Paola Dominici

Arabidopsis Nonsymbiotic Hemoglobin AHb1 Modulates Nitric Oxide Bioactivity

Nitric oxide (NO) is a widespread signaling molecule, and numerous targets of its action exist in plants. Whereas the activity of NO in erythrocytes, microorganisms, and invertebrates has been shown to be regulated by several hemoglobins, the function of plant hemoglobins in NO detoxification has not yet been elucidated. Here, we show that Arabidopsis thaliana nonsymbiotic hemoglobin AHb1 scavenges NO through production of S-nitrosohemoglobin and reduces NO emission under hypoxic stress, indicating its role in NO detoxification. However, AHb1 does not affect NO-mediated hypersensitive cell death in response to avirulent Pseudomonas syringae, suggesting that it is not involved in the removal of NO bursts originated from acute responses when NO mediates crucial defense signaling functions.

Structural insight into Parkinson's disease treatment from drug-inhibited DOPA decarboxylase.

DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of l-3,4-dihydroxyphenylalanine (l-DOPA) and l-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinsons disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.

A Common Structural Basis for pH- and Calmodulin-mediated Regulation in Plant Glutamate Decarboxylase

Glutamate decarboxylase (Gad) catalyzes glutamate to gamma-aminobutyrate conversion. Plant Gad is a approximately 340 kDa hexamer, involved in development and stress response, and regulated by pH and binding of Ca(2+)/calmodulin (CaM) to the C-terminal domain. We determined the crystal structure of Arabidopsis thaliana Gad1 in its CaM-free state, obtained a low-resolution structure of the calmodulin-activated Gad complex by small-angle X-ray scattering and identified the crucial residues, in the C-terminal domain, for regulation by pH and CaM binding. CaM activates Gad1 in a unique way by relieving two C-terminal autoinhibition domains of adjacent active sites, forming a 393 kDa Gad1-CaM complex with an unusual 1:3 stoichiometry. The complex is loosely packed: thanks to the flexible linkers connecting the enzyme core with the six C-terminal regulatory domains, the CaM molecules retain considerable positional and orientational freedom with respect to Gad1. The complex thus represents a prototype for a novel CaM-target interaction mode. Thanks to its two levels of regulation, both targeting the C-terminal domain, Gad can respond flexibly to different kinds of cellular stress occurring at different pH values.

Ligand Migration and Binding in Nonsymbiotic Hemoglobins of Arabidopsis thaliana

We have studied carbon monoxide (CO) migration and binding in the nonsymbiotic hemoglobins AHb1 and AHb2 of Arabidopsis thaliana using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures. Both proteins have similar amino acid sequences but display pronounced differences in ligand binding properties, at both physiological and cryogenic temperatures. Near neutral pH, the distal HisE7 side chain is close to the heme-bound ligand in the majority of AHb1-CO molecules, as indicated by a low CO stretching frequency at 1921 cm(-1). In this fraction, two CO docking sites can be populated, the primary site B and the secondary site C. When the pH is lowered, a high-frequency stretching band at approximately 1964 cm(-1) grows at the expense of the low-frequency band, indicating that HisE7 protonates and, concomitantly, moves away from the bound ligand. Geminate rebinding barriers are markedly different for the two conformations, and docking site C is not accessible in the low-pH conformation. Rebinding of NO ligands was observed only from site B of AHb1, regardless of conformation. In AHb2, the HisE7 side chain is removed from the bound ligand; rebinding barriers are low, and CO molecules can populate only primary docking site B. These results are interpreted in terms of differences in the active site structures and physiological functions.

Structural plasticity and functional implications of internal cavities in distal mutants of type 1 non-symbiotic hemoglobin AHb1 from Arabidopsis thaliana.

The increasing number of nonsymbiotic plant hemoglobins discovered in genomic studies in the past decade raises intriguing questions about their physiological role. Among them, the nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana deserves particular attention, as it combines an extremely high oxygen affinity with an internal hexacoordination of the distal histidine HisE7 to the heme iron in the absence of exogenous ligands. In order to gain insight into the structure-function relationships of the protein, the ligand binding properties of mutants of two conserved residues of the distal cavity, HisE7 --> Leu and PheB10 --> Leu, were investigated by experimental and computational studies and compared to results determined for the wild type (wt) protein. The Fe(2+)-deoxy HisE7 --> Leu mutant exists, as expected, in the pentacoordinated form, while a mixture of penta- and hexacoordinated forms is found for the PheB10 --> Leu mutant, with an equilibrium shifted toward the pentacoordinated form with respect to the wt protein. Spectroscopic studies of the complexes of CO and CN(-) with AHb1 and its mutants show a subtle interplay of steric and electrostatic effects by distal residues on the ligand binding to the heme. Moreover, stopped-flow and flash photolysis experiments reveal substantial kinetic differences triggered by those mutations, which are particularly manifested in the enhanced geminate rebinding and bimolecular association rate. These findings are discussed in light of the drastic alterations found by molecular dynamics simulations in the nature and distribution of internal cavities in the protein matrix of the mutants, revealing an extremely large sensitivity of the protein structure to changes in distal HisE7 and PheB10 residues. Overall, data are consistent with the putative NO-dioxygenase activity attributed to AHb1.

Histidine E7 dynamics modulates ligand exchange between distal pocket and solvent in AHb1 from Arabidopsis thaliana.

The distal His residue in type 1 nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana plays a fundamental role in stabilizing the bound ligand. This residue might also be important in regulating the accessibility to the distal cavity. The feasibility of this functional role has been examined using a combination of experimental and computational methods. We show that the exchange of CO between the solvent and the reaction site is modulated by a swinging motion of the distal His, which opens a channel that connects directly the distal heme pocket with the solvent. The nearby PheB10 aids the distal His in the stabilization of the bound ligand by providing additional protection against solvation. Overall, these findings provide evidence supporting the functional implications of the conformational rearrangement found for the distal His in AHb1, which mimics the gating role proposed for the same residue in myoglobin.

Oxygen Binding to Arabidopsis thaliana AHb2 Nonsymbiotic Hemoglobin: Evidence for a Role in Oxygen Transport

Nonsymbiotic hemoglobins AHb1 and AHb2 discovered in Arabidopsis thaliana are likely to carry out distinct physiological roles, in consideration of their differences in sequence, structure, expression pattern, and tissue localization. Despite a relatively fast autoxidation in the presence of O2, we were able to collect O2‐binding curves for AHb2 in the presence of a reduction enzymatic system. AHb2 binds O2 noncooperatively with a p50 of 0.021 ± 0.003 Torr, a value consistent with a recently proposed role in O2 transport. The analysis of the internal cavities derived from the structures sampled in molecular dynamics simulations confirms strong differences with AHb1, proposed to work as a NO deoxygenase in vivo. Overall, our results are consistent with a role for AHb2 as an oxygen carrier, as recently proposed on the basis of experiments on AHb2‐overexpressing mutants of A. thaliana.

Biochemical and biophysical characterization of a plant calmodulin: Role of the N- and C-lobes in calcium binding, conformational change, and target interaction

In plants, transient elevation of intracellular Ca(2+) concentration in response to abiotic stress is responsible for glutamate decarboxylase (GAD) activation via association with calmodulin (CaM), an EF-hand protein consisting of two homologous domains (N and C). An unusual 1:2 binding mode of CaM to CaM-binding domains of GAD has long been known, however the contribution of the two CaM domains in target recognition and activation remains to be clarified. Here, we explored the coupling between physicochemical properties of Arabidopsis CaM1 (AtCaM1) and Arabidopsis GAD1 activation, focusing on each AtCaM1 lobe. We found that the four EF-loops of AtCaM1 differently contribute to the ~20 μM apparent affinity for Ca(2+) and the C-lobe shows a ~6-fold higher affinity than N-lobe (Kd(app) 5.6 μM and 32 μM for C- and N-lobes, respectively). AtCaM1 responds structurally to Ca(2+) in a manner similar to vertebrate CaM based on comparison of Ca(2+)-induced changes in hydrophobicity exposure, secondary structure, and hydrodynamic behavior. Molecular dynamics simulations of AtCaM1 apo and Ca(2+)-bound reveal that the latter state is significantly less flexible, although regions of the N-lobe remain quite flexible; this suggests the importance of N-lobe for completing the transition to the extended structure of holoprotein, consistent with data from ANS fluorescence, CD spectroscopy, and SEC analysis. Moreover, enzymatic analysis reveal that mutations in the two lobes affect GAD1 activation in similar ways and only intact AtCaM1 can fully activate GAD1. Taken together, our data provide new insights into the CaM lobes role in interactions between CaM and plant GAD.

An essential arginine residue at the binding site of pig kidney 3,4-dihydroxyphenylalanine decarboxylase☆

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by the arginine-specific reagent phenylglyoxal. Under these experimental conditions, the reaction follows pseudo-first-order kinetics with a second-order rate constant of 25 m-1 min-1. Holo- and apo-enzyme were inactivated at the same rate. However, inactivation seems to be related to modification of 1 and 2 arginyl residues per mol of holo- and apo-enzyme, respectively. Only one of these two residues was essential to decarboxylase activity of the enzyme. Phenylglyoxal-modified apo-Dopa decarboxylase retained the capacity to bind pyridoxal-P. Neither this reconstituted species nor the phenylglyoxal-modified holoenzyme were able to form Schiff base intermediates with aromatic amino acids in L and D forms. These data together with protection experiments suggest that the susceptible arginine residue in holoenzyme may somehow perturb the substrate binding site. However, unlike in other pyridoxal-P enzymes, this critical arginine in Dopa decarboxylase does not seem to behave as an anionic recognition site for the phosphate group of the coenzyme or the carboxy group of the substrate. It is speculated that this guanidyl group could function in hydrogen bonding of substrate side chain.

Metal binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana.

In addition to the well‐known Ca2+ sensor calmodulin, plants possess many calmodulin‐like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca2+ and Mg2+ binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS‐based fluorescence, to evaluate the structural effects of metal binding and metal‐induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca2+ ion with micromolar affinity (Kd ∼ 12 µM) and the presence of 10 mM Mg2+ decreases the Ca2+ affinity by ∼5‐fold. Although binding of Ca2+ to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca2+ sensors, but causes only localized structural changes in the unique functional EF‐hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role.