Biomedical subjects

Martin Högbom

Publications and source records attributed to Martin Högbom.

10 recordsLinked to original sources

PubMed2005

Structural mechanics of the pH-dependent activity of beta-carbonic anhydrase from Mycobacterium tuberculosis.

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. Carbonic anhydrases are divided into at least three classes and are believed to share a zinc-hydroxide mechanism for carbon dioxide hydration. beta-carbonic anhydrases are broadly spread among the domains of life, and existing structures from different organisms show two distinct active site setups, one with three protein coordinations to the zinc (accessible) and the other with four (blocked). The latter is believed to be inconsistent with the zinc-hydroxide mechanism. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta-carbonic anhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. We have recently solved the structure of this protein, which was a dimeric protein with a blocked active site. Here we present the structure of the thiocyanate complexed protein in a different crystal form. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta-carbonic anhydrase can switch between the two states. A pH-dependent dimer to tetramer equilibrium was also demonstrated by dynamic light scattering measurements. The data presented here, therefore, suggest a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.

Binding Sites↗

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

Structure and function of carbonic anhydrases from Mycobacterium tuberculosis.

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the alpha-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three beta-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 A, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation.

Amino Acid Sequence↗

PubMed2004

The radical site in chlamydial ribonucleotide reductase defines a new R2 subclass.

Ribonucleotide reductase (RNR) synthesizes the deoxyribonucleotides for DNA synthesis. The R2 protein of normal class I ribonucleotide reductases contains a diiron site that produces a stable tyrosyl free radical, essential for enzymatic activity. Structural and electron paramagnetic resonance studies of R2 from Chlamydia trachomatis reveal a protein lacking a tyrosyl radical site. Instead, the protein yields an iron-coupled radical upon reconstitution. The coordinating structure of the diiron site is similar to that of diiron oxidases/monoxygenases and supports a role for this radical in the RNR mechanism. The specific ligand pattern in the C. trachomatis R2 metal site characterizes a new group of R2 proteins that so far has been found in eight organisms, three of which are human pathogens.

Amino Acid Sequence↗

PubMed2004

Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2.

The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior. The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site. In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains. We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography. The structures of the metal-free forms of wild-type C. ammoniagenes R2 and the mutant E. coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible. Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E. coli apo proteins as compared to wild-type apo R2. Further structural variations are revealed by the wild-type apo C. ammoniagenes R2 structure. The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues. Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.

Base Sequence↗

PubMed2004

A protein carboxylate coordinated oxo-centered tri-nuclear iron complex with possible implications for ferritin mineralization.

The crystal structure of an oxo-centered tri-nuclear iron complex formed on a protein surface is presented. The cluster forms when crystals of the class Ib ribonucleotide reductase R2 protein from Corynebacterium ammoniagenes are subjected to iron soaking. The tri-iron-oxo complex is coordinated by protein-derived carboxylate ligands arranged in a motif similar to the one found on the inner surface of ferritins and may mimic an early stage in the mineralization of iron in ferritins. In addition, the structure adds to the very limited data on protein-mineral interfaces.

Carboxylic Acids↗

PubMed2004

Crystals of the ribonucleotide reductase R2 protein from Chlamydia trachomatis obtained by heavy-atom co-crystallization.

Ribonucleotide reductases (RNRs) catalyse the conversion of ribonucleotides to deoxyribonucleotides, utilizing radical chemistry to carry out the reaction. Class I RNRs consist of R1 and R2 subunits: R1 contains the active site and R2 generates and stores a stable tyrosyl radical. The conserved tyrosine where the radical is stored until needed in R1 has previously been believed to be an absolute requirement for R2 activity. The Chlamydia trachomatis R2 lacks this tyrosine and a phenylalanine is present in its place, but the protein is still active. Here, the crystallization of C. trachomatis R2 is described. A heavy-atom co-crystallization approach was used to obtain crystals. Hopefully, the C. trachomatis R2 structure will provide key clues as to how this enzyme is able to function while lacking the features that have previously been believed to be essential for activity.

Binding Sites↗

PubMed2003

Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-A x-ray data.

The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122(*)). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-A x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122(*) obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography.

Crystallography, X-Ray↗

PubMed2003

Structural basis for recognition by an in vitro evolved affibody.

The broad binding repertoire of antibodies has permitted their use in a wide range of applications. However, some uses of antibodies are precluded due to limitations in the efficiency of antibody generation. In vitro evolved binding proteins, selected from combinatorial libraries generated around various alternative structural scaffolds, are promising alternatives to antibodies. We have solved the crystal structure of a complex of an all alpha-helical in vitro selected binding protein (affibody) bound to protein Z, an IgG Fc-binding domain derived from staphylococcal protein A. The structure of the complex reveals an extended and complementary binding surface with similar properties to protein-antibody interactions. The surface region of protein Z recognized by the affibody is strikingly similar to the one used for IgG(1) Fc binding, suggesting that this surface contains potential hot-spots for binding. The implications of the selected affibody binding-mode for its application as a universal binding protein are discussed.

Amino Acid Sequence↗

PubMed2002

Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes.

Ribonucleotide reductase (RNR) is the enzyme performing de novo production of the four deoxyribonucleotides needed for DNA synthesis. All mammals as well as some prokaryotes express the class I enzyme which is an alpha(2)beta(2) protein. The smaller of the homodimers, denoted R2, contains a di-iron carboxylate site which, upon reaction with molecular oxygen, generates a stable tyrosyl radical needed for catalysis. The three-dimensional structure of the oxidized class Ib RNR R2 from Corynebacterium ammoniagenes has been determined at 1.85 A resolution and refined to an R-value of 15.8% (R(free) = 21.3%). In addition, structures of both the reduced iron-containing, and manganese-substituted protein have been solved. The C. ammoniagenes R2 has been proposed to be manganese-dependent. The present structure provides evidence that manganese is not oxidized by the protein, in agreement with recent biochemical data, and that no obvious structural abnormalities are seen in the oxidized and reduced iron-containing forms, giving further support that the protein is indeed an iron-dependent RNR R2. The di-manganese structure also provides an explanation for the magnetic properties of this site. The structure of the oxidized C. ammoniagenes R2 also reveals an additional water molecule bridging the radical and the iron site, which has not previously been seen in any other R2 structure and which might have important mechanistic implications.

Amino Acid Sequence↗