Darío A. Estrin

When Galectins Recognize Glycans: From Biochemistry to Physiology and Back Again

In the past decade, increasing efforts have been devoted to the study of galectins, a family of evolutionarily conserved glycan-binding proteins with multifunctional properties. Galectins function, either intracellularly or extracellularly, as key biological mediators capable of monitoring changes occurring on the cell surface during fundamental biological processes such as cellular communication, inflammation, development, and differentiation. Their highly conserved structures, exquisite carbohydrate specificity, and ability to modulate a broad spectrum of biological processes have captivated a wide range of scientists from a wide spectrum of disciplines, including biochemistry, biophysics, cell biology, and physiology. However, in spite of enormous efforts to dissect the functions and properties of these glycan-binding proteins, limited information about how structural and biochemical aspects of these proteins can influence biological functions is available. In this review, we aim to integrate structural, biochemical, and functional aspects of this bewildering and ancient family of glycan-binding proteins and discuss their implications in physiologic and pathologic settings.

Ligand‐induced dynamical regulation of NO conversion in Mycobacterium tuberculosis truncated hemoglobin‐N

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is forced into latency by nitric oxide produced by macrophages during infection. In response to nitrosative stress M. tuberculosis has evolved a defense mechanism that relies on the oxygenated form of “truncated hemoglobin” N (trHbN), formally acting as NO‐dioxygenase, yielding the harmless nitrate ion. X‐ray crystal structures have shown that trHbN hosts a two‐branched protein matrix tunnel system, proposed to control diatomic ligand migration to the heme, as the rate‐limiting step in NO conversion to nitrate. Extended molecular dynamics simulations (0.1 μs), employed here to characterize the factors controlling diatomic ligand diffusion through the apolar tunnel system, suggest that O2 migration in deoxy‐trHbN is restricted to a short branch of the tunnel, and that O2 binding to the heme drives conformational and dynamical fluctuations promoting NO migration through the long tunnel branch. The simulation results suggest that trHbN has evolved a dual‐path mechanism for migration of O2 and NO to the heme, to achieve the most efficient NO detoxification. Proteins 2006.

Evidence for a ferryl intermediate in a heme-based dioxygenase

In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions.

Aromatic–Aromatic Interactions in Proteins: Beyond the Dimer

Aromatic residues are key widespread elements of protein structures and have been shown to be important for structure stability, folding, protein-protein recognition, and ligand binding. The interactions of pairs of aromatic residues (aromatic dimers) have been extensively studied in protein structures. Isolated aromatic molecules tend to form higher order clusters, like trimers, tetramers, and pentamers, that adopt particular well-defined structures. Taking this into account, we have surveyed protein structures deposited in the Protein Data Bank in order to find clusters of aromatic residues in proteins larger than dimers and characterized them. Our results show that larger clusters are found in one of every two unique proteins crystallized so far, that the clusters are built adopting the same trimer motifs found for benzene clusters in vacuum, and that they are clearly nonlocal brining primary structure distant sites together. We extensively analyze the trimers and tetramers conformations and found two main cluster types: a symmetric cluster and an extended ladder. Finally, using calmodulin as a test case, we show aromatic clsuters possible role in folding and protein-protein interactions. All together, our study highlights the relevance of aromatic clusters beyond the dimer in protein function, stability, and ligand recognition.

Role of Pre-A Motif in Nitric Oxide Scavenging by Truncated Hemoglobin, HbN, of Mycobacterium tuberculosis

Mycobacterium tuberculosis truncated hemoglobin, HbN, is endowed with a potent nitric-oxide dioxygenase activity and has been found to relieve nitrosative stress and enhance in vivo survival of a heterologous host, Salmonella enterica Typhimurium, within the macrophages. These findings implicate involvement of HbN in the defense of M. tuberculosis against nitrosative stress. The protein carries a tunnel system composed of a short and a long tunnel branch that has been proposed to facilitate diatomic ligand migration to the heme and an unusual Pre-A motif at the N terminus, which does not contribute significantly to the structural integrity of the protein, as it protrudes out of the compact globin fold. Strikingly, deletion of Pre-A region from the M. tuberculosis HbN drastically reduces its ability to scavenge nitric oxide (NO), whereas its insertion at the N terminus of Pre-A lacking HbN of Mycobacterium smegmatis improved its nitric-oxide dioxygenase activity. Titration of the oxygenated adduct of HbN and its mutants with NO indicated that the stoichiometric oxidation of protein is severalfold slower when the Pre-A region is deleted in HbN. Molecular dynamics simulations show that the excision of Pre-A motif results in distinct changes in the protein dynamics, which cause the gate of the tunnel long branch to be trapped into a closed conformation, thus impeding migration of diatomic ligands toward the heme active site. The present study, thus, unequivocally demonstrates vital function of Pre-A region in NO scavenging and unravels its unique role by which HbN might attain its efficient NO-detoxification ability.

pH-Dependent Conformational Changes in Proteins and Their Effect on Experimental pKas: The Case of Nitrophorin 4

The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pKas can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different “microscopic”, conformation-dependent pKa values. In those cases, interpretation of experimental measurements of the pKa is complicated by the coupling between pH, protonation state and protein conformation. We explored these issues using Nitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation where NO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open state from which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pKas: 8.5 in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopic values to the experimentally observed “apparent” pKa, obtaining a value of 6.5, in excellent agreement with experimental data. This value must be interpreted as the pH at which the closed to open population transition takes place. More generally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentally observed ensemble dependent apparent pKas and that the insight gained in the relatively simple case of NP4 can be useful in several more complex cases involving a pH dependent transition, of great biochemical interest.

Physiological concentrations of melatonin inhibit the nitridergic pathway in the Syrian hamster retina.

In the present work, the effect of melatonin on the hamster retinal nitridergic pathway was examined. When the retinas were incubated in the presence of low concentrations (1 pM–10 nM) of melatonin for 15 min, a significant decrease of nitric oxide synthase (NOS) activity was observed. However, when crude retinal homogenates were preincubated with melatonin for 15 min, no changes in NOS activity were detected, despite the fact that under the same conditions trifluoperazine, a calmodulin inhibitor, significantly decreased enzymatic activity. Kinetic analysis showed that melatonin decreased the Vmax of retinal NOS without changes in the Km. On the other hand, low concentrations (100 pM) of melatonin significantly reduced retinal L‐arginine influx. A decrease in the Vmax of L‐arginine uptake was observed in the presence of melatonin, whereas the Km remained unchanged. Melatonin significantly inhibited the accumulation of cyclic guanosine monophosphate (cGMP) levels induced by both L‐arginine and sodium nitroprusside (SNP). In summary, the present results indicate that melatonin could be a potent inhibitor of the retinal nitridergic pathway.

Modeling heme proteins using atomistic simulations

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.

Isomerization, melting, and polarity of model water clusters: (H2O)6 and (H2O)8

Energetics, structural features, polarity, and melting transitions in water clusters containing up to eight molecules were studied using ab initio methods and empirical force field models. Our quantum approach was based on density functional theory performed at the generalized gradient approximation level. For the specific case of (H2O)6, we selected five conformers of similar energy with different geometries and dipolar moments. For these cases, the cyclic arrangement was found to be the only nonpolar aggregate. For (H2O)8, the most stable structures corresponded to nonpolar, cubic-like, D2d and S4 conformers. Higher energy aggregates exhibit a large spectrum in their polarities. The static polarizability was found to be proportional to the size of the aggregates and presents a weak dependence with the number of hydrogen bonds. In order to examine the influence of thermal fluctuations on the aggregates, we have performed a series of classical molecular dynamics experiments from low temperature up to the ...

A Microscopic Study of the Deoxyhemoglobin-Catalyzed Generation of Nitric Oxide from Nitrite Anion†

There is recent evidence suggesting that nitrite anion (NO 2 (-)) represents the major intravascular NO storage molecule whose transduction to NO is facilitated by a reduction mechanism catalyzed by deoxygenated hemoglobin (deoxy-Hb). In this work, we provide a detailed microscopic study of deoxy-Hb nitrite reductase (NIR) activity by combining classical molecular dynamics and hybrid quantum mechanical-molecular mechanical simulations. Our results point out that two alternative mechanisms could be operative and suggest that the most energetic barriers should stem from either reprotonation of the distal histidine or NO dissociation from the ferric heme. In the first proposed mechanism, which is similar to that proposed for bacterial NIRs, nitrite anion or nitrous acid coordinates to the heme through the N atom. This pathway involves HisE7 in a one or two proton transfer process, depending on whether the active species is nitrite anion or nitrous acid, to yield an intermediate Fe(III)NO species which eventually dissociates leading to NO and methemoglobin. In the second mechanism, the nitrite anion coordinates to the heme through the O atom. This pathway requires only one proton transfer from HisE7 and leads directly to the formation of a hydroxo Fe(III) complex and NO.