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PubMed1996

Immunochemical characterization and transacting properties of upstream stimulatory factor isoforms.

The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was an efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter.

Amino Acid Sequence↗
PubMed1997

Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway.

In an effort to better understand the mechanism of toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, we employed an iterative search of human expressed sequence tags to identify novel basic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AHR) or the Ah receptor nuclear translocator (ARNT). We characterized five new "members of the PAS superfamily," or MOPs 1-5, that are similar in size and structural organization to the AHR and ARNT. MOPs 1-4 have N-terminal bHLH and PAS domains and C-terminal variable regions. MOP5 contained the characteristic PAS domain and a variable C terminus; it is possible that the cDNA contains a bHLH domain, but the entire open reading frame has yet to be completed. Coimmunoprecipitation studies, yeast two-hybrid analysis, and transient transfection experiments demonstrated that MOP1 and MOP2 dimerize with ARNT and that these complexes are transcriptionally active at defined DNA enhancer sequences in vivo. MOP3 was found to associate with the AHR in vitro but not in vivo. This observation, coupled with the fact that MOP3 formed tighter associations with the 90-kDa heat shock protein than the human AHR, suggests that MOP3 may be a conditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expression profiles of the AHR, MOP1, and MOP2 mRNAs, coupled with the observation that they all share ARNT as a common dimeric partner, suggests that the cellular pathways mediated by MOP1 and MOP2 may influence or respond to the dioxin signaling pathway.

Amino Acid Sequence↗
PubMed2000

MAP kinase pathways activated by stress: the p38 MAPK pathway.

A stress-activated serine/threonine protein kinase, p38 mitogen-activated protein kinase (p38 MAPK), belongs to the MAP kinase superfamily. Diverse extracellular stimuli, including ultraviolet light, irradiation, heat shock, high osmotic stress, proinflammatory cytokines and certain mitogens, trigger a stress-regulated protein kinase cascade culminating in activation of p38 MAPK through phosphorylation on a TGY motif within the kinase activation loop. p38 MAPK appears to play a major role in apoptosis, cytokine production, transcriptional regulation, and cytoskeletal reorganization, and has been causally implicated in sepsis, ischemic heart disease, arthritis, human immunodeficiency virus infection, and Alzheimer's disease. The availability of specific inhibitors helps to clarify the role that p38 MAPK plays in these processes, and may ultimately offer therapeutic benefit for certain critically ill patients.

Apoptosis↗
PubMed1996

NeuroD2 and neuroD3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family.

We have identified two new genes, neuroD2 and neuroD3, on the basis of their similarity to the neurogenic basic-helix-loop-helix (bHLH) gene neuroD. The predicted amino acid sequence of neuroD2 shows a high degree of homology to neuroD and MATH-2/NEX-1 in the bHLH region, whereas neuroD3 is a more distantly related family member. neuroD3 is expressed transiently during embryonic development, with the highest levels of expression between days 10 and 12. neuroD2 is initially expressed at embryonic day 11, with persistent expression in the adult nervous system. In situ and Northern (RNA) analyses demonstrate that different regions of the adult nervous system have different relative amounts of neuroD and neuroD2 RNA. Similar to neuroD, expression of neuroD2 in developing Xenopus laevis embryos results in ectopic neurogenesis, indicating that neuroD2 mediates neuronal differentiation. Transfection of vectors expressing neuroD and neuroD2 into P19 cells shows that both can activate expression through simple E-box-driven reporter constructs and can activate a reporter driven by the neuroD2 promoter region, but the GAP-43 promoter is preferentially activated by neuroD2. The noncongruent expression pattern and target gene specificity of these highly related neurogenic bHLH proteins make them candidates for conferring specific aspects of the neuronal phenotype.

Adult↗
PubMed1997

Protein-nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double-stranded DNA template-primer.

The conformation of the DNA and the interactions of the nucleic acid with the protein in a complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and 19-mer/18-mer double-stranded DNA template-primer (dsDNA) are described. The structure of this HIV-1 RT complex with dsDNA serves as a useful paradigm for studying aspects of nucleotide polymerases such as catalysis, fidelity, drug inhibition, and drug resistance. The bound dsDNA has a bend of approximately 41 degrees at the junction of an A-form region (first five base pairs near the polymerase active site) and a B-form region (the last nine base pairs toward the RNase H active site). The 41 degrees bend occurs smoothly over the four base pairs between the A-form portion and the B-form portion in the vicinity of helices alpha H and alpha I of the p66 thumb subdomain. The interactions between the dsDNA and protein primarily involve the sugar-phosphate backbone of the nucleic acid and structural elements of the palm, thumb, and RNase H of p66, and are not sequence specific. Amino acid residues from the polymerase active site region, including amino acid residues of the conserved Tyr-Met-Asp-Asp (YMDD) motif and the "primer grip," interact with 3'-terminal nucleotides of the primer strand and are involved in positioning the primer terminal nucleotide and its 3'-OH group at the polymerase active site. Amino acid residues of the "template grip" have close contacts with the template strand and aid in positioning the template strand near the polymerase active site. Helix alpha H of the p66 thumb is partly inserted into the minor groove of the dsDNA and helix alpha I is directly adjacent to the backbone of the template strand. Amino acid residues of beta 1', alpha A', alpha B', and the loop containing His539 of the RNase H domain interact with the primer strand of the dsDNA.

DNA↗
PubMed1996

A DNA structure is required for geminivirus replication origin function.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.

Base Sequence↗
PubMed1998

Cloning of a novel human testis mRNA specifically expressed in testicular haploid germ cells, having unique palindromic sequences and encoding a leucine zipper dimerization motif.

A novel gene coding for a human sperm surface protein (HSS) has been cloned from a human testis cDNA library. The 2.5 kb cDNA (hss) contains five direct repeats, three mirror repeats, three possible stem loop structures and three unique palindromic motifs located at functionally relevant positions. Open reading frame encodes a protein of 766 amino acids. The deduced protein analysis revealed a NH2 terminus extracellular domain having a leucine zipper (LZ) motif, a transmembrane helical domain and a cytoplasmic domain at the COOH terminus. Tissue specific expression studies demonstrated the presence of hss transcript only in testis. Cell type expression of hss mRNA was observed only on the round spermatids suggesting haploid germ cell expression.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp).

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.

Aspartate-tRNA Ligase↗
PubMed2004

The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5.

The human monoclonal antibody 2F5 neutralizes primary human immunodeficiency virus type 1 (HIV-1) with rare breadth and potency. A crystal structure of a complex of 2F5 and a peptide corresponding to its core epitope on gp41, ELDKWAS, revealed that the peptide interacts with residues at the base of the unusually long (22-residue) third complementarity-determining region of the heavy chain (CDR H3) but not the apex. Here, we perform alanine-scanning mutagenesis across CDR H3 and make additional substitutions of selected residues to map the paratope of Fab 2F5. Substitution of residues from the base of the H3 loop or from CDRs H1, H2, and L3, which are proximal to the peptide, significantly diminished the affinity of Fab 2F5 for gp41 and a short peptide containing the 2F5 core motif. However, nonconservative substitutions to a phenylalanine residue at the apex of the H3 loop also markedly decreased 2F5 binding to both gp41 and the peptide, suggesting that recognition of the core epitope is crucially dependent on features at the apex of the H3 loop. Furthermore, substitution at the apex of the H3 loop had an even more pronounced effect on the neutralizing activity of 2F5 against three sensitive HIV-1. These observations present a challenge to vaccine strategies based on peptide mimics of the linear epitope.

Amino Acid Sequence↗
PubMed2003

Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation.

Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates. Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis. Here we demonstrate that reducing expression of endogenous Akt1 and Akt2 by RNA interference (RNAi) reduces neuron generation in P19 cells transfected with a neural bHLH expression vector. The reduction in neuron generation from decreased Akt expression is not solely due to decreased cell survival, since addition of the caspase inhibitor z-VAD-FMK rescues cell death associated with loss of Akt function but does not restore neuron formation. This result indicates that Akt1 and Akt2 have additional functions during neuronal differentiation that are separable from neuronal survival. We show that activated Akt1 enhances complex formation between bHLH proteins and the transcriptional coactivator p300. Activated Akt1 also significantly augments the transcriptional activity of the bHLH protein neurogenin 3 in complex with the coactivators p300 or CBP. In addition, inhibition of endogenous Akt activity by the PI3K/Akt inhibitor LY294002 abolishes transcriptional cooperativity between the bHLH proteins and p300. We propose that Akt regulates the assembly and activity of bHLH-coactivator complexes to promote neuronal differentiation.

Amino Acid Chloromethyl Ketones↗
PubMed2001

NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development.

A key factor in the genetically programmed development of the nervous system is the death of massive numbers of neurons. Therefore, genetic mechanisms governing cell survival are of fundamental importance to developmental neuroscience. We report that inner ear sensory neurons are dependent on a basic helix-loop-helix transcription factor called NeuroD for survival during differentiation. Mice lacking NeuroD protein exhibit no auditory evoked potentials, reflecting a profound deafness. DiI fiber staining, immunostaining and cell death assays reveal that the deafness is due to the failure of inner ear sensory neuron survival during development. The affected inner ear sensory neurons fail to express neurotrophin receptors, TrkB and TrkC, suggesting that the ability of NeuroD to support neuronal survival may be directly mediated through regulation of responsiveness to the neurotrophins.

Animals↗
PubMed2002

The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1.

The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. Here we show that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, we define the role of SCL as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, we demonstrate by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. We therefore conclude that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, our observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells.

3T3 Cells↗
PubMed2004

Predicting interactions of winged-helix transcription factors with DNA.

Determining protein-DNA interactions is important for understanding gene regulation, DNA repair and chromatin structure. Unfortunately, the structures of DNA-bound complexes are often difficult to obtain experimentally, so the development of computational methods that provide good models of these complexes would be valuable. Here, we present a rigid-body docking approach using the computer program DOT. DOT performs a complete, six-dimensional search of all orientations for two rigid molecules and calculates the interaction energy as the sum of electrostatic and van der Waals terms. DOT was applied to three winged-helix transcription factors that share similar DNA-binding structural motifs but bind DNA in different ways. Docking with linear B-form DNA models accomplished several objectives; it (1) distinguished the different ways the transcription factors bind DNA, (2) identified each protein's DNA-binding site and the DNA orientation at the site and (3) gave at least one solution among the three best-ranked that shows the protein side chain-DNA base interactions responsible for recognition. Furthermore, the ensemble of top-ranked, docked linear B-DNA fragments indicated the DNA bending induced upon protein binding. Docking linear B-DNA to structures of the transcription factor FadR suggests that the allosteric, conformational change induced upon effector binding results in loss of the ability to bend DNA as well as loss of sequence-specific interactions with DNA. The electrostatic energy term calculated by DOT is comparable to the electrostatic binding energy calculated by Poisson-Boltzmann methods. Our results show rigid-body docking that includes a rigorous treatment of the electrostatic interaction energy can be effective in predicting protein-DNA interactions.

Bacterial Proteins↗
PubMed1997

Site-directed mutagenesis of alpha II spectrin at codon 1175 modulates its mu-calpain susceptibility.

Intracellular proteolysis by the calpains, a family of Ca2+ activated cysteine proteases, is a ubiquitous yet poorly understood process. Their action is implicated in an array of cellular and pathologic processes, including long-term potentiation, synaptic remodeling, protein kinase C and steroid receptor activation, ischemic cellular injury, and apoptosis. Unlike most proteases, the calpains display unusually strict substrate specificity, often cleaving only one or two bonds in proteins with hundreds of potential sites. Studies of synthetic peptides have defined sequences that modulate their specificity, but little data exist in the context of a bona fide protein. A prominent substrate for mu-calpain is alpha II spectrin (fodrin, brain spectrin), which is cleaved between Tyr1176 and Gly1177 within spectrin's 11th structural repeat unit. We have cloned and characterized human fetal brain alpha II spectrin (GenBank no. U26396) and identified a new Thr1300 to Ile polymorphism. From this clone, recombinant GST-fusion proteins representing repeat units 8-14 have been prepared and used to systematically explore the in vitro determinants of mu-calpain sensitivity. Twenty different amino acids were substituted by site-directed mutagenesis for wild-type Val1175, the penultimate (P2) residue flanking the major calpain cleavage site in alpha II spectrin. Gly, Pro, and Asp, and to a lesser extent Phe and Glu, substantively inhibited the susceptibility of this site to mu-calpain; other substitutions yielded lesser effects. Dynamic molecular modeling of the 11th structural repeat of human alpha II spectrin incorporating the various mutations suggests that the calpain cleavage site with its flanking calmodulin binding domain interrupts helix C of alpha II spectrin's 11th repetitive unit without significantly disrupting the repeat's triple-helical motif. This model predicts that the critical Tyr1176-Gly1177 bond occurs in a highly exposed loop juxtaposed between helix C and the calmodulin binding domain and that mutations at the P2 position subtly alter the conformation about this site. We conclude that secondary and tertiary conformational features surrounding the cleavage site, rather than the linear sequence itself, dominate the determinants that define alpha II spectrin's mu-calpain susceptibility.

Amino Acid Sequence↗
PubMed2005

Loss of circadian photoentrainment and abnormal retinal electrophysiology in Math5 mutant mice.

PURPOSE: To determine how the absence of retinal ganglion cells (RGCs) in Math5 (Atoh7) mutant mice affects circadian behavior and retinal function. METHODS: The wheel-running behavior of wild-type and Math5 mutant mice was measured under various light-dark cycle conditions. To evaluate retinal input to the suprachiasmatic nuclei (SCN) anatomically, the retinohypothalamic tracts were labeled in vivo. To assess changes in retinal function, corneal flash electroretinograms (ERGs) from mutant and wild-type mice were compared under dark- and light-adapted conditions. Alterations in retinal neuron populations were evaluated quantitatively and with cell-type-specific markers. RESULTS: The Math5-null mice did not entrain to light and exhibited free-running circadian behavior with a mean period (23.6 +/- 0.15 hours) that was indistinguishable from that of wild-type mice (23.4 +/- 0.19 hours). The SCN showed no anterograde labeling with a horseradish peroxidase-conjugated cholera toxin B (CT-HRP) tracer. ERGs recorded from mutant mice had diminished scotopic a- and b-wave and photopic b-wave amplitudes. The scotopic b-wave was more severely affected than the a-wave. The oscillatory potentials (OPs) and scotopic threshold response (STR) were also reduced. Consistent with these ERG findings, a pan-specific reduction in the number of bipolar cells and a smaller relative decrease in the number of rods in mutant mice were observed. CONCLUSIONS: Math5-null mice are clock-blind and have no RGC projections to the SCN. RGCs are thus essential for photoentrainment in mice, but are not necessary for the development or intrinsic function of the SCN clock. RGCs are not required to generate any of the major ERG waveforms in mice, including the STR, which is produced by ganglion cells in some other species. The diminished amplitude of b-wave, OPs, and STR components in Math5 mutants is most likely caused by the decreased abundance of retinal interneurons.

Animals↗
PubMed1994

Ectopic expression of myc or myn down-regulates immunoglobulin transcription.

Co-transfection of expression vectors for c-myc or myn down-regulated the expression from a reporter plasmid containing the chloramphenicol-acetyl-transferase (CAT) gene, under the control of an immunoglobulin kappa promoter and the immunoglobulin heavy-chain enhancer. The effect was dose-dependent and an additive effect was seen when both expression vectors were transfected simultaneously. Deletion mutants lacking the leucine zipper region of c-myc were unable to down-regulate the transcription from the reporter construct. Reporter genes where the immunoglobulin enhancer had been exchanged with a SV40 enhancer or an immunoglobulin minimal enhancer were still down-regulated by a cotransfection with c-myc expression vectors. A minimal promoter containing an octamer motif responded to myc expression while a minimal promoter containing a SP1 motif did not. No direct interactions between myc, myn and Oct proteins could be detected either by band-shift analysis, or by co-translation in vitro followed by precipitation with SP6 kappa promoter bound to magnetic carriers. Furthermore, B cells from mice transgenic for myc showed aberrant expression of transcription factors.

Animals↗
PubMed2000

Functional activities and immunoglobulin variable regions of human and murine monoclonal antibodies specific for the P1.7 PorA protein loop of Neisseria meningitidis.

The meningococcal PorA protein is considered a promising vaccine candidate. Although much is understood regarding the structure of PorA proteins, little is known about the structure-function relationships of PorA antibodies. The aim of this study was to compare the functional and molecular characteristics of a human monoclonal antibody (MAb) and three murine MAbs specific for the PorA P1.7 serosubtype. Murine MAbs 207,B-4 (immunoglobulin G2a [IgG2a]) and MN14C11.6 (IgG2a) were both bactericidal and opsonophagocytic for P1.7-expressing meningococci, whereas human MAb SS269 (IgG3) and murine MAb 208,D-5 (IgA) initiated neither effector function. Epitope mapping with synthetic peptides revealed that MAbs 207,B-4 and 208,D-5 recognized the sequence ASGQ, which is the same specificity motif that a previous study had established for SS269 and MN14C11.6. Nucleotide and amino acid sequence analyses of the variable regions of the four MAbs showed that the SS269 V(H) region belonged to the VH3 family and was approximately 70% homologous to those of the murine MAbs which were all from the 7183 family, whereas the SS269 V(L) region belonged to the Vlambda1-b family and was less than 40% homologous to those of the murine MAbs which were all members of the Vkappa1 family. The Fab fragment of SS269 was cloned and expressed in Escherichia coli and was shown by enzyme-linked immunosorbent assay analyses to bind as well as intact SS269 MAb to P1.7,16 serosubtype group B strain 44/76. We conclude that distinct differences exist in the effector function activities and variable region gene sequences of human and murine P1.7-specific MAbs despite their recognition of similar epitopes.

Amino Acid Sequence↗
PubMed2001

Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain.

The type I dockerin domain is responsible for incorporating its associated glycosyl hydrolase into the bacterial cellulosome, a multienzyme cellulolytic complex, via its interaction with a receptor domain (cohesin domain) of the cellulosomal scaffolding subunit. The highly conserved dockerin domain is characterized by two Ca(2+)-binding sites with sequence similarity to the EF-hand motif. Here, we present the three-dimensional solution structure of the 69 residue dockerin domain of Clostridium thermocellum cellobiohydrolase CelS. Torsion angle dynamics calculations utilizing a total of 728 NOE-derived distance constraints and 79 torsion angle restraints yielded an ensemble of 20 structures with an average backbone r.m.s.d. for residues 5 to 29 and 32 to 66 of 0.54 A from the mean structure. The structure consists of two Ca(2+)-binding loop-helix motifs connected by a linker; the E helices entering each loop of the classical EF-hand motif are absent from the dockerin domain. Each dockerin Ca(2+)-binding subdomain is stabilized by a cluster of buried hydrophobic side-chains. Structural comparisons reveal that, in its non-complexed state, the dockerin fold displays a dramatic departure from that of Ca(2+)-bound EF-hand domains. A putative cohesin-binding surface, comprised of conserved hydrophobic and basic residues, is proposed, providing new insight into cellulosome assembly.

Amino Acid Sequence↗