type" lectins but also somewhat functionally similar. The primary function of CBM13 is to bind the polysaccharide xylan, but it retains the ability of the R-type lectins to bind small sugars such as lactose and galactose. The association of CBM13 with xylan appears to involve cooperative and additive participation of three binding pockets in each of the three trefoil domains of CBM13, suggesting a novel mechanism of CBM-xylan interaction. Thus, the interaction of CBM13 with sugars displays considerable plasticity for which we provide a structural rationale. The high-resolution crystal structure of CBM13 was determined by multiple anomalous dispersion from a complex of CBM13 with a brominated ligand. Crystal structures of CBM13 in complex with lactose and xylopentaose revealed two distinct mechanisms of ligand binding. CBM13 has retained its specificity for lactose via Ricin-like binding in all of the three classic trefoil binding pockets. However, CBM13 has the ability to bind either the nonreducing galactosyl moiety or the reducing glucosyl moiety of lactose. The mode of xylopentaose binding suggests adaptive mutations in the trefoil sugar binding scaffold to accommodate internal binding on helical polymers of xylose.
Asunto(s)
Streptomyces/enzimología , Xilanos/química , Xilosidasas/química , Secuencia de Aminoácidos , Sitios de Unión , Carbohidratos/química , Cristalografía por Rayos X , Glucósidos/biosíntesis , Lectinas/química , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Xilano Endo-1,3-beta-XilosidasaRESUMEN
Family 6 carbohydrate-binding modules were amplified by polymerase chain reaction (PCR) from Clostridium stercorarium strain NCIB11754 genomic DNA as a triplet. Individually, these modules bound to xylooligosaccharides and cellooligosaccharides with affinities varying from approximately 3 x 10(3) M(-1) to approximately 1 x 10(5) M(-1). Tandem and triplet combinations of these modules bound co-operatively to soluble xylan and insoluble cellulose to give approximately 20- to approximately 40-fold increases in affinity relative to the individual modules. This co-operativity was an avidity effect resulting from the modules within the tandems and triplet interacting simultaneously with proximal binding sites on the polysaccharides. This occurred by both intrachain and interchain interactions. The duplication or triplication of modules appears to be linked to the growth temperature of the organism; co-operativity in these multiplets may compensate for the loss of affinity at higher temperatures.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Clostridium/enzimología , Xilosidasas/genética , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Celulosa/metabolismo , Clostridium/genética , ADN Bacteriano , Hidrolasas/metabolismo , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Solubilidad , Xilano Endo-1,3-beta-Xilosidasa , Xilanos/metabolismoRESUMEN
The C-terminal 191-residue module of Cel5A from the alkalophilic Bacillus sp. 1139 comprises a carbohydrate-binding module (CBM) belonging to a previously unidentified family that we have classified as CBM family 28. This example, called CBM28, bound specifically to cello-oligosaccharides and mixed beta-(1,3)(1,4)-glucans (barley beta-glucan) with association constants of approximately (1-4)x10(4) M(-1). Its binding to barley beta-glucan was remarkably insensitive to pH between 7.0 and 10.9, in keeping with its alkalophilic source. CBM28 bound to cellulose having a significant non-crystalline content with an association constant similar to that for its binding to soluble glucans. CBM17 (CBM family 17) and CBM28 modules naturally occur as tandems. The CBM17/CBM28 tandem from Cel5A bound with apparent co-operativity to barley beta-glucan. The association of CBM28 with cello-oligosaccharides was driven enthalpically and marked by the different thermodynamic contribution of three putative binding subsites that accommodate a cellohexaose molecule.