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Biomedical subjects

Deborah S Wuttke

Publications and source records attributed to Deborah S Wuttke.

At least 19 recordsLinked to original sources
PubMed2006

Empirical Bayes hierarchical models for regularizing maximum likelihood estimation in the matrix Gaussian Procrustes problem.

Procrustes analysis involves finding the optimal superposition of two or more "forms" via rotations, translations, and scalings. Procrustes problems arise in a wide range of scientific disciplines, especially when the geometrical shapes of objects are compared, contrasted, and analyzed. Classically, the optimal transformations are found by minimizing the sum of the squared distances between corresponding points in the forms. Despite its widespread use, the ordinary unweighted least-squares (LS) criterion can give erroneous solutions when the errors have heterogeneous variances (heteroscedasticity) or the errors are correlated, both common occurrences with real data. In contrast, maximum likelihood (ML) estimation can provide accurate and consistent statistical estimates in the presence of both heteroscedasticity and correlation. Here we provide a complete solution to the nonisotropic ML Procrustes problem assuming a matrix Gaussian distribution with factored covariances. Our analysis generalizes, simplifies, and extends results from previous discussions of the ML Procrustes problem. An iterative algorithm is presented for the simultaneous, numerical determination of the ML solutions.

Algorithms↗
PubMed2006

Themes in ssDNA recognition by telomere-end protection proteins.

The ends of eukaryotic linear chromosomes are unique structures that require special management by the cell. If left unattended, the ends are inappropriately processed, leading to genomic instability and problems with proliferation. Telomeres are specialized nucleoprotein structures that restore chromosome stability by protecting and maintaining chromosome ends. Proper telomere function is facilitated, in part, by the telomere-end protection (TEP) family of proteins, which targets the 3' single-stranded (ss) overhang region of the telomere via a specialized ssDNA-binding domain (DBD). With the recent availability of the structures of these DBDs, the ssDNA-binding characteristics of TEP proteins can be compared and the common underlying mechanisms of ssDNA recognition identified, thus providing insights into telomere function.

Animals↗
PubMed2006

A new model for Schizosaccharomyces pombe telomere recognition: the telomeric single-stranded DNA-binding activity of Pot11-389.

The protection of telomeres 1 (Pot1) proteins specifically recognize the single-stranded 3' end of the telomere, an activity essential for sustained cellular viability and proliferation. The current model for the telomeric single-stranded DNA (ssDNA) binding activity of Schizosaccharomyces pombe Pot1 is based on a 20 kDa fragment, Pot1pN. Recent biochemical studies suggest that SpPot1 contains a larger ssDNA-binding domain and we have identified a novel ssDNA-binding domain similar in size to the human Pot1 domain. This domain, Pot1(1-389), binds extremely tightly to an oligonucleotide consisting of two conserved hexameric S. pombe telomere repeats, d(GGTTACGGTTAC), with an affinity approximately 4000-fold tighter than Pot1pN binds its cognate ssDNA. The Pot1(1-389)/ssDNA complex exhibits a half-life of 53 min, consistent with that estimated for full-length SpPot1 and significantly longer than that of Pot1pN. Single nucleotide substitutions reveal that, in contrast to Pot1pN, tandem trinucleotide repeats (GTT) within d(GGTTACGGTTAC) are specifically recognized by Pot1(1-389). Interestingly, certain single nucleotide substitutions that impacted Pot1pN binding exhibited no effect on binding affinity by Pot1(1-389). However, these substitutions reduced binding affinity when simultaneously substituted in each hexameric repeat. The non-additive nature of these substitutions suggests that certain nucleotides are coupled through the ability of the flexible ssDNA oligonucleotide to adopt alternate, thermodynamically equivalent conformations. The biochemical behavior of Pot1(1-389) is more similar to that of the full-length SpPot1 protein than to that of Pot1pN, making Pot1(1-389) a valuable domain for the future study of how full-length SpPot1 interacts with telomeric ssDNA.

Amino Acid Sequence↗
PubMed2006

THESEUS: maximum likelihood superpositioning and analysis of macromolecular structures.

UNLABELLED: THESEUS is a command line program for performing maximum likelihood (ML) superpositions and analysis of macromolecular structures. While conventional superpositioning methods use ordinary least-squares (LS) as the optimization criterion, ML superpositions provide substantially improved accuracy by down-weighting variable structural regions and by correcting for correlations among atoms. ML superpositioning is robust and insensitive to the specific atoms included in the analysis, and thus it does not require subjective pruning of selected variable atomic coordinates. Output includes both likelihood-based and frequentist statistics for accurate evaluation of the adequacy of a superposition and for reliable analysis of structural similarities and differences. THESEUS performs principal components analysis for analyzing the complex correlations found among atoms within a structural ensemble. AVAILABILITY: ANSI C source code and selected binaries for various computing platforms are available under the GNU open source license from http://monkshood.colorado.edu/theseus/ or http://www.theseus3d.org.

Algorithms↗
PubMed2006

Identification of the determinants for the specific recognition of single-strand telomeric DNA by Cdc13.

The single-strand overhang present at telomeres plays a critical role in mediating both the capping and telomerase regulation functions of telomeres. The telomere end-binding proteins, Cdc13 in Saccharomyces cerevisiae, Pot1 in higher eukaryotes, and TEBP in the ciliated protozoan Oxytricha nova, exhibit sequence-specific binding to their respective single-strand overhangs. S. cerevisiae telomeres are composed of a heterogeneous mixture of GT-rich telomeric sequence, unlike in higher eukaryotes which have a simple repeat that is maintained with high fidelity. In yeast, the telomeric overhang is recognized by the essential protein Cdc13, which coordinates end-capping and telomerase activities at the telomere. The Cdc13 DNA-binding domain (Cdc13-DBD) binds these telomere sequences with high affinity (3 pM) and sequence specificity. To better understand the basis for this remarkable recognition, we have investigated the binding of the Cdc13-DBD to a series of altered DNA substrates. Although an 11-mer of GT-rich sequence is required for full binding affinity, only three of these 11 bases are recognized with high specificity. This specificity differs from that observed in the other known telomere end-binding proteins, but is well suited to the specific role of Cdc13 at yeast telomeres. These studies expand our understanding of telomere recognition by the Cdc13-DBD and of the unique molecular recognition properties of ssDNA binding.

Base Sequence↗
PubMed2005

Divergent evolution within protein superfolds inferred from profile-based phylogenetics.

Many dissimilar protein sequences fold into similar structures. A central and persistent challenge facing protein structural analysis is the discrimination between homology and convergence for structurally similar domains that lack significant sequence similarity. Classic examples are the OB-fold and SH3 domains, both small, modular beta-barrel protein superfolds. The similarities among these domains have variously been attributed to common descent or to convergent evolution. Using a sequence profile-based phylogenetic technique, we analyzed all structurally characterized OB-fold, SH3, and PDZ domains with less than 40% mutual sequence identity. An all-against-all, profile-versus-profile analysis of these domains revealed many previously undetectable significant interrelationships. The matrices of scores were used to infer phylogenies based on our derivation of the relationships between sequence similarity E-values and evolutionary distances. The resulting clades of domains correlate remarkably well with biological function, as opposed to structural similarity, indicating that the functionally distinct sub-families within these superfolds are homologous. This method extends phylogenetics into the challenging "twilight zone" of sequence similarity, providing the first objective resolution of deep evolutionary relationships among distant protein families.

Evolution, Molecular↗
PubMed2005

Soluble domains of telomerase reverse transcriptase identified by high-throughput screening.

Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high-throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using a GFP-fusion system. Many of the soluble protein domains identified do not coincide with domains inferred from multiple sequence alignment, so screening for fluorescent colonies provided information not otherwise readily obtained. The method revealed an essential, independently folded N-terminal domain that was expressed and purified with high yield and found to be suitable for structural analysis. These results provide a tool for future structural and biophysical studies of TERT.

Amino Acid Sequence↗
PubMed2004

Structural basis for telomeric single-stranded DNA recognition by yeast Cdc13.

The essential budding yeast telomere-binding protein Cdc13 is required for telomere replication and end protection. Cdc13 specifically binds telomeric, single-stranded DNA (ssDNA) 3' overhangs with high affinity using an OB-fold domain. We have determined the high-resolution solution structure of the Cdc13 DNA-binding domain (DBD) complexed with a cognate telomeric ssDNA. The ssDNA wraps around one entire face of the Cdc13-DBD OB-fold in an extended, irregular conformation. Recognition of the ssDNA bases occurs primarily through aromatic, basic, and hydrophobic amino acid residues, the majority of which are evolutionarily conserved among budding yeast species and contribute significantly to the energetics of binding. Contacting five of 11 ssDNA nucleotides, the large, ordered beta2-beta3 loop is crucial for complex formation and is a unique elaboration on the binding mode commonly observed in OB-fold proteins. The sequence-specific Cdc13-DBD/ssDNA complex presents a complementary counterpoint to the interactions observed in the Oxytricha nova telomere end-binding and Schizosaccharomyces pombe Pot1 complexes. Analysis of the Cdc13-DBD/ssDNA complex indicates that molecular recognition of extended single-stranded nucleic acids may proceed via a folding-type mechanism rather than resulting from specific patterns of hydrogen bonds. The structure reported here provides a foundation for understanding the mechanism by which Cdc13 recognizes GT-rich heterogeneous sequences with both unusually strong affinity and high specificity.

Amino Acid Sequence↗
PubMed2004

Prediction of multiple tandem OB-fold domains in telomere end-binding proteins Pot1 and Cdc13.

The heterodimeric Oxytricha nova telomere end binding protein, the original telomere end binding protein characterized, contains four OB-fold domains used for recognition of single-stranded telomeric DNA. In contrast, only solitary OB-fold domains have been found in the telomere end binding proteins from yeast and higher eukaryotes. Using a sliding-window algorithm coupled with sequence profile-profile analysis, we provide support for the existence of multiple OB-fold domains in two other telomeric ssDNA binding proteins, vertebrate Pot1 and budding yeast Cdc13. This common usage of multiple, tandem OB-fold domains in telomeric end binding proteins extends the known evolutionary conservation of eukaryotic end-protection mechanisms.

Algorithms↗
PubMed2003

Towards an understanding of the poliovirus replication complex: the solution structure of the soluble domain of the poliovirus 3A protein.

Poliovirus is a positive-strand RNA virus and the prototypical member of the Picornaviridae family. Upon infection, the viral RNA genome is translated from a single open reading frame into a polypeptide which undergoes a series of cleavages to ultimately form four structural and seven non-structural proteins. A replication complex is then formed which replicates the viral genome into negative and positive strands for further translation, replication, and packaging into viral progeny. Poliovirus 3A protein (3A) is a critical component of the viral replication complex and is the putative target of enviroxime, an antiviral drug shown to block viral replication. 3A also inhibits host cell endoplasmic reticulum-to-Golgi apparatus transport, a function which may play a key role in viral evasion from the host immune response. 3A, an 87-residue protein consisting of a soluble N terminus and a hydrophobic C terminus, is formed by the cleavage of the precursor protein 3AB into 3A and 3B (VPg). Although they differ by only 22 residues, the precursor protein 3AB and its cleavage product 3A have distinct functions in viral replication. We have determined the structure of the soluble, N-terminal domain of 3A (3A-N) using NMR spectroscopy. We show that 3A-N exists as a symmetric dimer, and each monomer consists of an alpha-helical hairpin with unstructured, yet functional, N- and C termini. We also show that the 3A-N structure contains a negatively charged surface patch and provides a context for interpreting the biochemical characteristics of a number of previously reported 3A and 3AB mutants.

Amino Acid Sequence↗
PubMed2003

Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13.

The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity. The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres. We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA). The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop. Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues. A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity. Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed. The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding. While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface. This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity.

Binding Sites↗
PubMed2003

Nucleic acid recognition by OB-fold proteins.

The OB-fold domain is a compact structural motif frequently used for nucleic acid recognition. Structural comparison of all OB-fold/nucleic acid complexes solved to date confirms the low degree of sequence similarity among members of this family while highlighting several structural sequence determinants common to most of these OB-folds. Loops connecting the secondary structural elements in the OB-fold core are extremely variable in length and in functional detail. However, certain features of ligand binding are conserved among OB-fold complexes, including the location of the binding surface, the polarity of the nucleic acid with respect to the OB-fold, and particular nucleic acid-protein interactions commonly used for recognition of single-stranded and unusually structured nucleic acids. Intriguingly, the observation of shared nucleic acid polarity may shed light on the longstanding question concerning OB-fold origins, indicating that it is unlikely that members of this family arose via convergent evolution.

Amino Acid Motifs↗
PubMed2003

Homology among telomeric end-protection proteins.

Telomere maintenance and end protection are essential for the survival and proliferation of eukaryotic cells, leading to the prediction that components of this system would be highly conserved. In practice, however, evidence for homology among these factors has been elusive, and, in the case of the known end-protection proteins, evolutionary relationships have been postulated largely on the basis of protein structural and functional similarity alone. Here we report support from sequence profile analyses for a significant and specific evolutionary relationship among OB-fold telomeric end-protection factors.

Databases, Protein↗
PubMed2003

Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments.

Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3'-kinase fold into an active native-like structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18 degrees C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.

Alanine↗
PubMed2002

Delineation of the high-affinity single-stranded telomeric DNA-binding domain of Saccharomyces cerevisiae Cdc13.

Cdc13 is an essential protein from Saccharomyces cerevisiae that caps telomeres by protecting the C-rich telomeric DNA strand from degradation and facilitates telomeric DNA replication by telomerase. In vitro, Cdc13 binds TG-rich single-stranded telomeric DNA with high affinity and specificity. A previously identified domain of Cdc13 encompassing amino acids 451-694 (the 451-694 DBD) retains the single-stranded DNA-binding properties of the full-length protein; however, this domain contains a large unfolded region identified in heteronuclear NMR experiments. Trypsin digestion and MALDI mass spectrometry were used to identify the minimal DNA-binding domain (the 497-694 DBD) necessary and sufficient for full DNA-binding activity. This domain was completely folded, and the N-terminal unfolded region removed was shown to be dispensable for function. Using affinity photocrosslinking to site-specifically modified telomeric single-stranded DNA, the 497-694 DBD was shown to contact the entire 11mer required for high-affinity binding. Intriguingly, both domains bound single-stranded telomeric DNA with much greater affinity than the full-length protein. The full-length protein exhibited the same rate of dissociation as both domains, however, indicating that the full-length protein contains a region that inhibits association with single-stranded telomeric DNA.

Binding Sites↗
PubMed2002

Conserved structure for single-stranded telomeric DNA recognition.

The essential Cdc13 protein in the yeast Saccharomyces cerevisiae is a single-stranded telomeric DNA binding protein required for chromosome end protection and telomere replication. Here we report the solution structure of the Cdc13 DNA binding domain in complex with telomeric DNA. The structure reveals the use of a single OB (oligonucleotide/oligosaccharide binding) fold augmented by an unusually large loop for DNA recognition. This OB fold is structurally similar to OB folds found in the ciliated protozoan telomere end-binding protein, although no sequence similarity is apparent between them. The common usage of an OB fold for telomeric DNA interaction demonstrates conservation of end-protection mechanisms among eukaryotes.

Binding Sites↗