Luis Alfonso Martínez-Cruz

Correlation between Gene Expression and GO Semantic Similarity

This research analyzes some aspects of the relationship between gene expression, gene function, and gene annotation. Many recent studies are implicitly based on the assumption that gene products that are biologically and functionally related would maintain this similarity both in their expression profiles as well as in their Gene Ontology (GO) annotation. We analyze how accurate this assumption proves to be using real publicly available data. We also aim to validate a measure of semantic similarity for GO annotation. We use the Pearson correlation coefficient and its absolute value as a measure of similarity between expression profiles of gene products. We explore a number of semantic similarity measures (Resnik, Jiang, and Lin) and compute the similarity between gene products annotated using the GO. Finally, we compute correlation coefficients to compare gene expression similarity against GO semantic similarity. Our results suggest that the Resnik similarity measure outperforms the others and seems better suited for use in Gene Ontology. We also deduce that there seems to be correlation between semantic similarity in the GO annotation and gene expression for the three GO ontologies. We show that this correlation is negligible up to a certain semantic similarity value; then, for higher similarity values, the relationship trend becomes almost linear. These results can be used to augment the knowledge provided by clustering algorithms and in the development of bioinformatic tools for finding and characterizing gene products.

Crystal structure of the β-glycosidase from the hyperthermophile Thermosphaera aggregans: Insights into its activity and thermostability

The glycosyl hydrolases are an important group of enzymes that are responsible for cleaving a range of biologically significant carbohydrate compounds. Structural information on these enzymes has provided useful information on their molecular basis for the functional variations, while the characterization of the structural features that account for the high thermostability of proteins is of great scientific and biotechnological interest. To these ends we have determined the crystal structure of the β‐glycosidase from a hyperthermophilic archeon Thermosphaera aggregans. The structure is a (β/α)8 barrel (TIM‐barrel), as seen in other glycosyl hydrolase family 1 members, and forms a tetramer. Inspection of the active site and the surrounding area reveals two catalytic glutamate residues consistent with the retaining mechanism and the surrounding polar and aromatic residues consistent with a monosaccharide binding site. Comparison of this structure with its mesophilic counterparts implicates a variety of structural features that could contribute to the thermostability. These include an increased number of surface ion pairs, an increased number of internal water molecules and a decreased surface area upon forming an oligomeric quaternary structure.

CBS domains: Ligand binding sites and conformational variability.

Cystathionine β-synthase (CBS) domains or CBS motifs are conserved structural domains that are present in thousands of non functionally-related proteins from all kingdoms of life. Their importance is underlined by the range of hereditary diseases associated with mutations in their amino acid sequence. CBS motifs associate in pairs referred to as Bateman modules. In contrast with initial assumptions, it is now well documented that CBS motifs and/or Bateman modules may suffer conformational changes upon binding of adenosine derivatives, metal ions or nucleic acids. The degree and direction of these structural changes depend on the type of ligand, the intrinsic features of the binding sites and the association manner of the Bateman modules. This review aims to provide a summary of the current knowledge on the structural basis of ligand recognition and on the structural effects caused by these ligands in CBS domain containing proteins.

Structural basis of regulation and oligomerization of human cystathionine β-synthase, the central enzyme of transsulfuration

Significance Cystathionine β-synthase (CBS), the pivotal enzyme of the transsulfuration pathway, regulates the flux through the pathway to yield compounds such as cysteine, glutathione, taurine, and H2S that control the cellular redox status and signaling. Our crystal structures of the full-length wild-type and D444N mutant human CBS enzymes show a unique arrangement of the regulatory CBS motifs, thus making it possible to infer how the enzyme is stimulated by its allosteric activator S-adenosyl-L-methionine and how native tetramers are formed. The structure will allow modeling of numerous mutations causing inherited homocystinuria and the design of compounds modulating CBS activity. Cystathionine β-synthase (CBS) controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H2S. CBS condenses serine and homocysteine to cystathionine with the help of three cofactors, heme, pyridoxal-5′-phosphate, and S-adenosyl-l-methionine. Inherited deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur metabolism. We present the structure of the human enzyme, discuss the unique arrangement of the CBS domains in the C-terminal region, and propose how they interact with the catalytic core of the complementary subunit to regulate access to the catalytic site. This arrangement clearly contrasts with other proteins containing the CBS domain including the recent Drosophila melanogaster CBS structure. The absence of large conformational changes and the crystal structure of the partially activated pathogenic D444N mutant suggest that the rotation of CBS motifs and relaxation of loops delineating the entrance to the catalytic site represent the most likely molecular mechanism of CBS activation by S-adenosyl-l-methionine. Moreover, our data suggest how tetramers, the native quaternary structure of the mammalian CBS enzymes, are formed. Because of its central role in transsulfuration, redox status, and H2S biogenesis, CBS represents a very attractive therapeutic target. The availability of the structure will help us understand the pathogenicity of the numerous missense mutations causing inherited homocystinuria and will allow the rational design of compounds modulating CBS activity.

Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S-adenosylmethionine

Significance Cystathionine β-synthase (CBS), the pivotal enzyme of the transsulfuration pathway, regulates flux through the pathway to yield compounds, such as cysteine, glutathione, taurine, and H2S, that control cellular redox status and signaling. Our crystal structure of an engineered human CBS construct bound to S-adenosylmethionine (AdoMet) reveals the unique binding site of the allosteric activator and the architecture of the human CBS enzyme in its activated conformation. Together with the basal conformation that we reported earlier, these structures unravel the molecular mechanism of human CBS activation by AdoMet. Current knowledge will allow for modeling of numerous pathogenic mutations causing inherited homocystinuria and for design of compounds modulating CBS activity. Cystathionine β-synthase (CBS) is a heme-dependent and pyridoxal-5′-phosphate–dependent protein that controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H2S. Deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur amino acid metabolism. In contrast to CBSs from lower organisms, human CBS (hCBS) is allosterically activated by S-adenosylmethionine (AdoMet), which binds to the regulatory domain and triggers a conformational change that allows the protein to progress from the basal toward the activated state. The structural basis of the underlying molecular mechanism has remained elusive so far. Here, we present the structure of hCBS with bound AdoMet, revealing the activated conformation of the human enzyme. Binding of AdoMet triggers a conformational change in the Bateman module of the regulatory domain that favors its association with a Bateman module of the complementary subunit to form an antiparallel CBS module. Such an arrangement is very similar to that found in the constitutively activated insect CBS. In the presence of AdoMet, the autoinhibition exerted by the regulatory region is eliminated, allowing for improved access of substrates to the catalytic pocket. Based on the availability of both the basal and the activated structures, we discuss the mechanism of hCBS activation by AdoMet and the properties of the AdoMet binding site, as well as the responsiveness of the enzyme to its allosteric regulator. The structure described herein paves the way for the rational design of compounds modulating hCBS activity and thus transsulfuration, redox status, and H2S biogenesis.

Crystal Structure of MJ1247 Protein from M. jannaschii at 2.0 Å Resolution Infers a Molecular Function of 3-Hexulose-6-Phosphate Isomerase

The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5′-mer U-rich RNA stretches through the solvent exposed side of its β-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain.

Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2.

Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine β-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

The CBS domain protein MJ0729 of Methanocaldococcus jannaschii binds DNA

The cystathionine beta‐synthase (CBS) domains function as regulatory motifs in several proteins. Elucidating how CBS domains exactly work is relevant because several genetic human diseases have been associated with mutations in those motifs. Here, we show, for the first time, that a CBS domain binds calf‐thymus DNA and E‐boxes recognized by transcription factors. We have carried out the DNA‐binding characterization of the CBS domain protein MJ0729 from Methanocaldococcus jannaschii by biochemical and spectroscopic techniques. Binding induces conformational changes in the protein, and involves the sole tryptophan residue. The apparent dissociation constant for the E‐boxes is ∼10 μM. These results suggest that CBS domains might interact with DNA.