Biomedical subjects

L Joshua-Tor

Publications and source records attributed to L Joshua-Tor.

16 recordsLinked to original sources

PubMed2001

Selective chemical inactivation of AAA proteins reveals distinct functions of proteasomal ATPases.

BACKGROUND: The 26S proteasome contains six highly related ATPases of the AAA family. We have developed a strategy that allows selective inhibition of individual proteasomal ATPases in the intact proteasome. Mutation of a threonine in the active site of Sug1/Rpt6 or Sug2/Rpt4 to a cysteine sensitizes these proteins to inactivation through alkylation by the sulfhydryl modifying agent NEM. Using this technique the individual contributions of Sug1 and Sug2 to proteasome function can be assessed. RESULTS: We show that both Sug1 and Sug2 can be selectively alkylated by NEM in the context of the intact 26S complex and as predicted by structural modeling, this inactivates the ATPase function. Using this technique we demonstrate that both Sug 1 and 2 are required for full peptidase activity of the proteasome and that their functions are not redundant. Kinetic analysis suggests that Sug2 may have an important role in maintaining the interaction between the 19S regulatory complex and the 20S proteasome. In contrast, inhibition of Sug1 apparently decreases peptidase activity of the 26S proteasome by another mechanism. CONCLUSIONS: These results describe a useful technique for the selective inactivation of AAA proteins. In addition, they also demonstrate that the functions of two related proteasomal AAA proteins are not redundant, suggesting differential roles of proteasomal AAA proteins in protein degradation.

Adenosine Triphosphatases↗

PubMed2000

Crystal structure of carboxypeptidase A complexed with D-cysteine at 1.75 A - inhibitor-induced conformational changes.

D-Cysteine differs from the antiarthritis drug D-penicillamine by only two methyl groups on the beta-carbon yet inhibits carboxypeptidase A (CPD) by a distinct mechanism: D-cysteine binds tightly to the active site zinc, while D-penicillamine catalyzes metal removal. To investigate the structural basis for this difference, we solved the crystal structure of carboxypeptidase A complexed with D-cysteine (D-Cys) at 1.75-A resolution. D-Cys binds the active site zinc with a sulfur ligand and forms additional interactions with surrounding side chains of the enzyme. The structure explains the difference in potency between D-Cys and L-Cys and provides insight into the mechanism of D-penicillamine inhibition. D-Cys binding induces a concerted motion of the side chains around the zinc ion, similar to that found in other carboxypeptidase-inhibitor crystal structures and along a limited path. Analysis of concerted motions of CPD and CPD-inhibitor crystal structures reveals a clustering of these structures into distinct groups. Using the restricted conformational flexibility of a drug target in this type of analysis could greatly enhance efficiency in drug design.

Binding Sites↗

PubMed2000

Conformational substates in different crystal forms of the photoactive yellow protein--correlation with theoretical and experimental flexibility.

The conformational changes during the photocycle of the photoactive yellow protein have been the subject of many recent studies. Spectroscopic measurements have shown that the photocycle also occurs in a crystalline environment, and this has been the basis for subsequent Laue diffraction and cryocrystallographic studies. These studies have shown that conformational changes during the photocycle are limited to the chromophore and its immediate environment. However, spectroscopic studies suggest the presence of large conformational changes in the protein. Here, we address this apparent discrepancy in two ways. First, we obtain a description of large concerted motions in the ground state of the yellow protein from NMR data and theoretical calculations. Second, we describe the high-resolution structure of the yellow protein crystallized in a different space group. The structure of the yellow protein differs significantly between the two crystal forms. We show that these differences can be used to obtain a description of the flexibility of the protein that is consistent with the motions observed in solution.

Bacterial Proteins↗

PubMed2000

Crystal structure of the DNA binding domain of the replication initiation protein E1 from papillomavirus.

Papillomaviral infection causes both benign and malignant lesions and is a necessary cause of cervical carcinoma. Replication of this virus requires the replication initiation proteins E1 and E2, which bind cooperatively at the origin of replication (ori) as an (E1)2-(E2)2-DNA complex. This is a precursor to larger E1 complexes that distort and unwind the ori. We present the crystal structure of the E1 DNA binding domain refined to 1.9 A resolution. Residues critical for DNA binding are located on an extended loop and an alpha helix. We identify the E1 dimerization surface by selective mutations at an E1/E1 interface observed in the crystal and propose a model for the (E1)2-DNA complex. These and other observations suggest how the E1 DNA binding domain orchestrates assembly of the hexameric helicase on the ori.

Amino Acid Sequence↗

PubMed1999

Crystal structure of Apaf-1 caspase recruitment domain: an alpha-helical Greek key fold for apoptotic signaling.

The caspase recruitment domain (CARD) of Apaf-1 binds to the CARD of caspase-9 to trigger a proteolytic cascade that leads to apoptotic cell death. We report the crystal structure of the Apaf-1 CARD at 1. 3 A resolution, solved in a two-element multiwavelength anomalous dispersion (MAD) X-ray diffraction experiment. This CARD adopts a six-helix bundle fold with Greek key topology surrounding an extensive hydrophobic core. This fold, which we call the "death fold", is found in other domains that mediate interactions in apoptotic signaling despite very low sequence identity. From a structure-based alignment, we identify conserved patterns that characterize the death fold and its subclasses. Like the Ig-fold, it provides a rigid structural scaffold upon which diverse recognition surfaces are assembled.

Amino Acid Motifs↗

PubMed1999

A structural snapshot of base-pair opening in DNA.

The response of double-helical DNA to torsional stress may be a driving force for many processes acting on DNA. The 1.55-A crystal structure of a duplex DNA oligonucleotide d(CCAGGCCTGG)(2) with an engineered crosslink in the minor groove between the central guanine bases depicts how the duplex can accommodate such torsional stress. We have captured in the same crystal two rather different conformational states. One duplex contains a strained crosslink that is stabilized by calcium ion binding in the major groove, directly opposite the crosslink. For the other duplex, the strain in the crosslink is relieved through partial rupture of a base pair and partial extrusion of a cytosine accompanied by helix bending. The sequence used is the target sequence for the HaeIII methylase, and this partially flipped cytosine is the same nucleotide targeted for extrusion by the enzyme. Molecular dynamics simulations of these structures show an increased mobility for the partially flipped-out cytosine.

Base Pairing↗

PubMed1999

Crystal structure of human bleomycin hydrolase, a self-compartmentalizing cysteine protease.

BACKGROUND: Bleomycin hydrolase (BH) is a cysteine protease that is found in all tissues in mammals as well as in many other eukaryotes and prokaryotes. Although its conserved cellular function is as yet unknown, human bleomycin hydrolase (hBH) has clinical significance in that it is thought to be the major cause of tumor cell resistance to bleomycin chemotherapy. In addition, it has been reported that an allelic variant of hBH is genetically linked to Alzheimer's disease. RESULTS: We have determined the crystal structures of wild-type hBH and of a mutant form of the enzyme. The overall structure is very similar to that of the previously determined yeast homolog, however, there is a striking difference in the charge distribution. The central channel, which has a strong positive electrostatic potential in the yeast protein, is slightly negative in hBH. We have determined that hBH does not have the DNA-binding activity of the yeast protein and that the enzyme is localized to the cytoplasm. CONCLUSIONS: The difference in charge distribution between the yeast and human BH enzymes is most likely responsible for the difference in DNA-binding activity. Nevertheless, the C-terminal autoprocessing activity and the role of the C terminus as a determinant for peptidase activity are conserved between the yeast and human forms. The structure of hBH suggests that the putative Alzheimer's disease linked variation does not directly alter the intrinsic peptidase activity. Rather, the position of the mutation suggests that it could affect interactions with another protein, which may modulate peptidase activity through repositioning of the C terminus.

Alzheimer Disease↗

PubMed1998

Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum.

The nitrogenase iron (Fe) protein performs multiple functions during biological nitrogen fixation, including mediating the mechanistically essential coupling between ATP hydrolysis and electron transfer to the nitrogenase molybdenum iron (MoFe) protein during substrate reduction, and participating in the biosynthesis and insertion of the FeMo-cofactor into the MoFe-protein. To establish a structural framework for addressing the diverse functions of Fe-protein, crystal structures of the Fe-proteins from Azotobacter vinelandii and Clostridium pasteurianum have been determined at resolutions of 2.2 A and 1.93 A, respectively. These two Fe-proteins are among the more diverse in terms of amino acid sequence and biochemical properties. As described initially for the A. vinelandii Fe-protein in a different crystal form at 2.9 A resolution, each subunit of the dimeric Fe-protein adopts a polypeptide fold related to other mononucleotide-binding proteins such as G-proteins, with the two subunits bridged by a 4Fe:4S cluster. The overall similarities in the subunit fold and dimer arrangement observed in the structures of the A. vinelandii and C. pasteurianum Fe-proteins indicate that they are representative of the conformation of free Fe-protein that is not in complex with nucleotide or the MoFe-protein. Residues in the cluster and nucleotide-binding sites are linked by a network of conserved hydrogen bonds, salt-bridges and water molecules that may conformationally couple these regions. Significant variability is observed in localized regions, especially near the 4Fe:4S cluster and the MoFe-protein binding surface, that change conformation upon formation of the ADP.AlF4- stabilized complex with the MoFe-protein. A core of 140 conserved residues is identified in an alignment of 59 Fe-protein sequences that may be useful for the identification of homologous proteins with functions comparable to that of Fe-protein in non-nitrogen fixing systems.

Amino Acid Sequence↗

PubMed1998

The unusual active site of Gal6/bleomycin hydrolase can act as a carboxypeptidase, aminopeptidase, and peptide ligase.

The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin. The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome. In Gal6 the C termini lie in the active site clefts. We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase. The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate. We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin.

Amino Acid Sequence↗

PubMed1998

Essential dynamics from NMR clusters: dynamic properties of the Myb DNA-binding domain and a hinge-bending enhancing variant.

Application of the "essential dynamics" method to the NMR cluster of structures for the R2R3 DNA-binding domain of the mouse c-Myb transcriptional activator is described. Using this method, large concerted fluctuations of atoms are extracted showing a hinge-bending motion between the two (R2 and R3) Myb repeats on the basis of NMR data alone. Molecular dynamics simulation of the same protein allowed quantitative comparison of the large concerted motions calculated from experimental and theoretical data, showing a significant degree of similarity. Detailed inspection of the motions reveals a conserved proline that plays a key role in determining hinge flexibility. The proline-to-alanine mutation at this position, which has previously been characterized biochemically, was subjected to molecular dynamics and subsequent essential dynamics analysis. The hinge-bending motion between the two repeats was found to be enhanced for the mutant. The approach described should have general applications, predicting the effect of mutations on protein dynamic properties of other proteins.

Binding Sites↗

PubMed1995

Crystal structure of a conserved protease that binds DNA: the bleomycin hydrolase, Gal6.

Bleomycin hydrolase is a cysteine protease that hydrolyzes the anticancer drug bleomycin. The homolog in yeast, Gal6, has recently been identified and found to bind DNA and to act as a repressor in the Gal4 regulatory system. The crystal structure of Gal6 at 2.2 A resolution reveals a hexameric structure with a prominent central channel. The papain-like active sites are situated within the central channel, in a manner resembling the organization of active sites in the proteasome. The Gal6 channel is lined with 60 lysine residues from the six subunits, suggesting a role in DNA binding. The carboxyl-terminal arm of Gal6 extends into the active site cleft and may serve a regulatory function. Rather than each residing in distinct, separable domains, the protease and DNA-binding activities appear structurally intertwined in the hexamer, implying a coupling of these two activities.

Amino Acid Sequence↗

PubMed1992

Three-dimensional structures of bulge-containing DNA fragments.

The three-dimensional structure of a DNA tridecamer d(CGCAGAATTCGCG)2 containing bulged adenine bases was determined by single crystal X-ray diffraction methods, at 120 K, to 2.6 A resolution. The structure is a B-DNA type double helix with a single duplex in the asymmetric unit. One of the bulged adenine bases loops out from the double helix, while the other stacks in to it. This is in contrast to our preliminary finding, which indicated that both adenine bases were looped out. This revised model was confirmed by the use of a covalently bound heavy-atom derivative. The conformation of the looped-out bulge hardly disrupts base stacking interactions of the bases flanking it. This is achieved by the backbone making a "loop-the-loop" curve with the extra adenine flipping over with respect to the other nucleotides in the strand. The looped-out base intercalates into the stacked-in bulge site of a symmetrically related duplex. The looped-out and stacked-in bases form an A.A reversed Hoogsteen base-pair that stacks between the surrounding base-pairs, thus stabilizing both bulges. The double helix is frayed at one end with the two "melted" bases participating in intermolecular interactions. A related structure, of the same tridecamer, after soaking the crystals with proflavin, was determined to 3.2 A resolution. The main features of this B-DNA duplex are basically similar to the native tridecamer but differ in detail especially in the conformation of the bulged-out base. Accommodation of a large perturbation such as that described here with minimal disruption of the double helix shows both the flexibility and resiliency of the DNA molecule.

Base Composition↗

PubMed1992

X-ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, have been determined by X-ray crystallography to a resolution of 1.8 A. Crystals of this rubredoxin grow in space group P2(1)2(1)2(1) with room temperature cell dimensions a = 34.6 A, b = 35.5 A, and c = 44.4 A. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X-PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 A and 2.06 degrees, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 A and in bond angles of 1.95 degrees. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen-bonding network in the beta-sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino-terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.

Amino Acid Sequence↗

PubMed1992

Comparison of the X-ray structure of native rubredoxin from Pyrococcus furiosus with the NMR structure of the zinc-substituted protein.

The three-dimensional X-ray structures of the oxidized and reduced forms of rubredoxin from Pyrococcus furiosus, determined at -161 degrees C, and the NMR structure of the zinc-substituted protein, determined in solution at 45 degrees C, are compared. The NMR and X-ray structures, which were determined independently, are very similar and lead to similar conclusions regarding the interactions that confer hyperthermostability.

Amino Acid Sequence↗

PubMed1992

A structural model for sequence-specific proflavin-DNA interactions during in vitro frameshift mutagenesis.

Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5' pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it. Because the most frequent mutations are opposite Pu in the template sequence 5' Py Pu 3', a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin. The model seeks to rationalize the roles of the 5' pyrimidine and proflavin in facilitating the bypass. Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5' pyrimidine; 4) hydrogen bonding with the phosphate backbone. These models point to the possibility that a number of proflavin-DNA interactions may be involved. In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.

Base Sequence↗

PubMed1988

The three-dimensional structure of a DNA duplex containing looped-out bases.

Unpaired bases in DNA have been assigned a possible role in the mechanism of frameshift mutagenesis in sequences with repeated base pairs. They also occur in quasipalindromic DNA sequences, which have been implicated in mutagenesis where there are no repeated base pairs, through the formation of single-stranded hairpin loops. The conformation of unpaired bases in DNA has been the subject of numerous thermodynamic as well as high resolution NMR (nuclear magnetic resonance) studies (reviewed in ref. 4). The NMR studies in solution have shown that the duplex of the tridecamer DNA fragment d(CGCAGAATTCGCG) remains intact, and that the unpaired adenosines are stacked into the duplex. Having crystallized this oligonucleotide and determined its structure, we find its conformation in the crystal is close to that of a B-DNA duplex, with the two additional adenosines looped out from the double helix and causing little disruption of the rest of the structure.

Base Composition↗