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1.  Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design 
Biochemical Journal  2016;473(19):3371-3383.
Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the β-subunit (F41). We therefore replaced this residue with a tyrosine (βF41Y, Hb Mequon). The βF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, βF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the β-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in βF41Y. NO bioavailability was enhanced in βF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the β-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.
PMCID: PMC5095908  PMID: 27470146
hemoglobin; oxidative stress; reactive oxygen species
Biochimica et biophysica acta  2015;1854(9):1184-1193.
O -acetylserine sulfhydrylase A (CysK) is the pyridoxal 5′-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This “cysteine synthase” complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40 years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional “moonlighting” activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode C. elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site.
PMCID: PMC4506874  PMID: 25731080
3.  Identification of a Small Molecule that Increases Hemoglobin Oxygen Affinity and Reduces SS Erythrocyte Sickling 
ACS Chemical Biology  2014;9(10):2318-2325.
Small molecules that increase the oxygen affinity of human hemoglobin may reduce sickling of red blood cells in patients with sickle cell disease. We screened 38 700 compounds using small molecule microarrays and identified 427 molecules that bind to hemoglobin. We developed a high-throughput assay for evaluating the ability of the 427 small molecules to modulate the oxygen affinity of hemoglobin. We identified a novel allosteric effector of hemoglobin, di(5-(2,3-dihydro-1,4-benzodioxin-2-yl)-4H-1,2,4-triazol-3-yl)disulfide (TD-1). TD-1 induced a greater increase in oxygen affinity of human hemoglobin in solution and in red blood cells than did 5-hydroxymethyl-2-furfural (5-HMF), N-ethylmaleimide (NEM), or diformamidine disulfide. The three-dimensional structure of hemoglobin complexed with TD-1 revealed that monomeric units of TD-1 bound covalently to β-Cys93 and β-Cys112, as well as noncovalently to the central water cavity of the hemoglobin tetramer. The binding of TD-1 to hemoglobin stabilized the relaxed state (R3-state) of hemoglobin. TD-1 increased the oxygen affinity of sickle hemoglobin and inhibited in vitro hypoxia-induced sickling of red blood cells in patients with sickle cell disease without causing hemolysis. Our study indicates that TD-1 represents a novel lead molecule for the treatment of patients with sickle cell disease.
PMCID: PMC4205001  PMID: 25061917
4.  From protein structure to function via single crystal optical spectroscopy 
The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic “artifacts,” including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5′-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms.
PMCID: PMC4428442  PMID: 25988179
protein crystal; microspectrophotometry; conformational changes; X-ray crystallography; metastable intermediate; structure-function relationship; synchrotron source
5.  Tertiary and quaternary allostery in HbII from Scapharca inaequivalvis 
Biochemistry  2013;52(12):10.1021/bi301620x.
The clam Scapharca inaequivalvis possesses two cooperative oxygen binding hemoglobins in its red cells: a homodimeric HbI and a heterotetrameric A2B2 HbII. Each AB dimeric half of HbII is assembled very similarly to that of the well studied HbI. This study presents crystal structures of HbII along with oxygen binding data both in the crystalline state and in wet nanoporous silica gels. Despite very similar ligand-linked structural transitions observed in HbI and HbII crystals, HbII in the crystal or encapsulated in silica gels apparently exhibits minimal cooperativity in oxygen binding, in contrast with the full cooperativity exhibited by HbI crystals. However, oxygen binding curves in the crystal indicate the presence of a significant functional inequivalence of A and B chains. When this inequivalence is taken into account, both crystal and R state gel functional data are consistent with the conservation of a tertiary contribution to cooperative oxygen binding, quantitatively similar to that measured for HbI, and are in keeping with the structural information. Furthermore, our results indicate that to fully express the cooperative ligand binding, HbII requires quaternary transitions hampered by crystal lattice and gel encapsulation, revealing greater complexity in cooperative function than the direct communication across a dimeric interface observed in HbI.
PMCID: PMC3742685  PMID: 23458680
X-ray crystallography; microspectrophotometry; silica gel; cooperativity; hemoglobin
6.  Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target 
PLoS ONE  2013;8(10):e77558.
The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.
PMCID: PMC3805590  PMID: 24167577
7.  Asymmetry of the Active Site Loop Conformation between Subunits of Glutamate-1-semialdehyde Aminomutase in Solution 
BioMed Research International  2013;2013:353270.
Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5′-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5′-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.
PMCID: PMC3747428  PMID: 23984351
8.  CO Rebinding Kinetics and Molecular Dynamics Simulations Highlight Dynamic Regulation of Internal Cavities in Human Cytoglobin 
PLoS ONE  2013;8(1):e49770.
Cytoglobin (Cygb) was recently discovered in the human genome and localized in different tissues. It was suggested to play tissue-specific protective roles, spanning from scavenging of reactive oxygen species in neurons to supplying oxygen to enzymes in fibroblasts. To shed light on the functioning of such versatile machinery, we have studied the processes supporting transport of gaseous heme ligands in Cygb. Carbon monoxide rebinding shows a complex kinetic pattern with several distinct reaction intermediates, reflecting rebinding from temporary docking sites, second order recombination, and formation (and dissociation) of a bis-histidyl heme hexacoordinated reaction intermediate. Ligand exit to the solvent occurs through distinct pathways, some of which exploit temporary docking sites. The remarkable change in energetic barriers, linked to heme bis-histidyl hexacoordination by HisE7, may be responsible for active regulation of the flux of reactants and products to and from the reaction site on the distal side of the heme. A substantial change in both protein dynamics and inner cavities is observed upon transition from the CO-liganded to the pentacoordinated and bis-histidyl hexacoordinated species, which could be exploited as a signalling state. These findings are consistent with the expected versatility of the molecular activity of this protein.
PMCID: PMC3537629  PMID: 23308092
9.  Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes 
BMC Biochemistry  2011;12:66.
Conjugation of human and animal hemoglobins with polyethylene glycol has been widely explored as a means to develop blood substitutes, a novel pharmaceutical class to be used in surgery or emergency medicine. However, PEGylation of human hemoglobin led to products with significantly different oxygen binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity. These recent findings call for the biotechnological development of stable, low-affinity PEGylated hemoglobins with low NO dioxygenase reactivity.
To investigate the effects of PEGylation on protein structure and function, we compared the PEGylation products of human hemoglobin and Trematomus bernacchii hemoglobin, a natural variant endowed with a remarkably low oxygen affinity and high tetramer stability. We show that extension arm facilitated PEGylation chemistry based on the reaction of T. bernacchii hemoglobin with 2-iminothiolane and maleimido-functionalyzed polyethylene glycol (MW 5000 Da) leads to a tetraPEGylated product, more homogeneous than the corresponding derivative of human hemoglobin. PEGylated T. bernacchii hemoglobin largely retains the low affinity of the unmodified tetramer, with a p50 50 times higher than PEGylated human hemoglobin. Moreover, it is still sensitive to protons and the allosteric effector ATP, indicating the retention of allosteric regulation. It is also 10-fold less reactive towards nitrogen monoxide than PEGylated human hemoglobin.
These results indicate that PEGylated hemoglobins, provided that a suitable starting hemoglobin variant is chosen, can cover a wide range of oxygen-binding properties, potentially meeting the functional requirements of blood substitutes in terms of oxygen affinity, tetramer stability and NO dioxygenase reactivity.
PMCID: PMC3268738  PMID: 22185675
10.  Bound Water at Protein-Protein Interfaces: Partners, Roles and Hydrophobic Bubbles as a Conserved Motif 
PLoS ONE  2011;6(9):e24712.
There is a great interest in understanding and exploiting protein-protein associations as new routes for treating human disease. However, these associations are difficult to structurally characterize or model although the number of X-ray structures for protein-protein complexes is expanding. One feature of these complexes that has received little attention is the role of water molecules in the interfacial region.
A data set of 4741 water molecules abstracted from 179 high-resolution (≤ 2.30 Å) X-ray crystal structures of protein-protein complexes was analyzed with a suite of modeling tools based on the HINT forcefield and hydrogen-bonding geometry. A metric termed Relevance was used to classify the general roles of the water molecules.
The water molecules were found to be involved in: a) (bridging) interactions with both proteins (21%), b) favorable interactions with only one protein (53%), and c) no interactions with either protein (26%). This trend is shown to be independent of the crystallographic resolution. Interactions with residue backbones are consistent for all classes and account for 21.5% of all interactions. Interactions with polar residues are significantly more common for the first group and interactions with non-polar residues dominate the last group. Waters interacting with both proteins stabilize on average the proteins' interaction (−0.46 kcal mol−1), but the overall average contribution of a single water to the protein-protein interaction energy is unfavorable (+0.03 kcal mol−1). Analysis of the waters without favorable interactions with either protein suggests that this is a conserved phenomenon: 42% of these waters have SASA ≤ 10 Å2 and are thus largely buried, and 69% of these are within predominantly hydrophobic environments or “hydrophobic bubbles”. Such water molecules may have an important biological purpose in mediating protein-protein interactions.
PMCID: PMC3178540  PMID: 21961043
11.  Design of O-acetylserine sulfhydrylase inhibitors by mimicking Nature 
Journal of medicinal chemistry  2010;53(1):345-356.
The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing L-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. 400 MNXXI pentapeptides were generated, docked into OASS active site using GOLD and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4, and to a lesser extent, at position P3, also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptides-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.
PMCID: PMC2804909  PMID: 19928859
12.  Haemoglobin-based oxygen carriers: research and reality towards an alternative to blood transfusions 
Blood Transfusion  2010;8(Suppl 3):s59-s68.
PMCID: PMC2897202  PMID: 20606751
haemoglobin; oxygen therapy; transfusion; red blood cells
13.  Identification of Xenoestrogens in Food Additives by an Integrated in Silico and in Vitro Approach 
In the search for xenoestrogens within food additives, we have analyzed the Joint FAO-WHO expert committee database, containing 1500 compounds, using an integrated in silico and in vitro approach. This analysis identified 31 potential estrogen receptor α ligands that were reduced to 13 upon applying a stringent filter based on ligand volume and binding mode. Among the 13 potential xenoestrogens, four were already known to exhibit an estrogenic activity, and the other nine were assayed in vitro, determining the binding affinity to the receptor and biological effects. Propyl gallate was found to act as an antagonist, and 4-hexylresorcinol was found to act as a potent transactivator; both ligands were active at nanomolar concentrations, as predicted by the in silico analysis. Some caution should be issued for the use of propyl gallate and 4-hexylresorcinol as food additives.
PMCID: PMC2758355  PMID: 19063592
14.  Energetics of the protein-DNA-water interaction 
To understand the energetics of the interaction between protein and DNA we analyzed 39 crystallographically characterized complexes with the HINT (Hydropathic INTeractions) computational model. HINT is an empirical free energy force field based on solvent partitioning of small molecules between water and 1-octanol. Our previous studies on protein-ligand complexes demonstrated that free energy predictions were significantly improved by taking into account the energetic contribution of water molecules that form at least one hydrogen bond with each interacting species.
An initial correlation between the calculated HINT scores and the experimentally determined binding free energies in the protein-DNA system exhibited a relatively poor r2 of 0.21 and standard error of ± 1.71 kcal mol-1. However, the inclusion of 261 waters that bridge protein and DNA improved the HINT score-free energy correlation to an r2 of 0.56 and standard error of ± 1.28 kcal mol-1. Analysis of the water role and energy contributions indicate that 46% of the bridging waters act as linkers between amino acids and nucleotide bases at the protein-DNA interface, while the remaining 54% are largely involved in screening unfavorable electrostatic contacts.
This study quantifies the key energetic role of bridging waters in protein-DNA associations. In addition, the relevant role of hydrophobic interactions and entropy in driving protein-DNA association is indicated by analyses of interaction character showing that, together, the favorable polar and unfavorable polar/hydrophobic-polar interactions (i.e., desolvation) mostly cancel.
PMCID: PMC1781455  PMID: 17214883

Results 1-14 (14)