Structural determinants of ligand migration in Mycobacterium tuberculosis truncated hemoglobin O

Mycobacterium tuberculosis is the causative agent of human tuberculosis, one of the most prevalent infectious diseases in the world. Its genome hosts the glbN and glbO genes coding for two proteins, truncated hemoglobin N (trHbN) and truncated hemoglobin O (trHbO), that belong to different groups (I and II, respectively) of the recently discovered trHb family of hemeproteins. The different expression pattern and kinetics rates constants for ligand association and NO oxidation rate suggest different functions for these proteins. Previous experimental and theoretical studies showed that, in trHbs, ligand migration along the internal tunnel cavity system is a key issue in determining the ligand-binding characteristics. The X-ray structure of trHbO has been solved and shows several internal cavities and secondary-docking sites. In this work, we present an extensive investigation of the tunnel/cavity system ofM. tuberculosis trHbO by means of computer-simulation techniques. We have computed the free-energy profiles for ligand migration along three found tunnels in the oxy and deoxy w.t. and mutant trHbO proteins. Our results show that multiple-ligand migration paths are possible and that several conserved residues such as TrpG8 play a key role in the ligand-migration regulation.     

A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase

Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC α1 subunit has been expressed in Escherichia coli and characterized for the first time. The heme binding and related NO/CO binding properties of both the α1 subunit and the β1 subunit were investigated via heme reconstitution, UV–vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the α1 subunit of human sGC, lacking the conserved axial ligand, is likely to interact with heme noncovalently. On the basis of the equilibrium and kinetics of CO binding to sGC, one possible CO binding model was proposed. CO binds to human sGCβ195 by simple one-step binding, whereas CO binds to human sGCα259, possibly from both axial positions through a more complex process. The kinetics of NO dissociation from human sGC indicated that the NO dissociation from sGC was complex, with at least two release phases, and human sGCα259 has a smaller k ₁ but a larger k ₂. Additionally, the role of the cavity of the α1 subunit of human sGC was explored, and the results indicate that the cavity likely accommodates heme. These results are beneficial for understanding the overall structure of the heme binding site of the human sGC and the NO/CO signaling mechanism.     

Heme reactivity of hemoglobins. Azide and fluoride binding equilibria of free and mercuriated ferri-gamma chains.

The free gamma chains, isolated from human foetal hemoglobin, are stable when oxidized and thus suitable for ligand binding and subunit equilibrium studies. The metaquo-ferri chains, with cysteine-F9 in the free state II ag gamma SH) possess several properties which are different from those of their p-mercuribenzoate derivative (III aq gammaSHgR); these are: stronger binding of a high-field ligand (N3- minus), altered spin equilibrium and an altered subunit equilibrium. A quantitative assessment of the free energy changes associated with all individual steps involved in changing the metaquo chains to their azide derivatives has been made. The results show that the higher apparent reactivity of III ag gammaSH (compared to IIIaq gammaSHgR) for the azide ion is not solely due to compensatory effects arising from differences of subunit dissociation or of spin equilibrium: other process(es) occurring in the ligand binding site have to be considered.     

Finding molecular dioxygen tunnels in homoprotocatechuate 2,3-dioxygenase: implications for different reactivity of identical subunits

Extradiol dioxygenases facilitate microbial aerobic degradation of catechol and its derivatives by activating molecular dioxygen and incorporating both oxygen atoms into their substrates. Experimental and theoretical studies have focused on the mechanism of the reaction at the active site. However, whether the catalytic rate is limited by O₂ access to the active site has not yet been explored. Here, we choose a recently solved X-ray structure of homoprotocatechuate 2,3-dioxygenase as a typical example to determine potential pathways for O₂ migration from the solvent into the enzyme center. On the basis of the trajectories of two 10-ns molecular dynamics simulations, implicit ligand sampling was used to calculate the 3D free energy map for O₂ inside the protein. The energetically optimal routes for O₂ diffusion were identified for each subunit of the homotetrameric protein structure. The O₂ tunnels formed because of thermal fluctuations were also characterized by connecting elongated cavities inside the protein. By superimposing the favorable O₂ tunnels on to the free energy map, both energetically and geometrically preferred O₂ pathways were determined, as also were the amino acids that may be critical for O₂ passage along these paths. Our results demonstrate that identical subunits possess quite distinct O₂ tunnels. The order of O₂ affinity of these tunnels is generally consistent with the order of the catalytic rate of each subunit. As a consequence, the probability of finding the reaction product is highest in the subunit containing the highest O₂ affinity pathway.     

A chemogenomic analysis of the transmembrane binding cavity of human G-protein-coupled receptors

The amino acid sequences of 369 human nonolfactory G-protein-coupled receptors (GPCRs) have been aligned at the seven transmembrane domain (TM) and used to extract the nature of 30 critical residues supposed--from the X-ray structure of bovine rhodopsin bound to retinal--to line the TM binding cavity of ground-state receptors. Interestingly, the clustering of human GPCRs from these 30 residues mirrors the recently described phylogenetic tree of full-sequence human GPCRs (Fredriksson et al., Mol Pharmacol 2003;63:1256-1272) with few exceptions. A TM cavity could be found for all investigated GPCRs with physicochemical properties matching that of their cognate ligands. The current approach allows a very fast comparison of most human GPCRs from the focused perspective of the predicted TM cavity and permits to easily detect key residues that drive ligand selectivity or promiscuity.     

Solution structure and functional ligand screening of HI0719, a highly conserved protein from bacteria to humans in the YjgF/YER057c/UK114 family

HI0719 belongs to a large family of highly conserved proteins with no definitive molecular function and is found in organisms ranging from bacteria to humans. We describe the NMR structure of HI0719, the first solution structure for a member of this family. The overall fold is similar to the crystal structures of two homologues, YabJ from Bacillus subtilis and YjgF from Escherichia coli, and all three structures are similar to that of chorismate mutase, although there is little sequence homology and no apparent functional connection. HI0719 is a homotrimer with a distinct cavity located at the subunit interface. Six of the seven invariant residues in the high identity group of proteins are located in this cavity, suggesting that this may be a binding site for small molecules. Using previously published observations about the biological role of HI0719 family members as a guide, over 100 naturally occurring small molecules or structural analogues were screened for ligand binding using NMR spectroscopy. The targeted screening approach identified six compounds that bind to HI0719 at the putative active site. Five of these compounds are either alpha-keto acids or alpha,beta-unsaturated acids, while the sixth compound is structurally similar. Previous studies have proposed that some HI0719 homologues may act on small molecules in the isoleucine biosynthetic path and, if this is correct, the ligand screening results presented here suggest that the interaction most likely occurs with 2-ketobutyrate and/or its unstable enamine precursor.     

Characterization of crystal water molecules in a high-affinity inhibitor and hematopoietic prostaglandin D synthase complex by interaction energy studies

Hematopoietic prostaglandin D synthase (H-PGDS) is one of the two enzymes that catalyze prostaglandin D₂ synthesis and a potential therapeutic target of allergic and inflammatory responses. To reveal key molecular interactions between a high-affinity ligand and H-PGDS, we designed and synthesized a potent new inhibitor (K_D: 0.14?nM), determined the crystal structure in complex with human H-PGDS, and quantitatively analyzed the ligand–protein interactions by the fragment molecular orbital calculation method. In the cavity, 10 water molecules were identified, and the interaction energy calculation indicated their stable binding to the surface amino acids in the cavity. Among them, 6 water molecules locating from the deep inner cavity to the peripheral part of the cavity contributed directly to the ligand binding by forming hydrogen bonding interactions. Arg12, Gly13, Gln36, Asp96, Trp104, Lys112 and an essential co-factor glutathione also had strong interactions with the ligand. A strong repulsive interaction between Leu199 and the ligand was canceled out by forming a hydrogen bonding network with the adjacent conserved water molecule. Our quantitative studies including crystal water molecules explained that compounds with an elongated backbone structure to fit from the deep inner cavity to the peripheral part of the cavity would have strong affinity to human H-PGDS.     

Theoretical Investigations of Nitric Oxide Channeling in Mycobacterium tuberculosis Truncated Hemoglobin N

Mycobacterium tuberculosis group I truncated hemoglobin trHbN catalyzes the oxidation of nitric oxide (•NO) to nitrate with a second-order rate constant k ≈ 745 μM⁻¹ s⁻¹ at 23°C (nitric oxide dioxygenase reaction). It was proposed that this high efficiency is associated with the presence of hydrophobic tunnels inside trHbN structure that allow substrate diffusion to the distal heme pocket. In this work, we investigated the mechanisms of •NO diffusion within trHbN tunnels in the context of the nitric oxide dioxygenase reaction using two independent approaches. Molecular dynamics simulations of trHbN were performed in the presence of explicit •NO molecules. Successful •NO diffusion from the bulk solvent to the distal heme pocket was observed in all simulations performed. The simulations revealed that •NO interacts with trHbN at specific surface sites, composed of hydrophobic residues located at tunnel entrances. The entry and the internal diffusion of •NO inside trHbN were performed using the Long, Short, and EH tunnels reported earlier. The Short tunnel was preferentially used by •NO to reach the distal heme pocket. This preference is ascribed to its hydrophobic funnel-shape entrance, covering a large area extending far from the tunnel entrance. This funnel-shape entrance triggers the frequent formation of solvent-excluded cavities capable of hosting up to three •NO molecules, thereby accelerating •NO capture and entry. The importance of hydrophobicity of entrances for •NO capture is highlighted by a comparison with a polar mutant for which residues at entrances were mutated with polar residues. A complete map of •NO diffusion pathways inside trHbN matrix was calculated, and •NO molecules were found to diffuse from Xe cavity to Xe cavity. This scheme was in perfect agreement with the three-dimensional free-energy distribution calculated using implicit ligand sampling. The trajectories showed that •NO significantly alters the dynamics of the key amino acids of Phe⁶²(E15), a residue proposed to act as a gate controlling ligand traffic inside the Long tunnel, and also of Ile¹¹⁹(H11), at the entrance of the Short tunnel. It is noteworthy that •NO diffusion inside trHbN tunnels is much faster than that reported previously for myoglobin. The results presented in this work shed light on the diffusion mechanism of apolar gaseous substrates inside protein matrix.     

Ligand-dependent and -independent integrin focal contact localization: the role of the alpha chain cytoplasmic domain.

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta 1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha 1 wild-type subunit (HA1 cells) or the cytoplasmic domain deleted alpha 1 subunit (CYT cells). Both cell lines express similar levels of the human alpha 1 subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha 1 beta 1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha 5 beta 1 but not for alpha 1 beta 1, there is a striking difference in the cellular localization of alpha 1 beta 1. The HA1 cells show only alpha 5 in focal contacts, without alpha 1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha 1 appears in focal contacts along with the alpha 5/beta 1. Thus, there is both ligand-dependent (alpha 5/beta 1) and ligand-independent (alpha 1/beta 1) focal contact localization in these cells. The truncated alpha 1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha 1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.     

Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis.

The X-ray crystal structure of a human copper/zinc superoxide dismutase mutant (G37R CuZnSOD) found in some patients with the inherited form of Lou Gehrig's disease (FALS) has been determined to 1.9 angstroms resolution. The two SOD subunits have distinct environments in the crystal and are different in structure at their copper binding sites. One subunit (subunit[intact]) shows a four-coordinate ligand geometry of the copper ion, whereas the other subunit (subunit[broken]) shows a three-coordinate geometry of the copper ion. Also, subunit(intact) displays higher atomic displacement parameters for backbone atoms ((B) = 30 +/- 10 angstroms2) than subunit(broken) ((B) = 24 +/- 11 angstroms2). This structure is the first CuZnSOD to show large differences between the two subunits. Factors that may contribute to these differences are discussed and a possible link of a looser structure to FALS is suggested.     

Radiation Inactivation Analysis of Progesterone Receptor in Human Uterine Cytosol

It is believed that human progesterone receptor (PR) contains a ligand binding subunit A (83 kDa) or subunit B (120 kDa) and 2 copies of heat shock proteins (hsp90) of molecular weight 90 kDa. To elucidate the mechanism of hormone binding, we employed radiation inactivation to determine its functional size. The functional masses determined in the presence of glycerol, molybdate and potassium chloride were 120 \pm 14, 124 \pm 13 and 130 \pm 20 kDa, respectively. From scatchard plot analysis, the radiation decreased the binding sites and increased the binding affinity of PR with ligand. The functional masses of PR dissolved in the three variant buffers were similar to the molecular weight of PR subunit B. The results implied that PR subunit B could bind with ligand despite hsp90 and hsp90 was not involved in the PR binding to progesterone.     

Synthetic site-directed ligands

Complexes of nucleotides, peptides and aromatic hapten-like compounds with immunoglobulin fragments were studied by X-ray analysis. After tri- or hexanucleotides of deoxythymidylate were diffused into triclinic crystals of a Fab (BV04-01) with specificity for single-stranded DNA, extensive changes were detected throughout the structure of the protein. The Fab co-crystallized with a tri- or pentanucleotide in a different space group (monoclinic), an observation sometimes correlated with alterations in the structure of the 'native' protein. Structural analyses of the co-crystals are in progress for direct comparisons with the unliganded Fab. In crystals of a human (Mcg) Bence-Jones dimer, synthetic opioid peptides, chemotactic peptides or dinitrophenyl (DNP) derivatives could be diffused into a large conical binding cavity. The conformations of both the ligand and the protein were usually altered during the binding process. At the base of the cavity tyrosine residues could be displaced like trap-doors to permit entry of some opioid peptides and DNP compounds into a deep binding pocket. In co-crystals of the dimer and bis(DNP)lysine, two ligand molecules were bound in tandem, one in the main cavity and the second in the deep pocket. One ligand adopted an extended conformation, with the epsilon-DNP ring near the floor of the main cavity and the alpha-DNP group in solvent outside the binding site. There were no significant conformational changes in the protein. In contrast, the second ligand was very compact, with DNP rings immersed in the deep pocket, and the binding site was expanded to accommodate the oversized ligand. Peptides designed to be specific for the main cavity were incrementally constructed from minimal binding units by M. Geysen, G. Trippick, S. Rodda and their colleagues. A pentapeptide optimized for binding by this method was diffused into a crystal of the dimer and found by Fourier difference analysis to lodge exclusively in the main cavity as predicted. Binding regions in the BV04-01 Fab and the Mcg dimer were markedly different in size and shape. The Fab had a groove-type site, in which a layer of sidechains acted like a false floor over regions analogous to the cavity and deep pocket of the Bence-Jones dimer.     

Identification of CRALBP ligand interactions by photoaffinity labeling, hydrogen/deuterium exchange, and structural modeling

Cellular retinaldehyde-binding protein (CRALBP) functions in the retinal pigment epithelium (RPE) as an acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a substrate carrier for 11-cis-retinol dehydrogenase. Toward a better understanding of CRALBP function, the ligand binding cavity in human recombinant CRALBP (rCRALBP) was characterized by photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal and by high resolution mass spectrometric topological analyses. Eight photoaffinity-modified residues were identified in rCRALBP by liquid chromatography tandem mass spectrometry, including Tyr(179), Phe(197), Cys(198), Met(208), Lys(221), Met(222), Val(223), and Met(225). Multiple different adduct masses were found on the photolabeled residues, and the molecular identity of each modification remains unknown. Supporting the specificity of photo-labeling, 50% of the modified residues have been associate with retinoid interactions by independent analyses. In addition, topological analysis of apo- and holo-rCRALBP by hydrogen/deuterium exchange and mass spectrometry demonstrated residues 198-255 incorporate significantly less deuterium when the retinoid binding pocket is occupied with 11-cis-retinal. This hydrophobic region encompasses all but one of the photo-labeled residues. A structural model of CRALBP ligand binding domain was constructed based on the crystal structures of three homologues in the CRAL-TRIO family of lipid-binding proteins. In the model, all of the photolabeled residues line the ligand binding cavity except Met(208), which appears to reside in a flexible loop at the entrance/exit of the ligand cavity. Overall, the results expand to 12 the number of residues proposed to interact with ligand and provide further insight into CRALBP ligand and protein interactions.     

One- and two-dimensional NMR investigations of the heme pocket in free alpha(CO) chains from human hemoglobin

Two-dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated alpha(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J-correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOSEY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu86, Leu-91, Val-93, Leu-101 and Leu-136. Starting from the known crystallographic structure of the alpha subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring-current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the alpha(CO) subunits in crystals of HbA(CO) and of the free alpha(CO) chains. The one-dimensional NMR spectra were used to monitor the pH-induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p-chloromercuribenzoate is bound to Cys-104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.     

Accessibility of cysteines in the native bovine rod cGMP-gated channel

Cyclic nucleotide-gated channels of photoreceptors and olfactory sensory neurons are tetramers consisting of A and B subunits. Here, the accessibility of the cysteines of the bovine rod cyclic nucleotide-gated channel is examined as a function of ligand binding. N-Ethylmaleimide-modified cysteines of both subunits were identified by mass spectrometry after trypsin digestion. In the absence of ligand, the intracellular carboxy-terminal cysteines of both subunits were accessible to N-ethylmaleimide. Activation of the channel abolished the accessibility of Cys505 of the A subunit and Cys1104 of the B subunit, with both being conserved cysteines of the cyclic nucleotide-binding sites. The cysteine of the pore loop of the B subunit was also found to be modified by this reagent in the absence of ligand. The total number of accessible cysteines of each subunit was determined by mass shifting upon modification with polyethylene glycol maleimide. In the absence of cyclic nucleotides, this hydrophilic reagent only weakly labeled cysteines of the A subunit but readily labeled at least three cysteines of the B subunit. Ligand binding exposed two cysteines of the A subunit and one cysteine of the B subunit to chemical modification. Double-modification experiments suggest that some of these cysteines are in or close to membrane-spanning domains. However, these cysteines could not yet be identified. Together, the cysteine accessibility of the native rod cyclic nucleotide-gated channel varies markedly upon ligand binding, thus indicating major structural rearrangements, which are of functional importance for channel activation.     

Chick integrin alpha V subunit molecular analysis reveals high conservation of structural domains and association with multiple beta subunits in embryo fibroblasts.

We have cloned and characterized a chick homologue of the human vitronectin receptor alpha subunit (alpha v) whose primary sequence is 83% identical with its human counterpart but less than 40% identical with any other known integrin alpha subunit. Comparison of the chick and human sequences reveals several highly conserved regions, including the cytoplasmic domain. The putative ligand binding domain contains alpha v-specific residues that may contribute to ligand binding specificity. These are concentrated in three regions that are located before and between the first three Ca2+ binding domains. Polyclonal antibodies raised against two peptides deduced from the putative cytoplasmic and extracellular domains of the chick alpha v sequence recognize specifically integrin heterodimers in chick embryo fibroblasts. At least three putative beta subunits coimmunoprecipitate with the chick alpha v subunit. In addition to a protein with the same molecular weight as beta 3 (94K), protein bands of Mr 84K and 110K are also coprecipitated. By successive immunodepletions, we demonstrate that this latter Mr 110K subunit is beta 1, which appears to be one of the alpha v-associated subunits in chick embryo fibroblasts.     

gamma-Aminobutyric acid/benzodiazepine receptor protein carries binding sites for both ligands on both two major peptide subunits

Affinity column-purified GABA-benzodiazepine receptor proteins from human, cow, and rat brain were photoaffinity labeled with both [3H]flunitrazepam and [3H]muscimol and examined by gel electrophoresis in sodium dodecyl sulfate. Using high receptor protein concentrations (1 microM), the benzodiazepine ligand [3H]flunitrazepam was incorporated covalently primarily into the expected 52 kiloDalton major subunit but also significantly into a second 57 kiloDalton peptide. Likewise the GABA ligand [3H]muscimol photolabeled primarily the 57 kiloDalton peptide but also to some extent the 52 kiloDalton peptide. This cross-labeling suggests strongly that both major subunits carry binding sites for both GABA and benzodiazepine.     

Escape of a Small Molecule from Inside T4 Lysozyme by Multiple Pathways

The T4 lysozyme L99A mutant is often used as a model system to study small-molecule binding to proteins, but pathways for ligand entry and exit from the buried binding site and the associated protein conformational changes have not been fully resolved. Here, molecular dynamics simulations were employed to model benzene exit from its binding cavity using the weighted ensemble (WE) approach to enhance sampling of low-probability unbinding trajectories. Independent WE simulations revealed four pathways for benzene exit, which correspond to transient tunnels spontaneously formed in previous simulations of apo T4 lysozyme. Thus, benzene unbinding occurs through multiple pathways partially created by intrinsic protein structural fluctuations. Motions of several α-helices and side chains were involved in ligand escape from metastable microstates. WE simulations also provided preliminary estimates of rate constants for each exit pathway. These results complement previous works and provide a semiquantitative characterization of pathway heterogeneity for binding of small molecules to proteins.     

The structure and function of plant hemoglobins.

Plants, like humans, contain hemoglobin. Three distinct types of hemoglobin exist in plants: symbiotic, non-symbiotic, and truncated hemoglobins. Crystal structures and other structural and biophysical techniques have revealed important knowledge about ligand binding and conformational stabilization in all three types. In symbiotic hemoglobins (leghemoglobins), ligand binding regulatory mechanisms have been shown to differ dramatically from myoglobin and red blood cell hemoglobin. In the non-symbiotic hemoglobins found in all plants, crystal structures and vibrational spectroscopy have revealed the nature of the structural transition between the hexacoordinate and ligand-bound states. In truncated hemoglobins, the abbreviated globin is porous, providing tunnels that may assist in ligand binding, and the bound ligand is stabilized by more than one distal pocket residue. Research has implicated these plant hemoglobins in a number of possible functions differing among hemoglobin types, and possibly between plant species.