Three dimensional (3D) structure prediction and substrate-protein interaction study of the chitin binding protein CBP24 from B. thuringiensis

Bacillus thuringiensis is an insecticidal bacterium whose chitinolytic system has been exploited to improve insect resistance in crops. In the present study, we studied the CBP24 from B. thuringiensis using homology modeling and molecular docking. The primary and secondary structure analyses showed CBP24 is a positively charged protein and contains single domain that belongs to family CBM33. The 3D model after refinement was used to explore the chitin binding characteristics of CBP24 using AUTODOCK. The docking analyses have shown that the surface exposed hydrophilic amino acid residues Thr-103, Lys-112 and Ser-162 interact with substrate through H-bonding. While, the amino acids resides Glu-39, Tyr-46, Ser-104 and Asn-109 were shown to have polar interactions with the substrate. The binding energy values evaluation of docking depicts a stable intermolecular conformation of the docked complex. The functional characterization of the CBP24 will elucidate the substrate-interaction pathway of the protein in specific and the carbohydrate binding proteins in general leading towards the exploration and exploitation of the prokaryotic substrate utilization pathways.     

Molecular characterization of the modular chitin binding protein Cbp50 from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> serovar <Emphasis Type="Italic">konkukian</Emphasis>

Bacillus thuringiensis is an insecticidal bacterium whose chitinolytic system may be exploited to improve the insecticidal system of Bt-crops. A nucleotide fragment of 1368 bp from B. thuringiensis serovar konkukian S4, containing the complete coding sequence of the chitin binding protein Cbp50, was cloned and sequenced. Analyses have shown the protein to contain a modular structure consisting of an N-terminal CBM33 domain, two copies of a fibronectin-like domain and a C-terminal chitin binding domain classified as CBM5. The Cbp50 protein was heterologously expressed in Escherichia coli, purified and assessed for chitin binding activity. A deletion mutant (CBD-N; containing only the N-terminal CBM33 domain) of Cbp50 was produced to determine the role of C-terminal domains in the binding activity of the protein. The full-length Cbp50 was shown to bind β-chitin most efficiently followed by α-chitin, colloidal chitin and cellulose. The polysaccharide binding activity of CBD-N was drastically decreased. The data demonstrate that both the N-terminal and C-terminal domains of Cbp50 are essential for the efficient binding of chitin. The purified Cbp50 showed antifungal activity against the phytopathogenic fungus Fusarium oxysporum and the opportunistic human pathogen Aspergillus niger. This is the first report of a modular chitin binding protein in bacteria.     

Molecular cloning,sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. PE2, and its domain structure

The pchA gene encoding chitinase A (PchA) from a Pythium porphyrae cell-wall-degrading marine bacterium, Pseudomonas sp. PE2, was cloned and characterized. The deduced PchA was a modular enzyme composed of an N-terminal signal peptide, a glycoside hydrolase family 18 catalytic domain that was responsible for the chitinase activity, the chitin-binding domains (ChBDs), and the carbohydrate-binding modules (CBM). The amino acid sequence of ChBD(PchA) was highly conserved in the CBM family 12 that also accommodates ChBDs without an AKWWTQG motif, a domain commonly found in bacterial chitinase and Streptomyces griseus protease C. Interestingly, CBM(PchA) showed significant sequence homology to the C-terminal region of endoglucanase B from Cellvibrio mixtus, which is a member of CBM family 6. This is the first report of a chitinase possessing a domain with high similarity to CBM family 6. Deletion analysis indicated clearly that ChBD(PchA) might play an important role in the binding of native chitin and chitosan, but not processed chitin. CBM(PchA) also appeared to play such a role in the binding of xylan and Avicel. These results suggest that the C-terminal region of PchA might be a key component in the binding of chitin in the cell walls of P. porphyrae or other structural components of marine organisms.     

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.     

Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin

Chemically prepared hevein domains (HDs), N-terminal domain of an antifungal protein from Nicotiana tabacum (CBP20-N) and an antimicrobial peptide from Amaranthus caudatus (Ac-AMP2), were examined for their affinity for chitin, a beta-1,4-linked polymer of N-acetylglucosamine. An intact binding domain, CBP20-N, showed a higher affinity than a C-terminal truncated domain, Ac-AMP2. The formation of a pyroglutamate residue from N-terminal Gln of CBP20-N increased the affinity. The single replacement of any aromatic residue of Ac-AMP2 with Ala resulted in a significant reduction in affinity, suggesting the importance of the complete set of three aromatic residues in the ligand binding site. The mutations of Phe18 of Ac-AMP2 to the residues with larger aromatic rings, i.e. Trp, beta-(1-naphthyl)alanine or beta-(2-naphthyl)alanine, enhanced the affinity, whereas the mutation of Tyr20 to Trp reduced the affinity. The affinity of an HD for chitin might be improved by adjusting the size and substituent group of stacking aromatic rings.     

Identification of functional domains of the Escherichia coli SeqA protein

The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-mA-T-C sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains. The N-terminal domain, which is not involved in DNA binding, has the self-association activity. In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-mA-T-C sequence. Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively.     

Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.     

Catalytic Efficiency of Chitinase-D on Insoluble Chitinous Substrates Was Improved by Fusing Auxiliary Domains

Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.     

Protein kinase C phosphorylation disrupts Na+/H+ exchanger regulatory factor 1 autoinhibition and promotes cystic fibrosis transmembrane conductance regulator macromolecular assembly

An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes.     

The Solution Structure and Dynamics of Full-length Human Cerebral Dopamine Neurotrophic Factor and Its Neuroprotective Role against α-Synuclein Oligomers

Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9–107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10–100 and 111–157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1–9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.     

Crystal structures of Urtica dioica agglutinin and its complex with tri-N-acetylchitotriose

Urtica dioica agglutinin is a small plant lectin that binds chitin. We purified the isolectin VI (UDA-VI) and crystal structures of the isolectin and its complex with tri-N-acetylchitotriose (NAG3) were determined by X-ray analysis. The UDA-VI consists of two domains analogous to hevein and the backbone folding of each domain is maintained by four disulfide bridges. The sequence similarity of the two domains is not high (42 %) but their backbone structures are well superimposed except some loop regions. The chitin binding sites are located on the molecular surface at both ends of the dumbbell-shape molecule. The crystal of the NAG3 complex contains two independent molecules forming a protein-sugar 2:2 complex. One NAG3 molecule is sandwiched between two independent UDA-VI molecules and the other sugar molecule is also sandwiched by one UDA-VI molecule and symmetry-related another one. The sugar binding site of N-terminal domain consists of three subsites accommodating NAG3 while two NAG residues are bound to the C-terminal domain. In each sugar-binding site, three aromatic amino acid residues and one serine residue participate to the NAG3 binding. The sugar rings bound to two subsites are stacked to the side-chain groups of tryptophan or histidine and a tyrosine residue is in face-to-face contact with an acetylamino group, to which the hydroxyl group of a serine residue is hydrogen-bonded. The third subsite of the N-terminal domain binds a NAG moiety with hydrogen bonds. The results suggest that the triad of aromatic amino acid residues is intrinsic in sugar binding of hevein-like domains.     

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.     

Amino acid sequence of yeast hemoglobin. A two-domain structure.

The complete amino acid sequence of a hemoglobin from yeast (Candida norvegensis) has been determined by peptide and cDNA sequence analyses. The protein is composed of 387 amino acid residues and its amino terminus was blocked by an acetyl group. A computer search showed that the sequence of 155 N-terminal residues has 39% homology with that of Vitreoscilla hemoglobin. On the other hand, the sequence of 230 C-terminal residues showed a small, but notable, degree of similarity with that of a methemoglobin reductase found in human erythrocyte, i.e. NADH-cytochrome b5 oxido-reductase. We therefore conclude that yeast hemoglobin consists of two distinct domains; one is a heme-containing oxygen binding domain of the N-terminal region and the other is an FAD-containing reductase domain found in the C-terminal region.     

Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells. Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.     

The Tra domain of the lactococcal CluA surface protein is a unique domain that contributes to sex factor DNA transfer

CluA is a cell surface-presented protein that causes cell aggregation and is essential for a high-efficiency conjugation process in Lactococcus lactis. We know from previous work that in addition to promoting cell-to-cell contact, CluA is involved in sex factor DNA transfer. To define the CluA domains involved in aggregation and in transfer, we first performed random mutagenesis of the cluA gene using a modified mini-Tn7 element which generated five amino acid insertions located throughout the encoded protein. Thirty independent cluA insertion mutants expressing modified CluA proteins at the cell surface were isolated and characterized further. The level of aggregation of each mutant was determined. The cell binding capacity of CluA was affected strongly when the protein had a mutation in its N-terminal region, which defined an aggregation domain extending from amino acid 153 to amino acid 483. Of the cluA mutants that still exhibited aggregation, eight showed an attenuated ability to conjugate, and six mutations were located in a 300-amino-acid C-terminal region of the protein defining a transfer domain (Tra). This result was confirmed by a phenotypic analysis of an additional five mutants obtained using site-directed mutagenesis in which charged amino acids of the Tra domain were replaced by alanine residues. Two distinct functional domains of the CluA protein were defined in this work; the first domain is involved in cell binding specificity, and the Tra domain is probably involved in the formation of the DNA transport machinery. This is the first report of a protein involved in conjugation that actively contributes to DNA transfer and mediates contact between donor and recipient strains.     

Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.     

Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21

Chitin proteins are commonly found in bacteria that utilize chitin as a source of energy. CBP21 is a chitin-binding protein from Serratia marcescens, a Gram-negative soil bacterium capable of efficient chitin degradation. When grown on chitin, S. marcescens secretes large amounts of CBP21, along with chitin-degrading enzymes. In an attempt to understand the molecular mechanism of CBP21 action, we have determined its crystal structure at 1.55 angstroms resolution. This is the first structure to be solved of a family 33 carbohydrate-binding module. The structure reveals a "budded" fibronectin type III fold consisting of two beta-sheets, arranged as a beta-sheet sandwich, with a 65-residue "bud" consisting of three short helices, located between beta-strands 1 and 2. Remarkably, conserved aromatic residues that have been suggested previously to play a role in chitin binding were mainly found in the interior of the protein, seemingly incapable of interacting with chitin, whereas the structure revealed a surface patch of highly conserved, mainly hydrophilic residues. The roles of six of these conserved surface-exposed residues (Tyr-54, Glu-55, Glu-60, His-114, Asp-182, and Asn-185) were probed by site-directed mutagenesis and subsequent binding studies. All single point mutations lowered the affinity of CBP21 for beta-chitin, as shown by 3-8-fold increases in the apparent binding constant. Thus, binding of CBP21 to chitin seems to be mediated primarily by conserved, solvent-exposed, polar side chains.