Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.     

Novel sugar-binding specificity of the type XIII xylan-binding domain of a family F/10 xylanase from Streptomyces olivaceoviridis E-86

The type XIII xylan-binding domain (XBD) of a family F/10 xylanase (FXYN) from Streptomyces olivaceoviridis E-86 was found to be structurally similar to the ricin B chain which recognizes the non-reducing end of galactose and specifically binds to galactose containing sugars. The crystal structure of XBD [Fujimoto, Z. et al. (2000) J. Mol. Biol. 300, 575-585] indicated that the whole structure of XBD is very similar to the ricin B chain and the amino acids which form the galactose-binding sites are highly conserved between the XBD and the ricin B chain. However, our investigation of the binding abilities of wt FXYN and its truncated mutants towards xylan demonstrated that the XBD bound xylose-based polysaccharides. Moreover, it was found that the sugar-binding unit of the XBD was a trimer, which was demonstrated in a releasing assay using sugar ranging in size from xylose to xyloheptaose. These results indicated that the binding specificity of the XBD was different from those of the same family lectins such as the ricin B chain. Somewhat surprisingly, it was found that lactose could release the XBD from insoluble xylan to a level half of that observed for xylobiose, indicating that the XBD also possessed the same galactose recognition site as the ricin B chain. It appears that the sugar-binding pocket of the XBD has evolved from the ancient ricin super family lectins to bind additional sugar targets, resulting in the differences observed in the sugar-binding specificities between the lectin group (containing the ricin B chain) and the enzyme group.     

Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain.

A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei. Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant. The hybrid was purified to homogeneity by affinity chromatography on cellulose. The relative association constants (Kr) for the binding of Cex, CexCBDCbhI, the catalytic domain of Cex (p33), and CbhI to bacterial microcrystalline cellulose (BMCC) were 14.9, 7.8, 0.8, and 10.6 liters g-1, respectively. Cex and CexCBDCbhI had similar substrate specificities and similar activities on crystalline and amorphous cellulose. Both released predominantly cellobiose and cellotriose from amorphous cellulose. CexCBDCbhI was two to three times less active than Cex on BMCC, but significantly more active than Cex on soluble cellulose and on xylan. Unlike Cex, the hybrid protein neither bound to alpha-chitin nor released small particles from dewaxed cotton fibers.     

Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.     

The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed-linkage beta-1,3/beta-1, 4-glucan

Thermotoga maritima XynA is an extremely thermostable modular enzyme with five domains (A1-A2-B-C1-C2). Its catalytic domain (-B-) is flanked by duplicated non-catalytic domains. The C-terminal repeated domains represent cellulose-binding domains (CBDs). Xylanase domains related to the N-terminal domains of XynA (A1-A2) are called thermostabilizing domains because their deletion normally leads to increased thermosensitivity of the enzymes. It was found that a glutathione-S-transferase (GST) hybrid protein (GST-A1A2) containing both A-domains of XynA can interact with various soluble xylan preparations and with mixed-linkage beta-1,3/beta-1,4-glucans. GST-A1A2 showed no affinity for insoluble microcrystalline cellulose, whereas, vice versa, GST-C2, which contains the C-terminal CBD of XynA, did not interact with soluble xylan. Another hybrid protein, GST-A2, displayed the same binding properties as GST-A1A2, indicating that A2 alone can also promote xylan binding. The dissociation constants for the binding of xylose, xylobiose, xylotriose, xylotetraose and xylopentaose by GST-A2, as determined at 20 degrees C by fluorescence quench experiments, were 8.1 x 10(-3) M, 2.3 x 10(-4) M, 2.3 x 10(-5) M, 2.5 x 10(-6)M and 1.1 x 10(-6) M respectively. The A-domains of XynA, which are designated as xylan binding domains (XBD), are, from the structural as well as the functional point of view, prototypes of a novel class of binding domains. More than 50 related protein segments with hitherto unknown function were detected in about 30 other multidomain beta-glycanases, among them putative plant (Arabidopsis thaliana) xylanases. It is argued that polysaccharide binding and not thermostabilization is the main function of A-like domains.     

De novo prediction of three-dimensional structures for major protein families

The family 10 xylanase from Streptomyces olivaceoviridis E-86 contains a (beta/alpha)(8)-barrel as a catalytic domain, a family 13 carbohydrate binding module (CBM) as a xylan binding domain (XBD) and a Gly/Pro-rich linker between them. The crystal structure of this enzyme showed that XBD has three similar subdomains, as indicated by the presence of a triple-repeated sequence, forming a galactose binding lectin fold similar to that found in the ricin toxin B-chain. Comparison with the structure of ricin/lactose complex suggests three potential sugar binding sites in XBD. In order to understand how XBD binds to the xylan chain, we analyzed the sugar-complex structure by the soaking experiment method using the xylooligosaccharides and other sugars. In the catalytic cleft, bound sugars were observed in the xylobiose and xylotriose complex structures. In the XBD, bound sugars were identified in subdomains alpha and gamma in all of the complexes with xylose, xylobiose, xylotriose, glucose, galactose and lactose. XBD binds xylose or xylooligosaccharides at the same sugar binding sites as in the case of the ricin/lactose complex but its binding manner for xylose and xylooligosaccharides is different from the galactose binding mode in ricin, even though XBD binds galactose in the same manner as in the ricin/galactose complex. These different binding modes are utilized efficiently and differently to bind the long substrate to xylanase and ricin-type lectin. XBD can bind any xylose in the xylan backbone, whereas ricin-type lectin recognizes the terminal galactose to sandwich the large sugar chain, even though the two domains have the same family 13 CBM structure. Family 13 CBM has rather loose and broad sugar specificities and is used by some kinds of proteins to bind their target sugars. In such enzyme, XBD binds xylan, and the catalytic domain may assume a flexible position with respect to the XBD/xylan complex, inasmuch as the linker region is unstructured.     

Evidence for synergy between family 2b carbohydrate binding modules in Cellulomonas fimi xylanase 11A

Glycoside hydrolases often contain multiple copies of noncatalytic carbohydrate binding modules (CBMs) from the same or different families. Currently, the functional importance of this complex molecular architecture is unclear. To investigate the role of multiple CBMs in plant cell wall hydrolases, we have determined the polysaccharide binding properties of wild type and various derivatives of Cellulomonas fimi xylanase 11A (Cf Xyn11A). This protein, which binds to both cellulose and xylan, contains two family 2b CBMs that exhibit 70% sequence identity, one internal (CBM2b-1), which has previously been shown to bind specifically to xylan and the other at the C-terminus (CBM2b-2). Biochemical characterization of CBM2b-2 showed that the module bound to insoluble and soluble oat spelt xylan and xylohexaose with K(a) values of 5.6 x 10(4), 1.2 x 10(4), and 4.8 x 10(3) M(-1), respectively, but exhibited extremely weak affinity for cellohexaose (<10(2) M(-1)), and its interaction with insoluble cellulose was too weak to quantify. The CBM did not interact with soluble forms of other plant cell wall polysaccharides. The three-dimensional structure of CBM2b-2 was determined by NMR spectroscopy. The module has a twisted "beta-sandwich" architecture, and the two surface exposed tryptophans, Trp 570 and Trp 602, which are in a perpendicular orientation with each other, were shown to be essential for ligand binding. In addition, changing Arg 573 to glycine altered the polysaccharide binding specificity of the module from xylan to cellulose. These data demonstrate that the biochemical properties and tertiary structure of CBM2b-2 and CBM2b-1 are extremely similar. When CBM2b-1 and CBM2b-2 were incorporated into a single polypeptide chain, either in the full-length enzyme or an artificial construct comprising both CBM2bs covalently joined via a flexible linker, there was an approximate 18-20-fold increase in the affinity of the protein for soluble and insoluble xylan, as compared to the individual modules, and a measurable interaction with insoluble acid-swollen cellulose, although the K(a) (approximately 6.0 x 10(4) M(-1)) was still much lower than for insoluble xylan (K(a) = approximately 1.0 x 10(6) M(-1)). These data demonstrate that the two family 2b CBMs of Cf Xyn11A act in synergy to bind acid swollen cellulose and xylan. We propose that the increased affinity of glycoside hydrolases for polysaccharides, through the synergistic interactions of CBMs, provides an explanation for the duplication of CBMs from the same family in some prokaryotic cellulases and xylanases.     

Cellulose-binding modules from extracellular matrix proteins of Dictyostelium discoideum stalk and sheath.

Cellulose-binding modules (CBMs) of two extracellular matrix proteins, St15 and ShD, from the slime mold Dictyostelium discoideum were expressed in Escherichia coli. The expressed proteins were purified to > 98% purity by extracting inclusion bodies at pH 11.5 and refolding proteins at pH 7.5. The two refolded CBMs bound tightly to amorphous phosphoric acid swollen cellulose (PASC), but had a low affinity toward xylan. Neither protein exhibited cellulase activity. St15, the stalk-specific protein, had fourfold higher binding affinity toward microcrystalline cellulose (Avicel) than the sheath-specific ShD CBM. St15 is unusual in that it consists of a solitary CBM homologous to family IIa CBMs. Sequence analysis of ShD reveals three putative domains containing: (a) a C-terminal CBM homologous to family IIb CBMs; (b) a Pro/Thr-rich linker domain; and (c) a N-terminal Cys-rich domain. The biological functions and potential role of St15 and ShD in building extracellular matrices during D. discoideum development are discussed.     

The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase

The solution structure is presented for the second family 4 carbohydrate binding module (CBM4-2) of xylanase 10A from the thermophilic bacterium Rhodothermus marinus. CBM4-2, which binds xylan tightly, has a beta-sandwich structure formed by 11 strands, and contains a prominent cleft. From NMR titrations, it is shown that the cleft is the binding site for xylan, and that the main amino acids interacting with xylan are Asn31, Tyr69, Glu72, Phe110, Arg115, and His146. Key liganding residues are Tyr69 and Phe110, which form stacking interactions with the sugar. It is suggested that the loops on which the rings are displayed can alter their conformation on substrate binding, which may have functional importance. Comparison both with other family 4 cellulose binding modules and with the structurally similar family 22 xylan binding module shows that the key aromatic residues are in similar positions, and that the bottom of the cleft is much more hydrophobic in the cellulose binding modules than the xylan binding proteins. It is concluded that substrate specificity is determined by a combination of ring orientation and the nature of the residues lining the bottom of the binding cleft.     

Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification.

The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the beta-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose.     

Binding site analysis of cellulose binding domain CBD(N1) from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis

Endoglucanase C (CenC), a beta1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBD(N1) and CBD(N2). In this work, the contributions of 10 amino acids within the binding cleft of CBD(N1) were evaluated by single site-directed mutations to alanine residues. Each isolated domain containing a single mutation was analyzed for binding to an insoluble amorphous preparation of cellulose, phosphoric acid swollen Avicel (PASA), and to a soluble glucopyranoside polymer, barley beta-glucan. The effect of any given mutation on CBD binding was similar for both substrates, suggesting that the mechanism of binding to soluble and insoluble substrates is the same. Tyrosines 19 and 85 were essential for tight binding by CBD(N1) as their replacement by alanine results in affinity decrements of approximately 100-fold on PASA, barley beta-glucan, and soluble cellooligosaccharides. The tertiary structures of unbound Y19A and Y85A were assessed by heteronuclear single quantum coherence (HSQC) spectroscopy. These studies indicated that the structures of both mutants were perturbed but that all perturbations are very near to the site of mutation.     

Properties and mutation analysis of the CelK cellulose-binding domain from the Clostridium thermocellum cellulosome

The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD(CelK)) was expressed in Escherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 micromol/g of cellulose and relative affinities (K(r)) of 2.33 and 9.87 liters/g, respectively. The CBD(CelK) is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD(CelK) also binds to the soluble polysaccharides lichenin, glucomannan, and barley beta-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBD(CelK) contains 1 mol of calcium per mol. The CBD(CelK) has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD(CelK) four highly conserved aromatic residues (Trp(56), Trp(94), Tyr(111), and Tyr(136)) and Asp(192) were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-N(CelK). The CBD-N(CelK) and D192A retained binding parameters close to that of the intact CBD(CelK), W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K(r) and Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBD(CelK) led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBD(CelK) and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD(CelK) are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBD(N1) Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD(CelK) correctly folded.     

Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene

At least three acetyl xylan esterases (AXE I, II and III) are secreted by Penicillium purpurogenum. This publication describes more detailed work on AXE I and its gene. AXE I binds cellulose but not xylan; it is glycosylated and inactivated by phenylmethylsulphonyl fluoride, showing that it is a serine esterase. The axe1 gene presents an open reading frame of 1278 bp, including two introns of 68 and 61 bp; it codes for a signal peptide of 31 residues and a mature protein of 351 amino acids (molecular weight 36,693). AXE I has a modular structure: a catalytic module at the amino terminus belonging to family 1 of the carbohydrate esterases, a linker rich in serines and threonines, and a family 1 carboxy terminal carbohydrate binding module (CBM). The CBM is similar to that of AXE from Trichoderma reesei, (with a family 5 catalytic module) indicating that the genes for catalytic modules and CBMs have evolved separately, and that they have been linked by gene fusion. The promoter sequence of axe1 contains several putative sequences for binding of gene expression regulators also found in other family 1 esterase gene promoters. It is proposed that AXE I and II act in succession in xylan degradation; first, xylan is attacked by AXE I and other xylanases possessing CBMs (which facilitate binding to lignocellulose), followed by other enzymes acting mainly on soluble substrates.     

Microbial starch-binding domain

Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.     

PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene.

We have previously defined the promoter elements, sites IIa and IIb, in the rice proliferating cell nuclear antigen (PCNA) gene that are essential for meristematic tissue-specific expression. In this study, we isolated and characterized cDNAs encoding proteins that specifically bind to sites IIa and IIb. The two DNA binding proteins, designated PCF1 and PCF2, share > 70% homology in common conserved sequences at the N-terminal regions. The conserved regions are responsible for DNA binding and homodimer and heterodimer formation, and they contain a putative basic helix-loop-helix (bHLH) motif. The structure and DNA binding specificity of the bHLH motif are distinguishable from those of other known bHLH proteins that bind to the E-box. The motif is > 70% homologous to several expressed sequence tags from Arabidopsis and rice, suggesting that PCF1 and PCF2 are members of a novel family of proteins that are conserved in monocotyledons and dicotyledons. A supershift assay using an anti-PCF2 antibody showed the involvement of PCF2 in site IIa (site IIb) binding activities in rice nuclear extracts, particularly in meristematic tissues. PCF1 and PCF2 were also more likely to form heterodimers than homodimers. Our results suggest that PCF1 and PCF2 are involved in meristematic tissue-specific expression of the rice PCNA gene through binding to sites IIa and IIb and formation of homodimers or heterodimers.     

Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A

The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.     

Diversity in the SH2 domain family phosphotyrosyl peptide binding site

Src homology 2 (SH2) domains are approximately 100 residue phosphotyrosyl peptide binding modules found in signalling proteins and are important targets for therapeutic intervention. The peptide binding site is evolutionarily well conserved, particularly at the two major binding pockets, pTyr and pTyr + 3. We present a computational analysis of diversity within the peptide binding region and discuss molecular recognition beyond the conventional binding motif, drawing attention to novel conserved ligand interaction sites which may be exploitable in ligand binding studies. The peptide binding site is defined by selecting crystal contacts and domains are clustered according to binding site residue similarity. Comparison with a classification based on experimental peptide screening reveals a high level of qualitative agreement, indicating that the method is able independently to generate functional information. A conservation scoring method reveals extensive patches of conservation in some groups not present across the whole family, challenging the notion that the domains recognise only a linear phosphopeptide sequence. Conservation difference maps determine group-dependent clusters of conserved residues that are not seen when considering a larger experimentally determined group. Many of these residues contact the peptide outside the pTyr to pTyr + 3 motif, challenging the conventional view that this motif is largely responsible for ligand recognition and discrimination.     

Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.     

Structure and exon to protein domain relationships of the mouse carbonic anhydrase II gene

We have isolated a cosmid clone containing the entire mouse (YBR strain) carbonic anhydrase (CA) II gene in 38 kilobase pairs of genomic DNA. The gene was found to be composed of seven exons and six introns. A TATA box (TATAAAA) and a possible CCAAT box (CCACT) have been located beginning 92 and 142 base pairs, respectively, upstream from the initiation codon ATG. When the regions encoded by exons and protein domains are examined, all but 1 of the 30 putative active site residues are encoded by four exons: exons 2 and 3 mainly code for hydrophilic residues and exons 4 and 6 mostly hydrophobic residues. Two intron splice positions, one between the codons for Glu-116 and Leu-117 and the other interrupting the codon for Gly-143, are located at the bottom of the active site cavity, and the former separates two of the three histidine residues forming ligands to the active site zinc ion. The other four splice sites map to the exterior of the molecule. Thus, except for the possible association of the 29 active site residues encoded by four exons, no obvious correspondence is seen between the regions coded by exons and the functional or secondary structural domains of the mouse CA II molecule. During this study, the possible basis for the two electrophoretic types, CA IIa and CA IIb, of inbred mouse strains was detected as a Gln/His interchange at position 38.     

X-ray crystallographic study of xylopentaose binding to Pseudomonas fluorescens xylanase A

The structure of the complex between a catalytically compromised family 10 xylanase and a xylopentaose substrate has been determined by X-ray crystallography and refined to 3.2 A resolution. The substrate binds at the C-terminal end of the eightfold betaalpha-barrel of Pseudomonas fluorescens subsp. cellulosa xylanase A and occupies substrate binding subsites -1 to +4. Crystal contacts are shown to prevent the expected mode of binding from subsite -2 to +3, because of steric hindrance to subsite -2. The loss of accessible surface at individual subsites on binding of xylopentaose parallels well previously reported experimental measurements of individual subsites binding energies, decreasing going from subsite +2 to +4. Nine conserved residues contribute to subsite -1, including three tryptophan residues forming an aromatic cage around the xylosyl residue at this subsite. One of these, Trp 313, is the single residue contributing most lost accessible surface to subsite -1, and goes from a highly mobile to a well-defined conformation on binding of the substrate. A comparison of xylanase A with C. fimi CEX around the +1 subsite suggests that a flatter and less polar surface is responsible for the better catalytic properties of CEX on aryl substrates. The view of catalysis that emerges from combining this with previously published work is the following: (1) xylan is recognized and bound by the xylanase as a left-handed threefold helix; (2) the xylosyl residue at subsite -1 is distorted and pulled down toward the catalytic residues, and the glycosidic bond is strained and broken to form the enzyme-substrate covalent intermediate; (3) the intermediate is attacked by an activated water molecule, following the classic retaining glycosyl hydrolase mechanism.