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1.
J Biol Chem ; 300(3): 105745, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38354784

RESUMEN

The NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at -31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.


Asunto(s)
Proteínas Hierro-Azufre , Proteínas Mitocondriales , Óxido Nítrico , Humanos , Peróxido de Hidrógeno/metabolismo , Hierro/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Óxido Nítrico/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Células HEK293 , Estabilidad Proteica
2.
Inorg Chem ; 63(23): 10713-10725, 2024 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-38805564

RESUMEN

Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure-activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.


Asunto(s)
Dominio Catalítico , Aprendizaje Automático , Metaloproteínas , Teoría Cuántica , Metaloproteínas/química , Humanos , Modelos Moleculares , Metaloproteinasa 12 de la Matriz/química , Metaloproteinasa 12 de la Matriz/metabolismo
3.
J Biomol NMR ; 77(5-6): 247-259, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37853207

RESUMEN

The robustness of NMR coherence transfer in proximity of a paramagnetic center depends on the relaxation properties of the nuclei involved. In the case of Iron-Sulfur Proteins, different pulse schemes or different parameter sets often provide complementary results. Tailored versions of HCACO and CACO experiments significantly increase the number of observed Cα/C' connectivities in highly paramagnetic systems, by recovering many resonances that were lost due to paramagnetic relaxation. Optimized 13C direct detected experiments can significantly extend the available assignments, improving the overall knowledge of these systems. The different relaxation properties of Cα and C' nuclei are exploited in CACO vs COCA experiments and the complementarity of the two experiments is used to obtain structural information. The two [Fe2S2]+ clusters containing NEET protein CISD3 and the one [Fe4S4]2+ cluster containing HiPIP protein PioC have been taken as model systems. We show that tailored experiments contribute to decrease the blind sphere around the cluster, to extend resonance assignment of cluster bound cysteine residues and to retrieve details on the topology of the iron-bound ligand residues.


Asunto(s)
Proteínas Hierro-Azufre , Resonancia Magnética Nuclear Biomolecular , Proteínas Hierro-Azufre/química , Espectroscopía de Resonancia Magnética , Imagen por Resonancia Magnética , Cisteína
4.
J Biol Inorg Chem ; 24(1): 91-101, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30470900

RESUMEN

Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.


Asunto(s)
Anhidrasa Carbónica II/química , Níquel/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica
5.
Biomol NMR Assign ; 17(1): 17-22, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36520264

RESUMEN

CISD3 is a mitochondrial protein that contains two [2Fe-2S] clusters. This protein is overexpressed in some types of cancer, so it has emerged as a potential drug target. A detailed characterization of this protein is crucial to understand how CISD3 is involved in these physiopathologies. In this study, isotopically labeled human CISD3 was expressed in Escherichia coli. A set of double and triple resonance experiments performed with standard parameters/datasets provided the assignment of 40% of the HN resonances, 47% of Cα, and 46% of C' resonances. Tailored paramagnetic HSQC, CON and CACO experiments extended up to 59% for HN, 70% for Cα and 69% for C'. The 1H, 13C and 15N NMR chemical shift assignment of human CISD3 is reported here.


Asunto(s)
Proteínas Hierro-Azufre , Humanos , Proteínas Hierro-Azufre/química , Resonancia Magnética Nuclear Biomolecular , Espectroscopía de Resonancia Magnética , Escherichia coli/metabolismo , Proteínas Mitocondriales/química
6.
J Inorg Biochem ; 244: 112222, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37068394

RESUMEN

Many proteins naturally carry metal centers, with a large share of them being in the active sites of several enzymes. Paramagnetic effects are a powerful source of structural information and, therefore, if the native metal is paramagnetic, or it can be functionally substituted with a paramagnetic one, paramagnetic effects can be used to study the metal sites, as well as the overall structure of the protein. One notable example is cobalt(II) substitution for zinc(II) in carbonic anhydrase. In this manuscript we investigate the effects of sodium thiocyanate on the chemical environment of the metal ion of the human carbonic anhydrase II. The electron paramagnetic resonance (EPR) titration of the cobalt(II) protein with thiocyanate shows that the EPR spectrum changes from A-type to C-type on passing from 1:1 to 1:1000-fold ligand excess. This indicates the occurrence of a change in the electronic structure, which may reflect a sizable change in the metal coordination environment in turn caused by a modification of the frozen solvent glass. However, paramagnetic nuclear magnetic resonance (NMR) data indicate that the metal coordination cage remains unperturbed even in 1:1000-fold ligand excess. This result proves that the C-type EPR spectrum observed at large ligand concentration should be ascribed to the low temperature at which EPR measurements are performed, which impacts on the structure of the protein when it is destabilized by a high concentration of a chaotropic agent.


Asunto(s)
Anhidrasas Carbónicas , Humanos , Anhidrasas Carbónicas/química , Tiocianatos , Ligandos , Cobalto/química , Sitios de Unión , Unión Proteica
7.
Chem Commun (Camb) ; 58(21): 3533-3536, 2022 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-35195626

RESUMEN

We present here how different iron-sulfide-glutathione ratios, applied in in vitro conditions comparable to those present in the mitochondrial matrix, affect the speciation of iron-sulfur cluster glutathione complexes. An excess of sulfide with respect to iron ions promotes the formation of a tetranuclear [FeII2FeIII2S4(GS)4]2- complex, while an excess of iron ions favors the formation of a dinuclear [FeIIFeIIIS2(GS)4]3- complex. These two complexes establish an interconversion equilibrium. The latter might play a role in the composition of the mitochondrial labile iron pool potentially contributing to the regulation of cellular iron homeostasis.

8.
J Phys Chem Lett ; 10(13): 3610-3614, 2019 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-31181162

RESUMEN

The recent derivation, based on pure quantum chemistry (QC) first-principles, of the pseudocontact shifts (PCSs) caused by a paramagnetic metal center on far away nuclei has cast doubts on the validity of the semiempirical (SE) theory, predicting PCSs to arise from the metal magnetic susceptibility anisotropy. The SE theory has been used and applied countless times, especially in the last 2 decades, to obtain structural information on proteins containing paramagnetic metal ions. We show here that the QC and SE predictions can be directly tested against experiments, provided a suitable macromolecular system is used. The SE approach yields a good prediction of the experimental PCSs while the QC one does not. It appears that the classic theory is able to grasp satisfactorily the underlying physics.

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