Biomedical subjects

Pär Nordlund

Publications and source records attributed to Pär Nordlund.

At least 19 recordsLinked to original sources

PubMed2006

Crystal structure of human inosine triphosphatase. Substrate binding and implication of the inosine triphosphatase deficiency mutation P32T.

Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide levels. It breaks down inosine and xanthine nucleotides generated by deamination of purine bases. Its enzymatic action prevents accumulation of ITP and reduces the risk of incorporation of potentially mutagenic inosine nucleotides into nucleic acids. Here we describe the crystal structure of human ITPA in complex with its prime substrate ITP, as well as the apoenzyme at 2.8 and 1.1A, respectively. These structures show for the first time the site of substrate and Mg2+ coordination as well as the conformational changes accompanying substrate binding in this class of enzymes. Enzyme substrate interactions induce an extensive closure of the nucleotide binding grove, resulting in tight interactions with the base that explain the high substrate specificity of ITPA for inosine and xanthine over the canonical nucleotides. One of the dimer contact sites is made up by a loop that is involved in coordinating the metal ion in the active site. We predict that the ITPA deficiency mutation P32T leads to a shift of this loop that results in a disturbed affinity for nucleotides and/or a reduced catalytic activity in both monomers of the physiological dimer.

Amino Acid Substitution↗

PubMed2006

An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries.

The implementation of generic and efficient technologies for the production of recombinant eukaryotic proteins remains an outstanding challenge in structural genomics programs. We have recently developed a new method for rapid identification of soluble protein expression in E. coli, the colony filtration blot (CoFi blot). In this study, the CoFi blot was used to screen libraries where the N-terminal translation start point was randomized. To investigate the efficiency of this strategy, we have attributed a large number of proteins to this process. In a set of 32 mammalian proteins, we were able to double the success rate (from 34 to 68%) of producing soluble and readily purifiable proteins in E. coli. Most of the selected constructs had their N-termini close to predicted domain borders and the method therefore provides a mean for experimental "domain foot printing." Surprisingly, for most of the targets, we also observed expressing constructs that were close to full-length. In summary this strategy constitutes a generic and efficient method for producing mammalian proteins for structural and functional studies.

Cloning, Molecular↗

PubMed2006

Structures of the hydrolase domain of human 10-formyltetrahydrofolate dehydrogenase and its complex with a substrate analogue.

10-Formyltetrahydrofolate dehydrogenase is a ubiquitously expressed enzyme in the human body. It catalyses the formation of tetrahydrofolate and carbon dioxide from 10-formyltetrahydrofolate, thereby playing an important role in the human metabolism of one-carbon units. It is a two-domain protein in which the N-terminal domain hydrolyses 10-formyltetrahydrofolate into formate and tetrahydrofolate. The high-resolution crystal structure of the hydrolase domain from human 10-formyltetrahydrofolate dehydrogenase has been determined in the presence and absence of a substrate analogue. The structures reveal conformational changes of two loops upon ligand binding, while key active-site residues appear to be pre-organized for catalysis prior to substrate binding. Two water molecules in the structures mark the positions of key oxygen moieties in the catalytic reaction and reaction geometries are proposed based on the structural data.

Binding Sites↗

PubMed2006

Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination.

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.

Animals↗

PubMed2006

GRETA, a new multifermenter system for structural genomics and process optimization.

As part of the Structural Proteomics In Europe (SPINE) project, the automated multifermenter system GRETA has been developed with structural genomics as a major application. GRETA comprises 6-24 parallel fermentation chambers, each with individual control of fermentation parameters such as temperature, stirring, pH, dissolved oxygen concentration and feed profiles. Six human proteins were used to optimize the GRETA fermentation processes and to compare these processes with typical baffled-flask protocols used in structural genomics projects. The optimized GRETA processes allows several times more protein to be produced per litre of culture with limited manual intervention and constitutes a potentially useful alternative both for scale-up production in structural proteomic projects and for fermentation-process optimization.

Calibration↗

PubMed2006

Thermofluor-based high-throughput stability optimization of proteins for structural studies.

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.

Buffers↗

PubMed2006

Crystal structure of a divalent metal ion transporter CorA at 2.9 angstrom resolution.

CorA family members are ubiquitously distributed transporters of divalent metal cations and are considered to be the primary Mg2+ transporter of Bacteria and Archaea. We have determined a 2.9 angstrom resolution structure of CorA from Thermotoga maritima that reveals a pentameric cone-shaped protein. Two potential regulatory metal binding sites are found in the N-terminal domain that bind both Mg2+ and Co2+. The structure of CorA supports an efflux system involving dehydration and rehydration of divalent metal ions potentially mediated by a ring of conserved aspartate residues at the cytoplasmic entrance and a carbonyl funnel at the periplasmic side of the pore.

Amino Acid Sequence↗

PubMed2006

The crystal structure of a human PP2A phosphatase activator reveals a novel fold and highly conserved cleft implicated in protein-protein interactions.

Protein phosphatase 2A (PP2A) is a heterotrimeric Ser/Thr phosphatase that is involved in regulating a plethora of signaling pathways in the cell, making its regulation a critical part of the well being of the cell. For example, three of the non-catalytic PP2A subunits have been linked to carcinogenic events. Therefore, the molecular basis for the complicated protein-protein interaction pattern of PP2A and its regulators is of special interest. The PP2A phosphatase activator (PTPA) protein is highly conserved from humans to yeast. It is an activator of PP2A and has been shown to be essential for a fully functional PP2A, but its mechanism of activation is still not well defined. We have solved the crystal structure of human PTPA to 1.6A. It reveals a two-domain protein with a novel fold comprised of 13 alpha-helices. We have identified a highly conserved cleft as a potential region for interaction with peptide segments of other proteins. Binding studies with ATP and its analogs are not consistent with ATP being a cofactor/substrate for PTPA as had previously been proposed. The structure of PTPA can serve as a basis for structure-function studies directed at elucidating its mechanism as an activator of PP2A.

Adenosine Triphosphate↗

PubMed2006

Structure of the synthetase domain of human CTP synthetase, a target for anticancer therapy.

Cytidine triphosphate synthetase (CTPS) is a key enzyme in nucleic acid and phospholipid biosynthesis and its activity is increased in certain human cancers, making it a promising drug target. The crystal structure of the synthetase domain of human CTPS, which represents the first structure of a CTPS from an eukaryote, has been determined. The structure is homotetrameric and each active site is formed by three different subunits. Sulfate ions bound to the active sites indicate the positions of phosphate-binding sites for the substrates ATP and UTP and the feedback inhibitor CTP. Together with earlier structures of bacterial CTPS, the human CTPS structure provides an extended understanding of the structure-function relationship of CTPS-family members. The structure also serves as a basis for structure-based design of anti-proliferative inhibitors.

Amino Acid Sequence↗

PubMed2006

The structure of the RNA m5C methyltransferase YebU from Escherichia coli reveals a C-terminal RNA-recruiting PUA domain.

Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S rRNA, is the modification product of the methyltransferase (MTase) YebU (also called RsmF). YebU requires S-adenosyl-l-methionine (SAM) and methylates C1407 within assembled 30 S subunits, but not in naked 16 S rRNA or within tight-couple 70 S ribosomes. Here, we describe the three-dimensional structure of YebU determined by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including RsmB and PH1374. The C-terminal one-third of YebU contains a domain similar to that in pseudouridine synthases and archaeosine-specific transglycosylases (PUA-domain), which was not predicted by sequence alignments. Furthermore, YebU is predicted to contain extended regions of positive electrostatic potential that differ from other RNA-MTase structures, suggesting that YebU interacts with its RNA target in a different manner. Docking of YebU onto the 30 S subunit indicates that the PUA and MTase domains make several contacts with 16 S rRNA as well as with the ribosomal protein S12. The ribosomal protein interactions would explain why the assembled 30 S subunit, and not naked 16 S rRNA, is the preferred substrate for YebU.

Amino Acid Sequence↗

PubMed2006

Expression, purification, crystallization and preliminary diffraction studies of the mammalian DAG kinase homologue YegS from Escherichia coli.

yegS is a gene encoding a 32 kDa cytosolic protein with unknown function but with strong sequence homology to a family of structurally uncharacterized eukaryotic non-protein kinases: diacylglycerol kinases, sphingosine kinases and ceramide kinases. Here, the overexpression, crystallization and preliminary diffraction analysis of Escherichia coli YegS are reported. The crystals belong to space group P2(1), with unit-cell parameters a = 42.4, b = 166.1, c = 48.5 A, beta = 96.97 degrees. The presence of a dimer in the asymmetric unit was estimated to give a Matthews coefficient (VM) of 2.5 A3 Da(-1) and a solvent content of 50.8%(v/v). Single-wavelength diffraction data were collected to a resolution of 1.9 A using synchrotron radiation.

Crystallization↗

PubMed2006

A simple strategy towards membrane protein purification and crystallization.

A simple and cost-efficient detergent screening strategy has been developed, by which a number of detergents were screened for their efficiency to extract and purify the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, hence selecting the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1 ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. Large-scale protein purification and subsequent crystallization screening resulted in AmtB crystals diffracting to low resolution with three detergents. This strategy allows exclusion of detergents with the lowest probability in yielding protein crystals and selecting those with higher probability, hence, reducing the number of detergents to be screened prior to large-scale membrane protein purification and perhaps also crystallization.

Cation Transport Proteins↗

PubMed2006

Ribonucleotide reductases.

Ribonucleotide reductases (RNRs) transform RNA building blocks to DNA building blocks by catalyzing the substitution of the 2'OH-group of a ribonucleotide with a hydrogen by a mechanism involving protein radicals. Three classes of RNRs employ different mechanisms for the generation of the protein radical. Recent structural studies of members from each class have led to a deeper understanding of their catalytic mechanism and allosteric regulation by nucleoside triphosphates. The main emphasis of this review is on regulation of RNR at the molecular and cellular level. Conformational transitions induced by nucleotide binding determine the regulation of substrate specificity. An intricate interplay between gene activation, enzyme inhibition, and protein degradation regulates, together with the allosteric effects, enzyme activity and provides the appropriate amount of deoxynucleotides for DNA replication and repair. In spite of large differences in the amino acid sequences, basic structural features are remarkably similar and suggest a common evolutionary origin for the three classes.

Allosteric Regulation↗

PubMed2005

Reaction mechanism of deoxyribonucleotidase: a theoretical study.

The reaction mechanism of human deoxyribonucleotidase (dN) is studied using high-level quantum-chemical methods. dN catalyzes the dephosphorylation of deoxyribonucleoside monophosphates (dNMPs) to their nucleoside form in human cells. Large quantum models are employed (99 atoms) based on a recent X-ray crystal structure. The calculations support the proposed mechanism in which Asp41 performs a nucleophilic attack on the phosphate to form a phospho-enzyme intermediate. Asp43 acts in the first step as an acid, protonating the leaving nucleoside, and in the second step as a base, deprotonating the lytic water. No pentacoordinated intermediates could be located.

5'-Nucleotidase↗

PubMed2005

A high throughput method for the detection of metalloproteins on a microgram scale.

Proteins that bind transition metals make up a substantial portion of the proteome, and the identification of a metal cofactor in a protein can greatly facilitate its functional assignment and help place it in the context of known cellular pathways. Existing methods for the detection of metalloproteins generally consume large amounts of protein, require expensive equipment, or are very labor intensive, rendering them unsuitable for use in high throughput proteomic initiatives. Here we present a method for the identification of metalloproteins that contain iron, copper, manganese, cobalt, nickel, and/or zinc that is sensitive, quick, robust, inexpensive, and can be performed with standard laboratory equipment. The assay is based on a combination of chemiluminescence and colorimetric detection methods, it typically consumes only 10 microg of protein, and most common chemical components of protein solutions do not interfere with metal detection. Analysis of 52 protein samples was compared with the results from inductively coupled plasma-atomic emission spectrometry to verify the accuracy and sensitivity of the method. The assay is conducted in a 384-well format and requires about 3 h for completion, including a 2-h wait; so whole proteomes can be assayed for metal content in a matter of days.

Colorimetry↗

PubMed2005

An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins.

The recombinant expression of integral membrane proteins is considered a major challenge, and together with the crystallization step, the major hurdle toward routine structure determination of membrane proteins. Basic methodologies for high-throughput (HTP) expression optimization of soluble proteins have recently emerged, providing statistically significant success rates for producing such proteins. Experimental procedures for handling integral membrane proteins are generally more challenging, and there have been no previous comprehensive reports of HTP technology for membrane protein production. Here, we present a generic and integrated parallel HTP strategy for cloning and expression screening of membrane proteins in their detergent solubilized form. Based on this strategy, we provide overall success rates for membrane protein production in Escherichia coli, as well as initial benchmarking statistics of parameters such as expression vectors, strains, and solubilizing detergents. The technologies were applied to 49 E. coli integral membrane proteins with human homologs and revealed that 71% of these proteins could be produced at sufficient levels to allow milligram amounts of protein to be relatively easily purified, which is a significantly higher success rate than anticipated. We attribute the high success rate to the quality and robustness of the methodology used, and to introducing multiple parameters such as different vectors, strains, and detergents. The presented strategy demonstrates the usefulness of HTP technologies for membrane protein production, and the feasibility of large-scale programs for elucidation of structure and function of bacterial integral membrane proteins.

Chromatography, Gel↗

PubMed2005

Structural basis for substrate specificity of the human mitochondrial deoxyribonucleotidase.

The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3'-monophosphate, thymidine 5'-monophosphate, deoxyuridine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine 5'-monophosphate, uridine 2'-monophosphate, and the 5'-monophosphate of the nucleoside analog 3'-deoxy 2'3'-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme's specificity for the deoxyribo form of nucleoside 5'-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2' position of the sugar and cause an energetically unfavorable environment for the 2'-hydroxyl group of ribonucleoside 5'-monophosphates. The close binding of the 3'-hydroxyl group of nucleoside 5'-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme.

5'-Nucleotidase↗

PubMed2005

Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli.

The implementation of efficient technologies for the production of recombinant mammalian proteins remains an outstanding challenge in many structural and functional genomics programs. We have developed a new method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level. The colony filtration (CoFi) blot is very well suited to screen libraries, and in the present work we used it to screen a deletion mutagenesis library.

Cell Culture Techniques↗