Biomedical subjects

Mark D Allen

Publications and source records attributed to Mark D Allen.

At least 19 recordsLinked to original sources

PubMed2006

The FtsK gamma domain directs oriented DNA translocation by interacting with KOPS.

The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, alpha, beta and gamma. FtsKalphabeta are necessary and sufficient for ATP hydrolysis-dependent DNA translocation, which is modulated by FtsKgamma through its interaction with KOPS. By solving the FtsKgamma structure by NMR, we show that gamma is a winged-helix domain. NMR chemical shift mapping localizes the DNA-binding site on the gamma domain. Mutated proteins with substitutions in the FtsKgamma DNA-recognition helix are impaired in DNA binding and KOPS recognition, yet remain competent in DNA translocation and XerCD-dif site-specific recombination, which facilitates the late stages of chromosome segregation.

Base Sequence↗

PubMed2006

Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase.

Methylation of CpG dinucleotides is the major epigenetic modification of mammalian genomes, critical for regulating chromatin structure and gene activity. The mixed-lineage leukaemia (MLL) CXXC domain selectively binds nonmethyl-CpG DNA, and is required for transformation by MLL fusion proteins that commonly arise from recurrent chromosomal translocations in infant and secondary treatment-related acute leukaemias. To elucidate the molecular basis of nonmethyl-CpG DNA recognition, we determined the structure of the human MLL CXXC domain by multidimensional NMR spectroscopy. The CXXC domain has a novel fold in which two zinc ions are each coordinated tetrahedrally by four conserved cysteine ligands provided by two CGXCXXC motifs and two distal cysteine residues. We have identified the CXXC domain DNA binding interface by means of chemical shift perturbation analysis, cross-saturation transfer and site-directed mutagenesis. In particular, we have shown that residues in an extended surface loop are in close contact with the DNA. These data provide a template for the design of specifically targeted therapeutics for poor prognosis MLL-associated leukaemias.

Amino Acid Sequence↗

PubMed2006

The PUB domain functions as a p97 binding module in human peptide N-glycanase.

The AAA ATPase p97 is a ubiquitin-selective molecular machine involved in multiple cellular processes, including protein degradation through the ubiquitin-proteasome system and homotypic membrane fusion. Specific p97 functions are mediated by a variety of cofactors, among them peptide N-glycanase, an enzyme that removes glycans from misfolded glycoproteins. Here we report the three-dimensional structure of the aminoterminal PUB domain of human peptide N-glycanase. We demonstrate that the PUB domain is a novel p97 binding module interacting with the D1 and/or D2 ATPase domains of p97 and identify an evolutionary conserved surface patch required for p97 binding. Furthermore, we show that the PUB and UBX domains do not bind to p97 in a mutually exclusive manner. Our results suggest that PUB domain-containing proteins constitute a widespread family of diverse p97 cofactors.

Adenosine Triphosphatases↗

PubMed2005

Solution structure of the C4 zinc finger domain of HDM2.

HDM2 is a ubiquitin E3 ligase that is a key negative regulator of the tumor suppressor p53. Here, we report the determination of the solution structure of the C4 zinc finger domain of HDM2 using multidimensional NMR. The HDM2 C4 zinc finger domain has a fold consisting of a 3(10) helix followed by four beta-strands, which shares significant structural similarity to the zinc ribbon protein family. Family based sequence analysis identified two putative binding sites, one of which resembles an RNA binding motif.

Amino Acid Sequence↗

PubMed2005

Functional analysis of the post-transcriptional regulator RsmA reveals a novel RNA-binding site.

The RsmA family of RNA-binding proteins are global post-transcriptional regulators that mediate extensive changes in gene expression in bacteria. They bind to, and affect the translation rate of target mRNAs, a function that is further modulated by one or more, small, untranslated competitive regulatory RNAs. To gain new insights into the nature of this protein/RNA interaction, we used X-ray crystallography to solve the structure of the Yersinia enterocolitica RsmA homologue. RsmA consists of a dimeric beta barrel from which two alpha helices are projected. From structure-based alignments of the RsmA protein family from diverse bacteria, we identified key amino acid residues likely to be involved in RNA-binding. Site-specific mutagenesis revealed that arginine at position 44, located at the N terminus of the alpha helix is essential for biological activity in vivo and RNA-binding in vitro. Mutation of this site affects swarming motility, exoenzyme and secondary metabolite production in the human pathogen Pseudomonas aeruginosa, carbon metabolism in Escherichia coli, and hydrogen cyanide production in the plant beneficial strain Pseudomonas fluorescens CHA0. R44A mutants are also unable to interact with the small untranslated RNA, RsmZ. Thus, although possessing a motif similar to the KH domain of some eukaryotic RNA-binding proteins, RsmA differs substantially and incorporates a novel class of RNA-binding site.

Amino Acid Sequence↗

PubMed2005

Ultra-fast barrier-limited folding in the peripheral subunit-binding domain family.

We have determined the solution structures, equilibrium properties and ultra-fast folding kinetics for three bacterial homologues of the peripheral subunit-binding domain (PSBD) family. The mesophilic homologue, BBL, was less stable than the thermophilic and hyper-thermophilic variants (E3BD and POB, respectively). The broad unfolding transitions of each PSBD, when probed by different techniques, were essentially superimposable, consistent with co-operative denaturation. Temperature-jump and continuous-flow fluorescence methods were used to measure the folding kinetics for E3BD, POB and BBL. E3BD folded fairly rapidly at 298K (folding half-time approximately 25 micros) and BBL and POB folded even faster (folding half-times approximately 3-5 micros). The variations in equilibrium and kinetic behaviour observed for the PSBD family resembles that of the homeodomain family, where the folding pattern changes from apparent two-state transitions to multi-state kinetics as the denatured state becomes more structured. The faster folding of POB may be a consequence of its higher propensity to form helical structure in the region corresponding to the folding nucleus of E3BD. The ultra-fast folding of BBL appears to be a consequence of residual structure in the denatured ensemble, as with engrailed homeodomain. We discuss issues concerning "one-state", downhill folding, and find no evidence for, and strong evidence against, it occurring in these PSBDs. The shorter construct used previously for BBL was destabilized significantly and the stability further perturbed by the introduction of fluorescent probes. Thermal titrations for 11 side-chains scattered around the protein, when probed by (13)C-NMR experiments, could be fit globally to a common co-operative transition.

Amino Acid Sequence↗

PubMed2005

The preservation of verb subcategory knowledge in a spoken language comprehension deficit.

Patient WBN has a lexical-semantic deficit resulting in impaired performance on language comprehension tasks that require access to verb meanings in both single-word and sentence contexts. However, WBN shows no such comprehension impairment with respect to lexical syntax. Specifically, he performs without error on comprehension tasks that rely on knowledge of lexical-specific verb subcategory requirements. This performance pattern supports theories of lexical representation and processing in which lexical syntactic information may be encoded and retrieved independently from lexical semantic information.

Aged, 80 and over↗

PubMed2005

The macro domain is an ADP-ribose binding module.

The ADP-ribosylation of proteins is an important post-translational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long-term memory formation. Yet no protein modules are known that specifically recognize the ADP-ribose nucleotide. We provide biochemical and structural evidence that macro domains are high-affinity ADP-ribose binding modules. Our structural analysis reveals a conserved ligand binding pocket among the macro domain fold. Consistently, distinct human macro domains retain their ability to bind ADP-ribose. In addition, some macro domain proteins also recognize poly-ADP-ribose as a ligand. Our data suggest an important role for proteins containing macro domains in the biology of ADP-ribose.

Adenosine Diphosphate↗

PubMed2005

Electrostatic interactions contribute to reduced heat capacity change of unfolding in a thermophilic ribosomal protein l30e.

The origin of reduced heat capacity change of unfolding (DeltaC(p)) commonly observed in thermophilic proteins is controversial. The established theory that DeltaC(p) is correlated with change of solvent-accessible surface area cannot account for the large differences in DeltaC(p) observed for thermophilic and mesophilic homologous proteins, which are very similar in structures. We have determined the protein stability curves, which describe the temperature dependency of the free energy change of unfolding, for a thermophilic ribosomal protein L30e from Thermococcus celer, and its mesophilic homologue from yeast. Values of DeltaC(p), obtained by fitting the free energy change of unfolding to the Gibbs-Helmholtz equation, were 5.3 kJ mol(-1) K(-1) and 10.5 kJ mol(-1) K(-1) for T.celer and yeast L30e, respectively. We have created six charge-to-neutral mutants of T.celer L30e. Removal of charges at Glu6, Lys9, and Arg92 decreased the melting temperatures of T.celer L30e by approximately 3-9 degrees C, and the differences in melting temperatures were smaller with increasing concentration of salt. These results suggest that these mutations destabilize T.celer L30e by disrupting favorable electrostatic interactions. To determine whether electrostatic interactions contribute to the reduced DeltaC(p) of the thermophilic protein, we have determined DeltaC(p) for wild-type and mutant T.celer L30e by Gibbs-Helmholtz and by van't Hoff analyses. A concomitant increase in DeltaC(p) was observed for those charge-to-neutral mutants that destabilize T.celer L30e by removing favorable electrostatic interactions. The crystal structures of K9A, E90A, and R92A, were determined, and no structural change was observed. Taken together, our results support the conclusion that electrostatic interactions contribute to the reduced DeltaC(p) of T.celer L30e.

Archaeal Proteins↗

PubMed2005

Simulation and experiment at high temperatures: ultrafast folding of a thermophilic protein by nucleation-condensation.

We used Phi-value analysis to characterise the transition state for folding of a thermophilic protein at the relatively high temperature of 325 K. PhiF values for the folding of the three-helix bundle, peripheral subunit binding domain from Bacillus stearothermophilus (E3BD) were determined by temperature-jump experiments in the absence of chemical denaturants. E3BD folded in microseconds through a highly diffuse transition state. Excellent agreement was observed between experiment and the results from eight (independent) molecular dynamics simulations of unfolding at 373 K. We used a combination of heteronuclear NMR experiments and molecular dynamics simulations to characterise the denatured ensemble, and found that it contained very little persistent, residual structure. However, those regions that adopt helical structure in the native state were found by simulation to be poised for helix formation in the denatured state. These regions also had significant structure in the transition state for folding. The overall folding pathway appears to be nucleation-condensation.

Bacterial Proteins↗

PubMed2005

Crystal structure of a hyperthermophilic archaeal acylphosphatase from Pyrococcus horikoshii--structural insights into enzymatic catalysis, thermostability, and dimerization.

Acylphosphatases catalyze the hydrolysis of the carboxyl-phosphate bond in acyl phosphates. Although acylphosphatase-like sequences are found in all three domains of life, no structure of acylphosphatase has been reported for bacteria and archaea so far. Here, we report the characterization of enzymatic activities and crystal structure of an archaeal acylphosphatase. A putative acylphosphatase gene (PhAcP) was cloned from the genomic DNA of Pyrococcus horikoshii and was expressed in Escherichia coli. Enzymatic parameters of the recombinant PhAcP were measured using benzoyl phosphate as the substrate. Our data suggest that, while PhAcP is less efficient than other mammalian homologues at 25 degrees C, the thermophilic enzyme is fully active at the optimal growth temperature (98 degrees C) of P. horikoshii. PhAcP is extremely stable; its apparent melting temperature was 111.5 degrees C and free energy of unfolding at 25 degrees C was 54 kJ mol(-)(1). The 1.5 A crystal structure of PhAcP adopts an alpha/beta sandwich fold that is common to other acylphosphatases. PhAcP forms a dimer in the crystal structure via antiparallel association of strand 4. Structural comparison to mesophilic acylphosphatases reveals significant differences in the conformation of the L5 loop connecting strands 4 and 5. The extreme thermostability of PhAcP can be attributed to an extensive ion-pair network consisting of 13 charge residues on the beta sheet of the protein. The reduced catalytic efficiency of PhAcP at 25 degrees C may be due to a less flexible active-site residue, Arg20, which forms a salt bridge to the C-terminal carboxyl group. New insights into catalysis were gained by docking acetyl phosphate to the active site of PhAcP.

Acid Anhydride Hydrolases↗

PubMed2005

Interaction of the E2 and E3 components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Use of a truncated protein domain in NMR spectroscopy.

A (15)N-labelled peripheral-subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2p) and the dimer of a solubilized interface domain (E3int) derived from the dihydrolipoyl dehydrogenase (E3) were used to investigate the basis of the interaction of E2p with E3 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Thirteen of the 55 amino acids in the PSBD show significant changes in either or both of the (15)N and (1)H amide chemical shifts when the PSBD forms a 1 : 1 complex with E3int. All of the 13 amino acids reside near the N-terminus of helix I of PSBD or in the loop region between helix II and helix III. (15)N backbone dynamics experiments on PSBD indicate that the structured region extends from Val129 to Ala168, with limited structure present in residues Asn126 to Arg128. The presence of structure in the region before helix I was confirmed by a refinement of the NMR structure of uncomplexed PSBD. Comparison of the crystal structure of the PSBD bound to E3 with the solution structure of uncomplexed PSBD described here indicates that the PSBD undergoes almost no conformational change upon binding to E3. These studies exemplify and validate the novel use of a solubilized, truncated protein domain in overcoming the limitations of high molecular mass on NMR spectroscopy.

Geobacillus stearothermophilus↗

PubMed2004

Solution structure of the Kaposi's sarcoma-associated herpesvirus K3 N-terminal domain reveals a Novel E2-binding C4HC3-type RING domain.

RING domains are found in a large number of eukaryotic proteins. Most function as E3 ubiquitin-protein ligases, catalyzing the terminal step in the ubiquitination process. Structurally, these domains have been characterized as binding two zinc ions in a stable cross-brace motif. The tumorigenic human gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus encodes a ubiquitin-protein ligase termed K3, which functions as an immune evasion molecule by ubiquitinating major histocompatibility complex class I. K3 possesses at its N terminus a domain related to cellular RING domains but with an altered zinc ligand arrangement. This domain was initially characterized as a plant homeodomain, a structure not previously known to function as an E3. Here, it is conclusively demonstrated that the K3 N-terminal domain is a variant member of the RING domain family and not a plant homeodomain. The domain is found to interact with the cellular ubiquitin-conjugating enzymes UbcH5A to -C and UbcH13, which dock to the equivalent surface as on classical cellular RING domains. Interaction with UbcH13 suggests a possible role for K3 in catalyzing Lys(63)-linked ubiquitination.

Amino Acid Sequence↗

PubMed2004

Crystallization and preliminary crystallographic analysis of an acylphosphatase from the hyperthermophilic archaeon Pyrococcus horikoshii.

Acylphosphatases catalyse the hydrolysis of the carboxyl phosphate bond in metabolites such as acetyl phosphate, 1,3-bisphosphoglycerate, succinoyl phosphate and carbamoyl phosphate. In this study, acylphosphatase (91 residues) from the hyperthermophilic archaeon Pyrococcus horikoshii has been cloned, overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method using sodium formate as a precipitant at 289 K. The crystals belong to space group P3(2)21, with unit-cell parameters a = b = 85.65, c = 75.51 A. The asymmetric unit contains two molecules of acylphosphatase, with a corresponding crystal volume per protein weight of 3.9 A Da(-1) and a solvent content of 68.6%. A data set diffracting to 1.6 A resolution was collected from a single crystal at 100 K.

Acid Anhydride Hydrolases↗

PubMed2004

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding.

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.

Alanine↗

PubMed2004

Crystallization and preliminary crystallographic analysis of a novel orange fluorescent protein from the Cnidaria tube anemone Cerianthus sp.

A novel orange fluorescent protein, with excitation and emission maxima at 548 and 565 nm, respectively, from the Cnidaria tube anemone Cerianthus sp. has been cloned and overexpressed in Escherichia coli. The orange fluorescent protein has been crystallized by the sitting-drop vapour-diffusion method at 290 K using polyethylene glycol 3350 as a precipitant. A complete set of diffraction data was collected to 2.0 A resolution at 100 K. The crystals belong to the space group R3, with hexagonal unit-cell parameters a = b = 216.947, c = 51.839 A. There are four protein molecules in the asymmetric unit, giving a Matthews coefficient of 2.3 A(3) Da(-1) and a solvent content of 47%.

Animals↗

PubMed2003

The structure of the AXH domain of spinocerebellar ataxin-1.

Spinocerebellar ataxia type 1 is a late-onset neurodegenerative disease caused by the expansion of a CAG triplet repeat in the SCA1 gene. This results in the lengthening of a polyglutamine tract in the gene product ataxin-1. This produces a toxic gain of function that results in specific neuronal death. A region in ataxin-1, the AXH domain, exhibits significant sequence similarity to the transcription factor HBP1. This region of the protein has been implicated in RNA binding and self-association. We have determined the crystal structure of the AXH domain of ataxin-1. The AXH domain is dimeric and contains an OB-fold, a structural motif found in many oligonucleotide-binding proteins, supporting its proposed role in RNA binding. By structure comparison with other proteins that contain an OB-fold, a putative RNA-binding site has been identified. We also identified a cluster of charged surface residues that are well conserved among AXH domains. These residues may constitute a second ligand-binding surface, suggesting that all AXH domains interact with a common yet unidentified partner.

Amino Acid Sequence↗

PubMed2003

Crystal structure of a superstable mutant of human p53 core domain. Insights into the mechanism of rescuing oncogenic mutations.

Most of the cancer-associated mutations in the tumor suppressor p53 map to its DNA-binding core domain. Many of them inactivate p53 by decreasing its thermodynamic stability. We have previously designed the superstable quadruple mutant M133L/V203A/N239Y/N268D containing the second-site suppressor mutations N239Y and N268D, which specifically restore activity and stability in several oncogenic mutants. Here we present the x-ray structure of this quadruple mutant at 1.9 A resolution, which was solved in a new crystal form in the absence of DNA. This structure reveals that the four point mutations cause only small local structural changes, whereas the overall structure of the central beta-sandwich and the DNA-binding surface is conserved. The suppressor mutation N268D results in an altered hydrogen bond pattern connecting strands S1 and S10, thus bridging the two sheets of the beta-sandwich scaffold in an energetically more favorable way. The second suppressor mutation N239Y, which is located in close proximity to the DNA-binding surface in loop L3, seems to reduce the plasticity of the structure in large parts of loop L3 as indicated by decreased crystallographic temperature factors. The same is observed for residues in the vicinity of the N268D substitution. This increase in rigidity provides the structural basis for the increase in thermostability and an understanding how N268D and N239Y rescue some of the common cancer mutants.

Amino Acid Sequence↗